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What regulates the broad, but less specific, virus-cell interaction in a hepatitis B infection?

The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).

Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively.

What regulates the broad, but less specific, virus-cell interaction in a hepatitis B infection?

The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).

Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture.

What regulates the broad, but less specific, virus-cell interaction in a hepatitis B infection?

The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

What regulates the broad, but less specific, virus-cell interaction in a hepatitis B infection?

The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

What regulates the broad, but less specific, virus-cell interaction in a hepatitis B infection?

The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).

Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection.

What regulates the broad, but less specific, virus-cell interaction in a hepatitis B infection?

The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).

For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades.

What regulates the broad, but less specific, virus-cell interaction in a hepatitis B infection?

The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).

NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection.

What regulates the broad, but less specific, virus-cell interaction in a hepatitis B infection?

The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).

Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] .

What regulates the broad, but less specific, virus-cell interaction in a hepatitis B infection?

The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).

Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture.

What regulates the broad, but less specific, virus-cell interaction in a hepatitis B infection?

The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).

More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors.

What regulates the broad, but less specific, virus-cell interaction in a hepatitis B infection?

The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).

They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] .

What regulates the broad, but less specific, virus-cell interaction in a hepatitis B infection?

The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).

have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments.

Which protein domain of the Hepatitis B envelope is necessary for infection?

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ). HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection.

Which protein domain of the Hepatitis B envelope is necessary for infection?

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).

Which protein domain of the Hepatitis B envelope is necessary for infection?

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] .

Which protein domain of the Hepatitis B envelope is necessary for infection?

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively.

Which protein domain of the Hepatitis B envelope is necessary for infection?

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules.

Which protein domain of the Hepatitis B envelope is necessary for infection?

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture.

Which protein domain of the Hepatitis B envelope is necessary for infection?

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection.

Which protein domain of the Hepatitis B envelope is necessary for infection?

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

Which protein domain of the Hepatitis B envelope is necessary for infection?

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments.

Which protein domain of the Hepatitis B envelope is necessary for infection?

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection.

Which protein domain of the Hepatitis B envelope is necessary for infection?

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

Which protein domain of the Hepatitis B envelope is necessary for infection?

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors.

Which protein domain of the Hepatitis B envelope is necessary for infection?

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] .

Which protein domain of the Hepatitis B envelope is necessary for infection?

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

Following the successful cloning of receptor for SARS coronavirus a few years ago, Dr. Wenhui Li and colleagues raised attention again by publishing a possible receptor for hepatitis B virus in eLife. We will briefly review the significance of this finding and the future prospects of hepatitis B research. Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture.

Where is NTCP located in the body?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] .

Where is NTCP located in the body?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

Where is NTCP located in the body?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.

Where is NTCP located in the body?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection.

Where is NTCP located in the body?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane.

Where is NTCP located in the body?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well.

Where is NTCP located in the body?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection.

Where is NTCP located in the body?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

Where is NTCP located in the body?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades.

Where is NTCP located in the body?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively.

Where is NTCP located in the body?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] .

Where is NTCP located in the body?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments.

Where is NTCP located in the body?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors.

Where is NTCP located in the body?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ). HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection.

What does the NTCP protein mediate?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

What does the NTCP protein mediate?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection.

What does the NTCP protein mediate?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane.

What does the NTCP protein mediate?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well.

What does the NTCP protein mediate?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] .

What does the NTCP protein mediate?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.

What does the NTCP protein mediate?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection.

What does the NTCP protein mediate?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

What does the NTCP protein mediate?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors. Yan et al. have taken a reasonable approach to fish out possible HBV receptor(s) [6] .

What does the NTCP protein mediate?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades.

What does the NTCP protein mediate?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ). HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection.

What does the NTCP protein mediate?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules. Among others, the heparan sulfates in the membrane proteins mediate the broad, but less specific, virus-cell interaction. However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ).

What does the NTCP protein mediate?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

have taken a reasonable approach to fish out possible HBV receptor(s) [6] . They engineered the first 2-47 amino acid peptide from PreS1 to increase its capacity to be cross-linked with proteins interacting with the cell membrane, without affecting its binding specificity. In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments.

What does the NTCP protein mediate?

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors.

Is NTCP sufficient to allow HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane.

Is NTCP sufficient to allow HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.

Is NTCP sufficient to allow HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

Is NTCP sufficient to allow HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection.

Is NTCP sufficient to allow HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection.

Is NTCP sufficient to allow HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] .

Is NTCP sufficient to allow HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades.

Is NTCP sufficient to allow HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

Is NTCP sufficient to allow HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively.

Is NTCP sufficient to allow HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ). HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection.

Is NTCP sufficient to allow HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules.

Is NTCP sufficient to allow HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors.

Is NTCP sufficient to allow HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection. In addition, they documented a 2-4-fold increase of viral RNA and DNA after infection in the cell line by real-time PCR. They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection.

Is NTCP sufficient to allow HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

Text: Among the five hepatotropic hepatitis viruses, only hepatitis B virus (HBV) and its satellite hepatitis D virus (HDV) still wait for the development of an in vitro infection system in cell culture. One hepatocellular carcinoma (HCC) cell line, HepaRG, can be infected at a modest efficiency after weeks of culture and induced differentiation [1] . Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture.

Why is NTCP thought to not be sufficient for HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

As a Chinese proverb says "a thousand-mile journey starts from one incremental step". As such, the identification of NTCP as a potential viral receptor for HBV may serve as an important initial step for this journey, leading to the development of an HBV infection system to facilitate the HBV research and hepatitis B treatment.

Why is NTCP thought to not be sufficient for HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process. This can be a critical and long-awaited discovery toward understanding HBV receptors and establishing an experimental HBV infection system. Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane.

Why is NTCP thought to not be sufficient for HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry. Such cooperative cellular or viral factors have to be discovered and demonstrated to enhance the efficiency of viral infection, at a level comparable to a natural one (hundreds or thousands fold viral amplification). For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection.

Why is NTCP thought to not be sufficient for HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] . Geographically, it is a good candidate for an HBV receptor. Moreover, the authors could convert the cell lines previously non-permissible to HBV infection to permissible by over-expression of NTCP, again supporting its possible role in the HBV infection process.

Why is NTCP thought to not be sufficient for HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

The same protein was pulled down from human hepatocytes as well. The authors further produced HCC cell lines stably expressing NTCP and subsequently infected them with HBV or HDV. Immunofluorescence staining clearly demonstrated the expression of HBV and HDV proteins in these cell lines, suggestive of a successful viral infection.

Why is NTCP thought to not be sufficient for HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

For example, the authors can use the NTCP-expressing cell lines as the starting materials to systemically identify other factors (maybe carboxypeptidase D) and make these cell lines more productive and permissive to HBV infection. In the near future, standard virological assays for HBV infections, including Northern or Western blots, are expected to demonstrate the successful HBV infections in vitro. The HBV research community has searched for HBV receptors for decades.

Why is NTCP thought to not be sufficient for HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

They also showed a Southern blot supporting the presence of HBV covalently closed circular DNA in the infected cell, a well-recognized marker for productive HBV infection. Finally, they identified a stretch of 10 amino acids in the NTCP transmembrane domain, as the motif directly interacting with the PreS1 peptide. NTCP is a transmembrane protein, usually located in the lateral surface (canalicular) of hepatocytes, which mediates bile acid transport [7] .

Why is NTCP thought to not be sufficient for HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

Even primary human hepatocytes rapidly lose the capacity for HBV infection after brief cell culture. The HBV infection demands both intracellular and cell-surface factors. The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively.

Why is NTCP thought to not be sufficient for HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection. This domain has been shown to function as a direct mediator for HBV by binding presumably cellular corresponding receptor(s) [3] . More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] .

Why is NTCP thought to not be sufficient for HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

The intracellular requirements appear less stringent, as after transfection of HBV DNA into many HCC cell lines or mouse liver, which cannot be infected naturally, the viral genome is expressed and replicates actively. Thus, the failure of HBV infection is considered largely to be due to strict restriction on the interaction between HBV virions and the cell membrane. The molecules on the cell membrane needed for HBV infection can be divided into two classes: low affinity and high affinity molecules.

Why is NTCP thought to not be sufficient for HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

More importantly, the myristoylated peptide is shown to effectively block HBV infection in primary human hepatocytes and in the human hepatocytechimera mouse at a nanomolar concentration [4] . In fact, a clinical trial testing the efficacy of this peptide in preventing HBV infection has been ongoing [5] . Clearly, this preS1 peptide can be a useful probe to pull out the interacting cellular factors, including specific viral receptors.

Why is NTCP thought to not be sufficient for HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

However, the high affinity membrane partners for HBV remain elusive (the carboxypeptidase D found for duck hepatitis B virus may be the only serious contender [2] ). HBV envelope protein, namely the surface antigens, plays an essential role in the infection process. Both genetic and functional examination identified one domain in the Nterminus of HBV preS1 (amino acids 1-47) necessary for infection.

Why is NTCP thought to not be sufficient for HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

In order to obtain sufficient materials after cross-linking, they adopted the Tupaia hepatocytes, instead of human hepatocytes, for the experiments. The strategies actually brought down many membrane proteins, but in comparison with the negative control (homologous peptide without specific binding), they identified one cellular protein, NTCP (sodium taurocholate cotransporting peptide) by LC/MS/MS. The same protein was pulled down from human hepatocytes as well.

Why is NTCP thought to not be sufficient for HBV infection?

Looking forward, we need to understand how NTCP interacts with both HBV envelope proteins and with other cellular proteins, especially through the motif embedded in the cell membrane. NTCP itself is not sufficient to allow HBV infection, as the majority of HepaRG cells were found to express NPCT but not to be infected [8] . NTCP might initiate or mediate molecular interactions that can overcome the cell-surface restrictions for viral entry.

The HBV research community has searched for HBV receptors for decades. Many candidates have been discovered and then discarded. The current study, however, took advantage of a well-documented viral peptide required for HBV entry in combination with a state-of-the-art proteomics platform. As a Chinese proverb says "a thousand-mile journey starts from one incremental step".

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.

The critical role of bacterial proteases in virulence was successfully demonstrated by eliminating the proteaseencoding gene in P. gingivalis [6] . Recently described cystatin superfamily of proteins comprises both eukaryotic and prokaryotic cysteine protease inhibitors [7] . Human cystatins C, D and S, rat cystatins A and S, chicken cystatin and oryza cystatin have been reported to inhibit the replication of certain viruses and bacteria [8] although it has not yet been directly demonstrated that these effects are due to the protease inhibitory capacity of the cystatins [9] .

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.

Pseudomonas, Serratia, Streptococcus, Staphylococcus and Bacteroides sp. have potent metallo-, cysteine and serine proteases with broad ranges of activities [5] . The critical role of bacterial proteases in virulence was successfully demonstrated by eliminating the proteaseencoding gene in P. gingivalis [6] .

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.

All the above mentioned bacterial species have been shown to secrete certain cysteine proteases which play very important role in the pathogenecity of different diseases caused by these microorganisms. The minimum inhibitory concentration (MICs) of compounds (3a-d) ( Table 4 ) against all tested bacteria except E. coli and P. vulgaris, were observed to be in great agreement with their respective inhibition constant (K i )/IC 50 values against papain (Table 3 ) which clearly indicates that these compounds have the potential to inhibit the papain like cysteine proteases of these pathogens. The partition coefficient (logP) is a well-established measure of the compound's lipophilicity.

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.

Furthermore, the calculated IC 50 values were also found to be 13.40, 21.17, 94.50 and 96.50 mM for compounds 3a, 3b, 3c and 3d respectively ( Table 3) . Except compound 3b, rest of the compounds showed non competitive, reversible inhibitions but all the compounds irrespective of types of binding, showed hydrophobic and entropically driven interaction. These derivatives (3a-j) were eventually evaluated for their antibacterial activities against seven clinically important microbes (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii).

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.

It has been proven that bacterial pathogens have a unique papain-like hydrolytic activity to block the normal host cell cycle progression as the core of an avirulence (Avr) protein (AvrPphB) from the plant pathogen Pseudomonas syringae, resembles the papain-like cysteine proteases. The similarity of this AvrPphB protein with papain includes the catalytic triad of Cys-98, His-212, and Asp-227 in the AvrPphB active site [30] . Turk et al.

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.

These derivatives (3a-j) were eventually evaluated for their antibacterial activities against seven clinically important microbes (S. aureus, P. vulgaris, Group D Streptococci, Bacillus sp., E. coli, P. aeruginosa and S. morganii). Here, we are showing the data of only four compounds (3a-d) because of their significant results ( Table 4 ). All the compounds strictly followed the pattern of antiprotease activity towards bacterial growth except P. vulgaris and E. coli at one instance each (Table 4) .

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.

(staphopain) [2] , Plasmodium falciparum (falcipain-1, -2, and -3) and Trypanosoma cruzi (cruzipain) [3] are some of the most widely studied members of papain family which have been reported to be linked with severity of infection and various pathological conditions caused by these microorganisms. The activation of the kallikrein-kinin pathway, which could be activated by more than sixteen bacterial proteases, is a mechanism that some pathogens exploit to ensure that there is a supply of nutrients to the site of infection by increasing vascular permeability. This has been shown to occur in infections with several microbial species, including Pseudomonas, Serratia, Clostridium, Candida, Bacteroides, Porphyromonas and Staphylococcus sp.

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.

All the compounds strictly followed the pattern of antiprotease activity towards bacterial growth except P. vulgaris and E. coli at one instance each (Table 4) . Since compound 3c & 3d do not have much difference in their IC50 values (3c-94.5 mM and 3d-96.5 mM) against cysteine protease, papain and hence in antibacterial activity in all instances except one. It might be random due to so close in IC50 values.

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.

The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC(50) values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs.

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.

Human cystatins C, D and S, rat cystatins A and S, chicken cystatin and oryza cystatin have been reported to inhibit the replication of certain viruses and bacteria [8] although it has not yet been directly demonstrated that these effects are due to the protease inhibitory capacity of the cystatins [9] . The key role of cysteine proteases in microbial infections, coupled with the relative lack of redundancy compared to mammalian systems has made microbial proteases attractive targets for the development of novel chemotherapeutic approaches [10, 11] . Imidazopyridine ring systems represent an important class of compounds not only for their theoretical interest but also from a pharmacological point of view.

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.

Three replicates were done for each compound and experiment was repeated two times. Bacteria use their cysteine proteases for pathogenecity as could be depicted from the structure of Cif homolog in Burkholderia pseudomallei (CHBP) which reveals a papain-like fold and a conserved Cys-His-Gln catalytic triad [29] . It has been proven that bacterial pathogens have a unique papain-like hydrolytic activity to block the normal host cell cycle progression as the core of an avirulence (Avr) protein (AvrPphB) from the plant pathogen Pseudomonas syringae, resembles the papain-like cysteine proteases.

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.

This has been shown to occur in infections with several microbial species, including Pseudomonas, Serratia, Clostridium, Candida, Bacteroides, Porphyromonas and Staphylococcus sp. [4] . Many bacteria secrete several nonspecific proteases e.g. Pseudomonas, Serratia, Streptococcus, Staphylococcus and Bacteroides sp.

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.

The cysteine proteases from Streptococcus sp. (streptopain) [1] , Staphylococcus sp. (staphopain) [2] , Plasmodium falciparum (falcipain-1, -2, and -3) and Trypanosoma cruzi (cruzipain) [3] are some of the most widely studied members of papain family which have been reported to be linked with severity of infection and various pathological conditions caused by these microorganisms.

What enzymes have been reported to be linked with severity of infection and various pathological conditions caused by microorganisms?

Among all the tested compounds, compound 3a was the most potent whose MIC was the lowest among all the tested compounds and showed maximum drug score and positive values for drug likeness. In summary, the results of the present study have established that 1-substituted pyridylimidazo[1,5-a]pyridine derivatives could be candidate for novel and potent inhibitors of papain like cysteine proteases, which play deleterious role in the progression of different diseases caused by diverse microorganisms. Therefore, this group of compounds could be the subject of future research to confront the challenges with resistant microorganisms that is a major threat globally.

Although, E. coli does contain six major cysteine proteases but none belong to the CA clan of papain. It is argued that these compounds also inhibited the cysteine proteases of other clan than papain but with low efficacy. Since, pyridylimidazo[1,5-a]pyridine derivatives is absolutely new scaffold towards antibacterial agents and hence, not any standard compound(s) of same scaffold is available for reference.

At what temperatures was the assay completed?

The entropy change was obtained from the equation, The assay was done at different temperatures (32uC, 37uC, 42uC) calculating various K i of 1-pyridylimidazo[1,5-a]pyridine derivatives with papain as model enzyme. The disk diffusion method [25] was used for the preliminary antibacterial evaluation of 1-pyridylimidazo[1,5-a]pyridine derivatives. The MIC 50 of these derivatives, showing inhibition in the preliminary tests, were further determined by the microtitre plate technique using micro dilution method [26] .

After incubation at 37uC overnight, the MICs were taken as the lowest inhibitor concentration at which the bacterial growth was inhibited. The average of three values was calculated and that was the MIC for the test material and bacterial strain. For the agar plate count method [27] , 25 mL aliquots of bacteria at 1610 5 CFU/ml in SPB containing 0.03% LB broth were incubated with 25 mL of diluted compounds for 2 h at 37uC.

At what temperatures was the assay completed?

The entropy change was obtained from the equation, The assay was done at different temperatures (32uC, 37uC, 42uC) calculating various K i of 1-pyridylimidazo[1,5-a]pyridine derivatives with papain as model enzyme. The disk diffusion method [25] was used for the preliminary antibacterial evaluation of 1-pyridylimidazo[1,5-a]pyridine derivatives. The MIC 50 of these derivatives, showing inhibition in the preliminary tests, were further determined by the microtitre plate technique using micro dilution method [26] .

Temperature dependence of the inhibition constants was used to determine the thermodynamic parameters. Changes in enthalpy (DH) were determined from the Van't Hoff plots by using the equation, Where DH is enthalpy change, R is gas constant, DS is entropy change and T is the absolute temperature. The entropy change was obtained from the equation, The assay was done at different temperatures (32uC, 37uC, 42uC) calculating various K i of 1-pyridylimidazo[1,5-a]pyridine derivatives with papain as model enzyme.

At what temperatures was the assay completed?

The entropy change was obtained from the equation, The assay was done at different temperatures (32uC, 37uC, 42uC) calculating various K i of 1-pyridylimidazo[1,5-a]pyridine derivatives with papain as model enzyme. The disk diffusion method [25] was used for the preliminary antibacterial evaluation of 1-pyridylimidazo[1,5-a]pyridine derivatives. The MIC 50 of these derivatives, showing inhibition in the preliminary tests, were further determined by the microtitre plate technique using micro dilution method [26] .

To solutions of active papain (final concentration: 0.05 mM) were added concentrated solutions of the different derivatives to final concentrations of 0.2 mM. After incubation for 30 min at 37uC, the substrate solution was added and after a further incubation for 20 min the reaction was stopped by the addition of 5% trichloric acid (TCA) acidified with 2.25% HCl and the absorbance of the reaction mixture was determined at a wavelength of 410 nm by Microplate Manager 4.0 (Bio-Rad laboratories). The same procedure was used at 32uC and 42uC for thermodynamics studies.

At what temperatures was the assay completed?

The entropy change was obtained from the equation, The assay was done at different temperatures (32uC, 37uC, 42uC) calculating various K i of 1-pyridylimidazo[1,5-a]pyridine derivatives with papain as model enzyme. The disk diffusion method [25] was used for the preliminary antibacterial evaluation of 1-pyridylimidazo[1,5-a]pyridine derivatives. The MIC 50 of these derivatives, showing inhibition in the preliminary tests, were further determined by the microtitre plate technique using micro dilution method [26] .

For the agar plate count method [27] , 25 mL aliquots of bacteria at 1610 5 CFU/ml in SPB containing 0.03% LB broth were incubated with 25 mL of diluted compounds for 2 h at 37uC. The mixtures of bacteria and compounds were serially diluted 10-fold with SPB and plated on LB plates that were incubated at 37uC overnight. Bacterial colonies were enumerated the following day.