We tested the effect of sampling location , general feeding strategy , phylogeny, and standard length on δ13C and δ15N. The reef fish communities of the artificial and natural reef tracts were significantly different; the artificial sites also exhibited more variability. For all samples, δ13C and δ15N ranged from -19.5 to -13.1‰ and 6.7–13.3‰, respectively. Significant effects were detected for both general feeding strategy and phylogeny. Significant differences were also seen in δ13C and δ15N profiles between artificial and natural reefs; however, these changes were primarily driven by differences in fish community structure, rather than by changes in the feeding strategy or trophic relationships of individual fish taxa. The trophic guild invertivore was the only group of fish to demonstrate significant isotopic differences between both reef tracts (inner and outer) and reef types . The artificial reef may act more as a foraging corridor between the natural first and second reef tracts for omnivores and carnivores. If the function of artificial reefs is to provide additional foraging habitat for fishes, then perhaps more time is needed for the trophically important, infaunal invertebrate community to develop similarly to the natural reef environment.Artificial reefs may enhance the biological production of reef-associated flora and fauna, but their trophic structure relative to that of natural reefs remains understudied. We assessed trophic relationships by 1) comparing reef fish communities and 2) comparing δ Reef fish, Stable isotopes, Artificial reef, Trophic dynamics In resp12/C13 and N14/N15 are most frequently used for trophic studies of marine fauna  between trophic steps  to study the trophic behavior of reef-associated fish is well-established ; however, we did detect significant differences in community species composition between natural and artificial reefs . Further analysis indicated that artificial reef communities were largely frequented by a subset of the overall community, most notably by Tomtate, Slippery Dick, Porkfish, Lionfish, Sharpnose Puffer, Purple Reef Fish, and French Grunt , nor did it affect species richness or evenness .Analysis of visual survey data revealed that artificial and natural reefs did not differ in their fish species richness , and reef subtype (inner vs. outer reef tract) on δ15N and δ13C values separately using ANOVA  and to a lesser extent, of reef type  but not by the artificial or natural state of the reef where it was collected – in other words, the stable isotope profiles of each fish taxon reflected their feeding ecology rather than reef type. We saw no evidence that fishes adjusted their feeding preference depending on the reef type from which they were sampled. Lastly, δ13C was significantly influenced by both trophic guild and by the distance from shore at which the individual was sampled, suggesting that there are differences between these communities  in their basal carbon sources.Our primary conclusion, based on comparative analysis of the catch and survey data, is that fish community composition differed significantly among reef types, and that this in turn shaped the overall stable isotope profiles at each location . Further3.1Species composition was significantly influenced by reef type, suggesting that the fish communities of the artificial reef sites and natural reef sites differed in their species composition. Previous studies in the region have shown that the fish assemblages of the first and second reef tracts are different . Additio3.215N and δ13C values of muscle tissue were primarily influenced by family and trophic guild. The results are also consistent with prior studies which found dietary habits directly affect δ15N and δ13C, and that higher trophic level feeding behavior results in enrichment of δ15N and δ13C  most closely resembles the δ13C of benthic marine algae, which makes it difficult to distinguish the two primary producers  was too depleted, so mangroves were excluded. Seagrasses were also considered as a possible food source, but the exhibited δ13C range (-13 to -7‰) was much more enriched than the herbivores collected in this study, with the exception of the Bicolor Damselfish. 15N of 5.6‰, which was too enriched to be the basal carbon source for the Bicolor Damselfish of this study. 13C by as much as 9‰. It may be that the complex structure of the artificial reef piles where the Bicolor Damselfish were sampled reduced water movement enough to cause the algal food source to become more enriched in δ13C.The calculated δ feeders . The avene algae ; this agroducers . It is u3.4omnivore were slightly more enriched in δ15N (9.3‰ ± 0.5) and more depleted in δ13C (-17.0‰ ± 0.85) when compared to those in the trophic guild herbivore  of trophic guild planktivore suggests that phytoplankton is the source of primary production in the diet of these species. Additionally, the δ15N-based trophic position estimates using phytoplankton as the food web base more closely matches the stomach content-based trophic position when compared to the other primary producers. Phytoplankton tends to exhibit δ15N that is less enriched when compared to marine benthic algae  relative to the other trophic guilds in this study, with the exception of the trophic guild carnivore, which is consistent with higher trophic level feeding habits relative to the other trophic guilds of this study. Species within the trophic guild invertivore are known to feed primarily on marine invertebrate fauna, and 15N-based trophic position estimates using marine benthic algae as the food web base were closest to the stomach content-based trophic positions.The trophic guild 3.6carnivore consists of reef-associated fish species that exhibit a diet of both marine invertebrates and teleost fishes. Piscivory (exclusive consumption of fishes) is associated with higher trophic level feeding, and it was expected for this reason that individuals within this guild would exhibit the highest levels of enrichment in δ15N (15N (10.7‰ ± 1.01), it is only slightly more enriched compared to the mean δ15N of the trophic guild invertivore (10.5‰ ± 0.74). Additionally, the mean δ13C of the trophic guild carnivore (15.5‰ ± 1.4) is similar to the mean δ13C of the trophic guild invertivore (15.1‰ ± 1.07), suggesting that the individuals of these two trophic guilds share similar feeding habits. The mean δ13C of species within this guild suggest that marine benthic algae are the major carbon source for their diets  and distance from shore (inner versus outer). The mean δ13C for these locations  increased slightly with seaward movement. This trend is consistent with prior studies that reported increasingly depleted δ13C values of sampled fauna with seaward movement and depth  had the lowest δThe stable isotopic trends observed in the reef fish communities of the artificial reef and the adjacent first and second reef tract suggest that the artificial reefs are acting as a corridor between the first and second reef, with opportunistic feeding occurring at the artificial reef sites. It is well established that connectivity between reef habitats is important for overall reef health and in this capacity, the artificial reefs studied here seem to increase connectivity and simultaneously provide opportunistic foraging habitat.4Fish community composition data collection was limited to two surveys completed within one year. During these surveys, two members of a three-person dive team identified 83 different fish species across 17 families. Spearguns were used to acquire fish samples for stable isotope analysis, which inherently limited the size range of the fishes that could be sampled.Christopher Blanar: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Joseph Hornbeck & David William Kerstetter: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Amy Hirons: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.10.13039/100006596Florida Fish and Wildlife Conservation Commission (FWC-11229).This work was supported by Data will be made available on request.The authors declare no conflict of interest.No additional information is available for this paper.


Viral infections cause a host of fatal diseases and seriously affect every form of life from bacteria to humans. Although most viral infections can receive appropriate treatment thereby limiting damage to life and livelihood with modern medicine and early diagnosis, new types of viral infections are continuously emerging that need to be properly and timely treated. As time is the most important factor in the progress of many deadly viral diseases, early detection becomes of paramount importance for effective treatment. Aptamers are small oligonucleotide molecules made by the systematic evolution of ligands by exponential enrichment (SELEX). Aptamers are characterized by being able to specifically bind to a target, much like antibodies. However, unlike antibodies, aptamers are easily synthesized, modified, and are able to target a wider range of substances, including proteins and carbohydrates. With these advantages in mind, many studies on aptamer-based viral diagnosis and treatments are currently in progress. The use of aptamers for viral diagnosis requires a system that recognizes the binding of viral molecules to aptamers in samples of blood, serum, plasma, or in virus-infected cells. From a therapeutic perspective, aptamers target viral particles or host cell receptors to prevent the interaction between the virus and host cells or target intracellular viral proteins to interrupt the life cycle of the virus within infected cells. In this paper, we review recent attempts to use aptamers for the diagnosis and treatment of various viral infections. A virus is an organism at the boundary between living and non-living. Viruses are infectious pathogens and proliferate by infecting living organisms. Simple in form, viruses consist of nothing but some genetic material and an encompassing protein shell. Viruses have no means of self-reproduction, and therefore must invade host cells in order to use the host’s molecular machinery to reproduce, after which the newly created viruses go on to infect other host cells. Proliferation in this way can cause either acute disease or chronic disease depending on how long the virus leaves the host cell intact. Various diseases such as coronavirus 19 (COVID19), acquired immune deficiency syndrome (AIDS), and hepatitis are caused by viruses. Viral diseases are often associated with various symptoms such as fever, cough, and weakness, but some present with few or no symptoms, making identification and treatment harshly difficult. Therefore, methods to either improve diagnostic accuracy or provide viable treatment options are of intense interest within the healthcare community.Viruses sometimes cause fatal damage to the human body and without proper treatment, can lead to death. Therefore, many drugs and vaccines have been developed for the treatment of viral diseases. When a host comes in contact with a viral particle, the processes of attachment, uncoating, genome replication, assembly, and release quickly follow. Currently, most drugs target this process at some level to suppress the life cycle of the virus, thereby limiting host damage and ideally restricting the spread of infection. In addition, some treatments attempt to treat the virus by inducing the host’s own immune response, however, as viruses tend to mutate quickly, many easily avoid the effects of highly targeted drugs and the host’s immune system. In cases such as these, it becomes difficult to treat the viral infection without causing toxicity to host cells.There have been many attempts to minimize side effects and accurately select specific targets for the treatment of different viral infections. In this review, we address aptamers as a potential alternative for both the treatment and diagnoses of viral infections. Aptamers are small single-stranded RNA or DNA molecules that can be rapidly selected through SELEX . Aptamers can be identified against various targets with high affinity and specificity and are easy to synthesize and modify. Due to these clear advantages, many attempts have been made to develop aptamers as antiviral drugs and diagnostic substances.Aptamers are small single-stranded oligonucleotides first developed by Szostak and Ellington through an in vitro selection of RNA molecules that bound to various organic dyes . Aptamer14–1015 nucleic acid variants of 30–100 random nucleotides flanked by constant sequences at both ends. The randomized oligonucleotides have unique sequences that acquire a three-dimensional structure depending on the experimental conditions , or the presence of specific ligands [Aptamers are developed by Systematic Evolution of Ligands by EXponential enrichment (SELEX), a method developed by Gold and Tuerk . The fir ligands . These c ligands . During Human papillomavirus (HPV) is a non-enveloped DNA virus of the family Papilomaviridae. Most HPV infections show no symptoms and resolve within two years, however, in some cases, infection can generate warts or precancerous lesions and these sites increase the risk of cancer in the cervix, vulva, vagina, penis, and anus . HPV is Toscano-Garibay et al. isolated an RNA aptamer against the HPV-16 E7 oncoprotein. The isolated RNA aptamer is specifically bound to E7 in a clamp-like manner and might be useful for the detection of HPV infection . Leija-MAiming to create treatments specific for HPV infection, several groups have developed various aptamer-based therapeutics. Nicol et al. isolated RNA aptamers against the HPV-16 E7 oncoprotein. They showed that the isolated aptamers could inhibit the interaction between E7 and RB  and the Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are linear double-stranded DNA-enveloped viruses that belong to the Herpesviridae family. They can infect epithelial tissues and invade the nervous system where they enter a latent stage of infection. Both viruses persist in the human body by hiding in the cell bodies of neurons and when activated move from neurons to skin where virus replication and shedding occurs . Most HSIn attempts to develop antiviral agents against HSV, several groups have isolated aptamers against the HSV envelope glycoprotein (gD). Moore et al. developed DNA aptamers against HSV-2 gD. These aptamers could bind to gD and block HSV2 infection by blocking the interaction of gD and the various entry receptors (Nectin1 and HSV1 target cell receptors (HVEM)) . GopinatHCV is a small, enveloped virus of the family Flaviviridae. HCV is the cause of hepatitis C and can also cause liver fibrosis, cirrhosis, and liver cancer, making HCV infection a major global public health concern. HCV has positive-sense single-stranded RNA as a genome that contains about 9.6 kilobases which has an untranslated (UTR) region (at 5′ and 3′ ends) and a coding region for a viral polyprotein. This polyprotein is composed of four structural proteins , and six non-structural proteins . The first stage of HCV infection is viral attachment to the target cells through their glycoproteins and the cell-surface molecules CD81, LDL receptor, SR-BI, DC-SIGN, Claudin-1, and Occludin . After a−10 M to 10−13 M [Aptamers to be used as HCV diagnostics tools have been developed for HCV detection in both the early stages and in immunosuppressed patients. Lee et al. developed a chip-based detection system for HCV using RNA aptamers that are specifically bound to the HCV core antigen. They immobilized 2′-F RNA aptamers in a 96-well plate and detected the HCV core antigen from the patient’s sera by Cy3-labeled secondary antibodies . Chen et 10−13 M ,29.There are currently no effective vaccines or treatments for HCV. Generally, HCV treatments combine protease inhibitors, like Telaprevir (TVR) and Boceprevir (BOC), with pegylated-interferon (PEG-IFN) and Ribavirin. Fortunately, newly developed treatments using pan-genotypic direct-acting antivirals (DAAs) are short duration (12~24 weeks) with few side effects and have about a 90% of cure rate, regardless of HCV genotype . UnfortuThe 5′ and 3′ UTR of the HCV genome have highly conserved sequences and structured regions which are strongly related to the translation and replication of HCV. The 5′ UTR contains the Internal Ribosome Entry Site (IRES) domain which plays an important role in translation initiation by facilitating the ribosome’s recognition of the HCV viral genome. Additionally, the 3′ UTR has a region called the cis-acting replication element (CRE) which is also essential for viral replication.Kikuchi et al. selected an RNA aptamer that is specifically bound to the HCV IRES. The selected aptamer bound to the target sequences via RNA–RNA interactions and the aptamers that targeted domain IIId showed strong inhibition of translation ,33. In aFukuda et al. isolated RNA aptamers specific for the 3′ X tail of the HCV RNA which had the potential to block HCV replication . Marton Another target is the minus-IRES ((-) IRES) at 3′ end of the negative strand of the HCV RNA. This structure is essential for HCV replication because the viral RNA polymerase NS5B recognizes this site as the initiation site for the synthesis of the positive-strand RNA. Konno et al. isolated RNA aptamers specific for HCV (−) IRES domain I. They showed that this aptamer inhibited up to 50% of the NS5B-mediated positive-strand RNA synthesis ,41,42.Aptamers for targeting HCV Structural Proteins The HCV structural proteins E1 and E2 are good targets for HCV therapeutics because of their essential role in target recognition during HCV entry. Chen et al. developed DNA aptamers specific for HCV glycoprotein E2 that showed a high affinity for genotypes 1a, 1b, and 2a, and strongly inhibited HCV infection . Yang etAptamers for targeting HCV Nonstructural Protein 2 (NS2)The HCV NS2 protein is a viral autoprotease that plays a key role in the life cycle of HCV by mediating the cleavage between NS2 and NS3, making NS2 an important target for HCV treatments. Gao et al. developed DNA aptamers specific for the HCV NS2 protein. They showed that selected aptamers had an anti-viral effect through binding to the N-terminus of NS2 that blocked the interaction of NS2 with NS5A .Aptamers for targeting HCV Nonstructural protein 3 (NS3)The HCV NS3 is a multifunctional protein that has three enzymatic activities. First, NS3 has serine protease activity which is encoded at its N-terminal region. In addition, NS3 has nucleoside triphosphatase (NTPase) and helicase activities at its C-terminal region. Therefore, the HCV NS3 protein is an essential protein for the viral life cycle and an important target for HCV treatments. Kumar et al. developed RNA aptamers specific for HCV NS3 protein and showed that selected aptamers inhibited both the proteolytic and helicase activity of HCV NS3 . Hwang eAptamers for targeting HCV Nonstructural Protein 5A (NS5A)HCV NS5A is a dimeric zinc-binding metalloprotein capable of binding viral RNA, Core, and host factors like lipid droplets, and is essential for HCV production and replication. Yu et al. developed HCV NS5A-specific DNA aptamers and showed that selected aptamers inhibited HCV RNA replication and infectious virus production by blocking the interaction of NS5A with the Core protein. Importantly, the aptamers did not induce interferon beta (IFN-β) or interferon-stimulated genes (ISGs) .Aptamers for targeting HCV Nonstructural Protein 5B (NS5B)HCV NS5B is an RNA-dependent RNA polymerase protein (RdRp) which is essential for the synthesis of positive-sense HCV genomic RNA and negative-sense RNA templates. Because of NS5B’s role in HCV replication, NS5B is the main target for HCV treatments using aptamers. Biroccio et al. developed an RNA aptamer against a truncated HCV NS5B∆55 that did not have a C-terminal region. They showed that the HCV NS5B polymerase was efficiently inhibited by the selected aptamer. Interestingly, the mechanism of inhibition turned out to be noncompetitive with template RNA, suggesting that the aptamer and template RNA did not bind to the same site . BellecaThe Zika virus (ZIKV) is a member of the virus family Flaviviridae. ZIKV is an enveloped virus with a genome of single-stranded positive-sense RNA about 10 kilobases in size. This genome is directly translated into a viral polyprotein that encodes three structural proteins and seven non-structural proteins. Zika’s replication depends on the synthesis of double-stranded RNA from its single-stranded positive-sense RNA genome, after which the replication of the new single-stranded positive-sense RNA proceeds . ZIKV ca4 tissue culture infective dose (TCID) 50/mL [7 ZIKV [Lee et al. developed an aptamer-based ELISA assay specific for ZIKV using anti-ZIKV non-structural protein 1 (NS1) DNA aptamers. ZIKV NS1 is essential for RNA replication and immune evasion . In the D) 50/mL . Dolai e [7 ZIKV .Dengue virus (DENV) is a single-stranded positive-sense RNA enveloped virus also of the family Flaviviridae. Based on the type of envelope proteins, DENV has four genotypes called DENV-1, DENV-2, DENV-3, and DENV-4. DENV envelope proteins bind to cellular receptors which allows the virus to enter the cell and release its viral RNA. Like other Flaviviridae viruses, DENV viral RNA is also translated into a polyprotein  and is processed by cellular or viral proteases. After that, processes such as replication, maturation, and release take place. While DENV is the known cause of dengue fever, many DENV infections are understood to be asymptomatic or subclinical . Interes2O3 (SAMN) nanoparticles and modified with gold nanoparticles (AuNPs) conjugated to aptamers [For DENV diagnostics, Fletcher et al. developed an aptamer-based biosensor using DNA aptamers for the DENV genome. They showed that the biosensor was able to detect sequences derived from each of the four DENV serotypes with high specificity . For theaptamers . This meTo target DENV viral proteins, Gandham et al. developed DNA thioaptamers that specifically bound DENV-2 envelope protein domain III (ED3). They showed that the aptamers could bind to adjacent neutralizing antibody binding sites and expected that the aptamers inhibited infection by blocking DENV entry into the host cells . Chen etThe Japanese encephalitis virus (JEV) is a single-stranded positive-sense RNA enveloped virus of the family Flaviviridae. Based on the type of envelope proteins, JEV has five genotypes. The JEV viral genome, much like others in the same family, encodes several structural and non-structural proteins and the JEV life cycle is similar to other Flaviviridae viruses. JEV infects the central nervous system and causes Japanese encephalitis (JE). About 67,900 cases of JEV-related JE are reported annually and its mortality ranges from 20 to 30% . CurrentThe tick-borne Encephalitis Virus (TBEV) is a positive-sense single-stranded RNA virus also of the family Flaviviridae. Its 11 kilobase genome contains a 5′-cap, 5′-UTR, 3′-UTR, and a single open reading frame (ORF). The ORF encodes three structural proteins and seven non-structural proteins and the life cycle of this virus is again similar to other Flaviviridae viruses. TBEV is the cause of tick-borne encephalitis (TBE) and this disease typically targets the central nervous system (CNS) . There aNorovirus (NoV) is a non-enveloped virus of the family Caliciviridae and has a single-stranded positive-sense RNA as a genome. Its RNA genome encodes a viral polyprotein that is processed into six non-structural proteins and one structural protein. It has at least seven genogroups  including various genotypes within each group, and among them, GI and GIV can infect humans . NoV is Escudero-Abarca et al. developed DNA aptamers against human NoV. Isolated aptamers bound to NoV GII.2 and GII.4 strains with high affinity and the virus was found in contaminated lettuce through an aptamer magnetic capture (AMC) method with RT-qPCR. They demonstrated that the isolated aptamers were potentially useful for NoV capture and detection in a variety of sample types . The samTo create a system for diagnosing NoV, Giamberadino et al. developed an aptasensor using DNA aptamers. They developed aptamers against murine NoV, but found it could cross-react with human NoV strain GII.3. Aptamers were immobilized on the electrochemical sensor using a gold nanoparticle-modified screen-printed carbon electrode (GNPs-SPCE) and could detect NoV with a detection limit of approximately 180 viral particles . Chand eCoronavirus (CoV) is a positive-sense single-stranded RNA virus that belongs to the family Coronaviridae with a genome of about 30 kilobases in size. CoV’s genome is organized as 5′-leader-UTR-replicase (ORF1ab)—spike (S)—envelope (E)—membrane (M)—nucleocapsid (N)—3′UTR-poly (A)—tail, and thus can act like a messenger RNA (mRNA) . The virIn 2003, an outbreak of severe acute respiratory syndrome (SARS) begun in Asia and quickly spread. The virus named SARS-CoV infected more than 8000 people, about 10% of whom died . In SeptJang et al. developed RNA aptamers against the SARS-CoV NTPase/Helicase (nsP10). They showed that the isolated aptamers efficiently inhibited the double-stranded DNA unwinding activity of the helicase by up to 85%, but showed only a slight effect on ATPase activity . Shum etWith the ongoing pandemic in mind, Song et al. developed DNA aptamers against the SARS-CoV2 herpes simplex virus receptor-binding domain (RBD) of the spike glycoprotein (S protein). They isolated two aptamers that may have partially identical binding sites at the human SARS-CoV2 receptor angiotensin-converting enzyme 2 (ACE2) on the SARS-CoV2 RBD, and predicted the structure of the aptamer–RBD binding complex. The structure showed that aptamer could bind to RBD by hydrogen bonding. They also demonstrated that these aptamers were useful for the diagnosis and treatment of SARS-CoV2  Zhang etThe influenza virus is a member of the family Orthomyxoviridae. Those in this family are enveloped viruses with a segmented negative single-stranded RNA as the genome. There are four types of this virus, type A, type B, type C, and type D, however, only types A and B are clinically relevant to humans . The infThe high infectivity and variability of the influenza virus have caused serious pandemics to occur several times throughout the 20th and 21st centuries. In 1918, the Spanish influenza virus killed 20–50 million people. In 1957, the Asian influenza virus recorded approximately 1.1 million deaths. Additionally, in 1968, the Hong Kong influenza virus caused about 1 million people to die worldwide ,103. FinRapid diagnosis of influenza and accurate classification of a subtype are vital due to its high infectivity and mutagenicity. While antibodies are typically used for the detection of these viruses, most current antibodies differentiate only between influenza types A and B. In cases like this, the advantages of aptamers become quickly apparent  and offer a potential alternative method of identification. Anti-influenza aptamers mainly target viral particles or hemagglutinin (HA), and more than 40 aptamers have been developed to date. Especially in recent years, due to the rapid spread of the H1N1 virus and the high fatality rate of the H5N1 subtype of the avian influenza virus (AIV), the influenza virus has become the main target for the development of aptamers. Aptamers targeting H1, H3, H5, H9, or Ha (type B) for HA and an aptamer targeting the H5N1 viral particle have been developed just in recent years .3 times the sensitivity of conventional serological diagnosis. Wang et al. also developed a microfluidic device using DNA aptamers [Because of its high infectivity and mutagenicity, most of the aptamers developed thus far are anti-influenza A aptamers. Cui et al. developed a diagnostic system using Quantum dots (QD) . They coaptamers . To do taptamers . They idaptamers . They usaptamers . To do t−4 hemagglutinin units (HAU)/probe.Le et al. developed an aptamer-based biosensor using a specific RNA aptamer for the H3N2 virus  which coThe H5N1 virus, a subtype of AIV, also requires a fast and accurate diagnosis because it is potentially fatal and spreads quickly within human populations. Therefore, many diagnostic systems have been developed using various aptamers. Bai et al. developed a portable Surface Plasmon Resonance (SPR) biosensor for the rapid detection of AIV H5N1 using a specific DNA aptamer . This biNguyen et al. developed a specific sandwich-type SPR-based biosensor for the detection of H5Nx whole viruses . They usAnti-viral aptamer therapeutics can target viral proteins or related host cellular factors. Most influenza therapeutics being currently developed also target these molecules, and most developed aptamers mainly target hemagglutinin (HA) . HA playAnother aptamer target is the NS1 protein, a viral nonstructural protein encoded by the NS gene segments of the influenza virus. NS1 prevents the polyadenylation of cellular mRNAs in order to escape the antiviral responses of the host cells. Moreover, the RNA binding domain of NS1 is able to target RIG-I, which is responsible for the cellular interferon (IFN) response. Finally, NS1 inhibits the host’s innate immune response by suppressing the induction of IFNs. Therefore, blocking NS1 activity could be a potential strategy for antiviral agents against the influenza virus. Woo et al. developed a specific DNA aptamer for the influenza A virus NS1 protein . In theiAnother target for the development of influenza virus aptamers is the PA endonuclease. PA plays an essential role in viral RNA transcription and replication by forming the heterotrimeric polymerase complex together with the PB1 and PB2 subunits. PA cleaves host mRNAs downstream of their mRNA cap structures, which are then recognized and bound by PB2 . The N-tAnother strategy is to target influenza virus-related host cellular factors. The mRNAs of the influenza virus possess a 5′ cap structure and a 3′ poly (A) tail that makes them structurally indistinguishable from cellular mRNAs. However, selective translation of viral mRNAs occurs in infected cells through a discriminatory mechanism, whereby the viral polymerase and NS1 interact with components of the translation–initiation complex, such as the eIF4GI and PABP1 proteins ,142,143.The rift valley fever virus (RVFV) belongs to the family of Phenuiviridae and is an enveloped negative single-stranded RNA virus. RVFV has three segmented RNAs as a genome and they encode six major viral proteins. This virus has an outer lipid envelope with two glycoproteins (G(N) and G(C)) that are required for cell entry. RVFV infection is the cause of several severe diseases such as hemorrhagic fever syndrome, meningoencephalitis, hepatitis, and can severely affect the eyes . CurrentThe severe Fever with Thrombocytopenia Syndrome Virus (SFTSV), also called Dabie bandavirus, is a tick-borne virus in the family Phenuiviridae . It has The Ebola virus (EBOV) is an RNA virus of the family Filoviridae and belongs to the genus Ebolavirus. It has a negative-sense single-stranded RNA as a genome and the viral genome contains seven genes arranged as follows: 3′-UTR-NP-VP35-VP40-GP-VP30-VP24-L-5′-UTR . Five viBinning et al. developed RNA aptamers against EBOV viral protein 35 (eVP35). eVP35 is a multifunctional double-strand RNA binding protein that is essential for viral replication, innate immune evasion, and pathogenesis. eVP35 has a dsRNA binding interferon (IFN) inhibitory domain (IID) at its C-terminal for eVP35-mediated IFN antagonism and viral polymerase co-factor functions. The authors showed that the isolated aptamer could bind to eVP35 IID and disrupt the eVP35–nucleoprotein (NP) interaction. In addition to its ability to antagonize the eVP35–NP interaction, the isolated aptamer also inhibited the EBOV polymerase complex . Tanaka Shubham et al. developed a 2′fluoro pyrimidine (2′FY)-modified RNA aptamer against the soluble glycoprotein (sGP) of EBOV. sGP is a good biomarker for EBOV because it is secreted in abundance into the bloodstream, even during the early stages of infection. The isolated aptamer was specifically bound to sGP with high affinity and was demonstrated to be an excellent diagnostic tool for the detection of EBOV infection . Hong etHIV is a lentivirus (a subgroup of retroviruses) that causes acquired immunodeficiency syndrome (AIDS) . It is aUsing aptamers may also be a good option to diagnose HIV. Various attempts to use aptamers for the diagnosis of HIV have been reported. For instance, Pavski et al. developed an affinity capillary electrophoresis/laser-induced fluorescence (CE/LIF) assay using a specific DNA aptamer for HIV-1’s reverse transcriptase (RT) as a probe . An aptaThere is no complete cure for HIV/AIDS, however, current treatments do manage to slow the progression of the disease and reduce the risk of death. These treatments are called antiretroviral therapies and are created through the use of a combination of several antiretroviral drugs (cART). cART is usually a combination of three or more medications from several different drug classes. Unfortunately, cART has numerous shortcomings such as severe side effects and high cost. Thus, aptamers have been considered as potential alternative agents in cART to overcome these limitations. Many aptamers have been developed specifically targeting HIV genomes, HIV proteins, or cellular factors related to HIV infection.The HIV genome consists of two identical single-stranded RNAs. Each RNA has a long terminal repeat (LTR) region at its 5′ and 3′ end. The 5′ LTR region acts as a promotor for the transcription of viral genes. If the functions of HIV LTR are inhibited by disrupting their structure or by blocking their interaction with viral or cellular factors, it may affect HIV’s life cycle. Srisawat et al. developed RNA aptamers that could bind to the LTRs of HIV-1 DNA . The aptAptamers for targeting HIV Reverse Transcriptase (RT)The RT of HIV plays an important role in viral genome replication. RT has DNA polymerase activity and also plays a role as an RNase H. RT can copy the genome to either a DNA or an RNA template and can cleave RNA only when the RNA is part of an RNA/DNA duplex. Therefore, these two enzymatic activities convert the RNA into a linear double-stranded DNA . Many apChaloin et al. developed a pseudoknot RNA aptamer specific for the HIV-1 RT . TransieFor specifically targeting RNase H activity, Andreola et al. developed two DNA aptamers . The selAptamers for targeting HIV Integrase (IN)Integrase (IN), which catalyzes the integration of newly synthesized double-stranded viral DNA into the host’s genome, is also a good target for anti-HIV aptamer therapeutics . MétifioAptamers for targeting HIV Protease (PR)HIV protease (PR) has a dual purpose; the precursor HIV PR is responsible for catalyzing its own production into mature PR through PR auto-processing, where the mature PR then hydrolyzes peptide bonds on the Gag-Pol polyproteins at nine specific sites and produces functional proteins  . Few PR Aptamers for targeting HIV Nucleocapsid (NC) proteinThe nucleocapsid (NC) protein of HIV-1 plays an important role in the encapsidation of viral RNAs and assembly of viral particles . The NC Aptamers for targeting HIV Surface Envelope Glycoprotein (gp120)Gp120 is essential for the HIV virus to enter target cells as it plays a vital role in HIV’s attachment to specific cell surface receptors. Interestingly, many neutralizing antibodies bind to variable regions of gp120, which is likely the reason this protein has such a high mutation rate . For bloAptamers for targeting the HIV Gag proteinAnother target viral protein is the HIV Gag polyprotein. Gag polyproteins are processed by protease into matrix proteins, capsid proteins, spacer peptides, and nucleocapsid proteins. Two aptamers have been developed targeting the Gag protein. Lochrie et al. developed RNA aptamers against the HIV-1 gag protein , which bAptamers for targeting the HIV Rev proteinThe HIV Rev protein mediates nuclear export of unspliced and partially spliced viral RNAs. Rev assembles on a 351-nt Rev response element (RRE) within viral transcripts and induces the host’s nuclear export mechanism . AptamerAnother strategy for blocking HIV infection is to target the host’s cellular factors which to date, has mainly meant targeting host cell receptors. HIV uses C–C chemokine receptor type 5 (CCR5) or C–X–C chemokine receptor type 4 (CXCR-4) as co-receptors to invade target cells. Of note, mutations in CCR5 confer immunity to HIV in humans. Therefore, CCR5 is an essential factor for HIV’s entry and likely a good target for the treatment of HIV. Zhou et al. developed RNA aptamers against CCR5 . They shAnother target is nucleolin (NCL). NCL is a multifunctional nucleolar protein. In cellular fractions, NCL complexes with CD4, CXCR4, and CCR5, thus, NCL is most heavily involved in the early stage of HIV’s entry. If there are no cellular receptors like CD4 and CXCR4/CCR5 on the cell surface, HIV-1 attachment occurs through interactions with heparan sulfate proteoglycans and cell-surface-expressed NCL. Perrone et al. developed a G-rich aptamer against NCL that formed a stable G-quadruplex structure and showed that this aptamer efficiently inhibited HIV-1 attachment/entry into host cells .Another potential target is human cyclin T1 (CycT1). The regulatory cyclin, CycT1, is a host factor essential for HIV-1 replication in CD4 T cells and macrophages. CycT1 complexes with Positive Transcription Elongation Factor b (P-TEFb) to facilitate HIV replication . Um et aHepatitis B virus (HBV) is a partially double-stranded DNA virus that belongs to the Hepadnaviridae family and is classified into eight genotypes from A to H. The HBV viral DNA genome is rendered fully double-stranded DNA by cellular DNA polymerases and transcribed by cellular RNA polymerases. The transcribed viral RNAs are translated into the viral proteins essential for the virus’s life cycle. HBV is the cause of hepatitis B, which can be either acute or become chronic leading to liver cirrhosis and hepatocellular carcinoma. Despite an effective vaccine that prevents HBV infection, it is still a global health concern due to the lack of adequate treatments.For effectively detecting HBV, Suh et al. developed a detection system for the HBV surface antigen (HBsAg) using specific RNA aptamers. HBsAg is the main element of the HBV particle and thus the main target of diagnostic tests. To create their system, they combined the conventional competitive binding assay with fluorescence resonance energy transfer (FRET) and aptamers. They demonstrated that their method was approximately 40-fold better than that of the most widely used conventional detection assays . Xi et aFor developing effective aptamer therapeutics, Feng et al. generated RNA aptamers against the HBV polymerase protein (P protein). During viral replication, the interaction between the P protein and the RNA stem-loop on the pregenomic (pg) RNA is essential. They showed that the isolated aptamers inhibited viral pgRNA packaging and DNA synthesis by blocking P protein binding to pgRNA . Zhang eIn the past 20 years, many studies have been conducted that attempted to treat or diagnose specific diseases using aptamers. In particular, the advantages of aptamers have enabled many studies on viral diseases that would have been otherwise difficult to conduct using older methods . OverallIn order to effectively treat viral diseases, a quick and accurate diagnosis in the early stages of infection is essential. There have been many attempts to use aptamers for virus detection which represent potentially simpler, faster, and cheaper systems than those otherwise available. These aptamers systems require more intensive development and larger-scale trials to ensure their effectiveness and safety, and improvements over time are rapidly converging on these goals. Take the detection systems using field-effect transistors as an example, these systems started as complex and expensive systems using diamond . HoweverCompared with antibodies, the ability to bind to various targets with lower probabilities of immune response means that aptamers have major potential advantages for the treatment of viral diseases. Of note, the chemical synthesizability of aptamer confers one of the major advantages against antibody as therapeutics. In particular, with aptamers, it is possible to block various processes by specifically developing them to target various factors involved in the viral life cycle . In the With consideration of recent pandemics including the pandemic caused by COVID-19, the development of fast and accurate diagnosis methods, appropriate treatments after diagnosis that are effective against a wide range of variants, and effective vaccines is urgently required. Due to advantages such as rapid development, high specificity, affinity, and stability, easy manufacturability, and convenient storage, aptamers could represent an excellent alternative to other current methods for the urgent purpose of controlling viral pandemics. However, as with all methods, there are obvious problems to be overcome. Aptamers can be selected by targeting various molecules, but there are cases where selection is prohibitively difficult. In addition, even if aptamers are appropriately selected, there are cases in which the results of aptamer methods differ between experimental testing and when used in clinical practice. Researchers are making ongoing efforts to overcome these shortcomings, such as modifying aptamers in various ways or developing improved SELEX methods . Providi


Several life-threatening viruses have recently appeared, including the coronavirus, infecting a variety of human and animal hosts and causing a range of diseases like human upper respiratory tract infections. They not only cause serious human and animal deaths, but also cause serious public health problems worldwide. Currently, seven species are known to infect humans, namely SARS-CoV-2, MERS-CoV, SARS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1. The coronavirus nonstructural protein 16 (NSP16) structure is similar to the 5′-end capping system of mRNA used by eukaryotic hosts and plays a vital role in evading host immunity response and protects the nascent viral mRNA from degradation. NSP16 is also well-conserved among related coronaviruses and requires its binding partner NSP10 to activate its enzymatic activity. With the continued threat of viral emergence highlighted by human coronaviruses and SARS-CoV-2, mutant strains continue to appear, affecting the highly conserved NSP16: this provides a possible therapeutic approach applicable to any novel coronavirus. To this end, current information on the 2′-O-MTase activity mechanism, the differences between NSP16 and NSP10 in human coronaviruses, and the current potential prevention and treatment strategies related to NSP16 are summarized in this review. A high-incidence respiratory illness that is coronavirus disease 2019 (COVID-19) is caused by person-to-person transmission of the novel coronavirus, now termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the ICTV Coronaviridae Study Group on 12 February 2020, and was identified in December 2019 . The perSARS-CoV was first identified in 2002 and diagnosed in Southern China , with a Nidovirales, family Coronaviridae, and subfamily Orthocoronavirinae) are divided into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus, with the human coronaviruses under Alphacoronavirus (HCoV-229E and -NL63) and Betacoronavirus .Coronaviruses are enveloped, positive-sense RNA viruses containing a single-stranded RNA genome with a 5′-terminal cap structure and 3′-polyadenylation, and can infect a wide variety of mammalian and avian species ,17. The The virus particles are formed by four major structural proteins: spike, envelope, membrane, and nucleocapsid. The envelope and membrane proteins with spike protein form a viral envelope, with the spike protein allowing the virus to attach to the host cell membrane, and the nucleocapsid protein holding the virus RNA genome . The repPatients infected with SARS-CoV, MERS-CoV, or SARS-CoV-2 usually develop acute lung injury, systemic inflammatory response syndrome, acute respiratory distress syndrome, and renal failure, eventually leading to death . When coCoronaviruses activate the release of various pro-inflammatory cytokines in the host during the infection process, such as interleukin (IL)-1β, IL-2, IL-6, IL-7, granulocyte colony-stimulating factor, interferon, and tumor necrosis factor ,33,34. SEukaryotic hosts utilize a 5′-terminal capping system to promote efficient nuclear export, robust translation, and enhanced stability of mRNA. In addition, mRNA capping also helps to distinguish between self and nonself RNA and can lead to initiation of the host immune response. However, many viruses that replicate in the cytoplasm of eukaryotes have evolved 2′-O-methyltransferase (2′O-MTase) to autonomously modify their mRNAs, thus being similar to the 5′-cap structures of higher eukaryote mRNAs that have ribose 2′-O-methylation . Thus, sThe usual viral RNA cap structure, the guanosine cap core structure, the N7 methylation, and the 2′-O-methylation respectively protect the 5′-triphosphate from activating the host innate immune response ,43; ensuThe mRNA cap for coronaviruses is completed by NSP16, an m7GpppA-specific, S-adenosyl-L-methionine (SAM)-dependent 2′-O-MTase ,50. The The presence of these host and virus effectors suggests the importance of NSP16 (2′-O-methylation) as a virulence determinant during coronavirus infection ,57. For In heterodimeric complexes, NSP10 is essential by acting as a cofactor for NSP16 methylase ,53,60,65The structure of 2′-O-MTase reveals the critical residues involved in the interaction between NSP10 and NSP16 which might contribute to the binding of cofactor SAM (such as K93 of NSP10 with S105 of NSP16) in SARS-CoV , SARS-CoA more robust replication induced early during coronavirus infection may improve overall immune performance ,69. EnhaThe mutual regulation of NSP10 and NSP16 maintains 2′-O-MTase activity. The critical KDKE motif is highly conserved, which can predict the functional disruption of the 2′-O-MTase mutation of NSP16 . For exaTherefore, to change the 2′-O-MTase activity of coronaviruses, candidate vaccines such as attenuated vaccines have been developed. Previous research confirmed that the dNSP16 mutant (D130A) MERS-CoV had a type I interferon-based attenuation and had a protective effect upon challenging with a mouse-adapted MERS-CoV strain . The NSPAs highlighted earlier, NSP16 needs to be combined with NSP10 to maintain stability and 2′-O-MTase activity . Based oIn addition to targeting the 2′-O-MTase active site, other approaches also utilize indirect factors necessary for coronavirus NSP16 activity. The specificity of the 2′-O-MTase active site is one of the potential methods, and can significantly impact viral infection with minimal toxicity . CoronavOver the past few centuries, coronaviruses have repeatedly emerged from zoonotic reservoirs and are important emerging human pathogens. The complex interaction between NSP16 and NSP10 is necessary for their stability and 2′-O-MTase activity. In the absence of functional NSP16, enhanced recognition of viral RNA by host sensor molecules occurs, such as by the IFIT family, which cause significant attenuation of infections. Notably, the KDKE motif mutation of NSP16 can change the activity of 2′-O-MTase. The targeted SAM-binding site is also expected to interact with 2′-O-MTase encoded by other viruses, thereby providing a wide range of antiviral therapeutics. Importantly, SARS-CoV-2 mutant strains continue to appear, thus the need for coronavirus prevention and treatment methods towards the highly conserved NSP16 activity.


Yersinia pestis, is a rapidly progressing lethal infection. The various phases of pneumonic plague are yet to be fully understood. A well-established way to address the pathology of infectious diseases in general, and pneumonic plague in particular, is to conduct concomitant transcriptomic analysis of the bacteria and the host. The analysis of dual RNA by RNA sequencing technology is challenging, due the difficulties of extracting bacterial RNA, which is overwhelmingly outnumbered by the host RNA, especially at the critical early time points post-infection (prior to 48 h). Here, we describe a novel technique that employed the infusion of an RNA preserving reagent (RNAlater) into the lungs of the animals, through the trachea, under deep anesthesia. This method enabled the isolation of stable dual mRNA from the lungs of mice infected with Y. pestis, as early as 24 h post-infection. The RNA was used for transcriptomic analysis, which provided a comprehensive gene expression profile of both the host and the pathogen.Pneumonic plague, caused by  Yersinia pestis, the causative agent of plague, has been responsible, in the past, for major pandemics, which caused the death of over 200 million people and still represent a significant public health concern in endemic regions, particularly in the third world. According to the report of the World Health Organization (WHO), since 2010, there have been hundreds of people that have died of plague. In 2017, 2348 cases of plague were reported in Madagascar, including 1791 cases of pneumonic plague, which caused 200 deaths. In the wild, plague mostly affects rodent populations, by flea bites. In some cases, Yersinia pestis afflicts populated areas and may manifest in humans as bubonic plague; although, in some cases, it causes primary septicemia, without detectable characteristic bubos. Secondary pneumonia may evolve from bubonic and septicemic plague [Y. pestis to invade the mammalian host, colonize internal organs, and overcome the immune response is attributed to the combined activities of multiple virulence pathways that are activated in a timely manner during infection, in response to the host milieu signals [Yersinia infection, it may be applied to study other pulmonary infections.Bacteria have successfully evolved sophisticated mechanisms that allow them to sense, cope with, and adapt to varying conditions in their immediate surroundings. Often, these mechanisms are essential for the manifestation of virulence in bacterial pathogens . The rap signals ,6. Some Y. pestis strain Kimberley53 (Kim53) [Y. pestis bacteria were grown on BHIA plates  for 48 h at 28 °C, and several colonies were suspended in HIB  supplemented with 0.2% (+) xylose and 2.5 mM CaCl2  and incubated overnight at 28 °C. The culture was diluted in saline solution to achieve the required infection dose.The bacterial strain examined in this study was the virulent  (Kim53) . The bac (Kim53) . BrieflyAll animal experiments were approved by the IIBR Committee for Animal Research (protocols M-63-2017 and M-31-2017). The experimental animals were handled according to the National Research Council 1996 Guide for the Care and Use of Laboratory Animals and regulations of the IIBR Animal Use Committee.50 of Y. pestis Kim53 is ~550 CFU [50 . Of note, 24 hpi the bacterial load in the lungs was 4 × 107 CFU/animal, and 48 hpi, the load was 1.8 × 109 CFU/animal. The infectious dose was verified by plating diluted bacterial suspensions onto BHIA plates. To collect lung tissue, mice were anesthetized with 0.1 mL (10% ketamine (100 mg/kg) + 2% xylazine (10 mg/kg))/10 g body weight.Female OF1 mice (5–6 weeks old) were purchased from Charles River (UK). Prior to infection, mice were anaesthetized by intraperitoneal injection of a mixture of 0.5% ketamine HCl and 0.1% xylazine, then subjected to intranasal infection with 35 μL/mouse of the bacterial suspension. Under these conditions, the LD~550 CFU ,12. Mice6 or 107 CFU of Y. pestis bacteria. RNA was extracted to 50% of the initial volume using the RNeasy Mini Kit .The lungs of naive mice were mashed in 1 mL of RLT buffer  and spiked with 10We implemented a novel method that employed in situ infusion of the RNA preserving reagent RNAlater  into the lungs of the animals through the trachea under deep anesthesia. A puncture tracheotomy technique ,14 enablThe RNA samples were subjected to rRNA removal using the Ribo-Zero Gold rRNA Removal Kit  according to the manufacturer’s instructions. The RNA quality was evaluated by RIN (RNA integrity number) analysis with the BioAnalyzer device  using the RNA high-sensitivity chip.Y. pestis CO92, available in the NCBI database (accession numbers: NC_003143 (chromosome), NC_003131 (pCD1), NC_003132 (pPCP1), NC_003134 (pMT1)). This genome, also belonging to the Orientalis biovar (as the Kim 53 strain used for infection) served as a reference for Y pestis genomic sequence analysis. Mapping was performed using Bowtie 2, version 2.2.0 [Y. pestis CO92 feature file, downloaded from the NCBI database.RNA-seq was performed at the JP Sulzberger Columbia Genome Center . Following RiboZero rRNA depletion, libraries were constructed using the TruSeq RNA library preparation kit (Illumina) without the poly A pulldown step. Whole transcriptome sequencing  using an Illumina HiSeq was performed producing paired-end 100 nt reads. Quality control was performed using fastQC version 0.11.5, checking for per-base sequence quality, per-sequence quality scores, and adapter content. All reads of the bacterial transcriptome were mapped to the reference genome on 2.2.0 , with deon 2.2.0 , using GThe reproducibility of the biological replicates was evaluated by principal component analysis (PCA) using the R package DESeq2, version 1.26.0 (13). Similarity matrix and hierarchical clustering were generated using the R packages gplots and RColorBrewer.Y. pestis infection. The RNA-seq raw data were deposited in the NCBI public domain and are available under accession number GSE181680.The generated data will enable the dual transcriptome to be profiled and insights to be gained into the processes involved during To evaluate the consistency of the method and the compatibility of the sequences for further analysis, the various samples were examined using heatmap and principal component analysis (PCA).Y. pestis. The tissue embedment was used for RNA extraction, and approximately 50 ng/µL of RNA could be extracted from the tissue spiked with 106 CFU, and 200 ng/µL from the tissue spiked with 107 CFU. The RIN (RNA integrity number) of the RNA samples was >8 for all the samples, indicating that the RNA quality was adequate for downstream RNA-seq analysis (data not shown). Next, RNA was extracted from the dissected lungs of the Y. pestis-infected mice, under the same conditions. The Y. pestis load in the lungs at 24 h post-infection (hpi) was 107 CFU, which was shown to be adequate for RNA extraction (see above). Nevertheless, the initial attempts to obtain high-quality RNA from the early time points after infection (24 hpi), in a sufficient quantity, were unsuccessful by us and others (for example, see [To evaluate the efficiency of extracting dual RNA from the lungs of plague-infected mice, by conventional methods, the mashed lungs of naïve mice were spiked with ple, see ,18.Y. pestis reads/sample (This limitation, probably owing to the overwhelming excess of host RNA and the poor stability of the bacterial RNA, could be alleviated by the implementation of the novel protocol, which includes pre-infusion of the lungs with an RNA preserving reagent. The protocol enabled the extraction of high-quality RNA . The sams/sample , as wells/sample .Y. pestis infection.Cluster analysis, including PCA and corroborated by the heatmap depiction of highly modulated genes, evaluated possible pairwise structural dissimilarities between the datasets generated by the inspection of similar samples. PCA provides information pertaining to the interrelationship among variables, and the heatmap depicts a two-way trait and accession classification. The results depicted in Y. pestis, were made publicly available for further identification of the bacterial process and host responses occurring in the course of the infection. While the protocol was demonstrated in the murine pneumonic plague model, it may be applied to other pneumonic bacterial infections.Previous attempts to extract RNA from the airways of infected animals, for in vivo dual pathogen–host RNA-seq analysis, generated inferior results ,6,19. Th


The ongoing COVID-19/Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2) pandemic has become a significant threat to public health and has hugely impacted societies globally. Targeting conserved SARS-CoV-2 RNA structures and sequences essential for viral genome translation is a novel approach to inhibit viral infection and progression. This new pharmacological modality compasses two classes of RNA-targeting molecules: 1) synthetic small molecules that recognize secondary or tertiary RNA structures and 2) antisense oligonucleotides (ASOs) that recognize the RNA primary sequence. These molecules can also serve as a “bait” fragment in RNA degrading chimeras to eliminate the viral RNA genome. This new type of chimeric RNA degrader is recently named ribonuclease targeting chimera or RIBOTAC. This review paper summarizes the sequence conservation in SARS-CoV-2 and the current development of RNA-targeting molecules to combat this virus. These RNA-binding molecules will also serve as an emerging class of antiviral drug candidates that might pivot to address future viral outbreaks. SARS-CoV-2 belongs to the betacoronavirus genus and is an enveloped ssRNA (+) virus with a genome length of about 30,000 nucleotides (RefSeq NC_045512) . The virpro) is also an attractive drug target. PF-07321332 (Paxlovoid) was developed as an oral drug targeting Mpro and is being tested in a Phase 3 clinical trial   . Other red cells . RdRP ined cells . In thised cells . Compareed cells . We envied cells . RNA-tar−3 substitutions per base (30 nucleotides/genome) per year under neutral genetic drift conditions (−5–1×10−4 substitutions per base (0.3–3 nucleotides/genome) in each transmission events from population phylodynamic studies (https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/), including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and B.1.1.529 (Omicron) variants. Among these variants, the spike protein (S) harbors most of the nucleotide mutations compared to the original genomic sequence isolated from Wuhan, China, in December 2019 , which encodes the viral RdRP (nsp12), requires a PFS that shifts the ORF by –1 nucleotide via a “slippery sequence” to circumvent the ORF1a stop codon  Figure . AlthougIn the 5’ UTR (1–265), there are five stem-loops identified, SL1–5 . SL1 wasIn the 3’ UTR, three main secondary structures were elucidated by chemical probing: bulged stem-loop (BSL), SL-1, and the highly variable region (HVR) . BioinfoThe HVR in the 3' UTR is not essential to betacoronavirus. The HVR can be deleted without affecting viral propagation in cell culture, albeit the HVR-deleted MHV strain has lower pathogenicity in mice . NeverthAmong the 106 predicted conserved structured RNA regions by the Das group , two locDe novo design of nucleic acid ligands has been pursued for more than 35 years. The field was first pioneered by the Dervan group in optimizing DNA-binding molecules , and thein vitro and in vivo  to interact with a highly structured transactivation response (TAR) hairpin in its RNA to enhance the viral transcription . Peptoid in vivo .The discovery of RNA-targeting anti-SARS-CoV or CoV-2 small molecules primarily focused on the PFS element. MTDB was first identified by virtual screening and 3-dimensional (3D) modeling. MTBD can potently bind to the pseudoknot in the SARS-CoV PFS element and inhibit the PFS function in a dual luciferase system  Figure . The dua50) in the dual fluorescent protein reporter cells at 19 μM and SARS-CoV-2-infected cells at 2.4 μM  in the host cells  were predicted to bind to the SARS-CoV-2 RNA genome . Some FDvia Watson-Crick base-pairing to a target RNA and modulate RNA splicing or gene expression (via ASO-induced ribonuclease (RNase) H1 activity and 2) masking the target RNA from interaction with the human RBPs or the ribosome.ASOs are RNA or DNA sequences with 15–25 natural or modified nucleotides , which hpression . ASOs geThe ASOs used to induce RNase H1 activation are also termed “gapmers”. Gapmers usually contain a central DNA sequence (> 6 nucleotides) that hybridizes with the target RNA . RNase H“Masking” ASOs are commonly used as a steric block in the target RNA and, thereby, to modulate RNA splicing and suppress translation. The FDA has approved several ASOs acting through this mechanism to treat a variety of human diseases . For exain vivo due to high serum protein binding and nuclease resistance (Tm) by 2 °C (via a constrained methylene bridge (Tm per LNA substitution)  . One or itution) . A populitution) . PMOs haitution) .By using 3D antisense modeling, a PMO named PRF3p was optimized to target the Stem 3 region in the PFS element  Figure . The PRFA PMO named SBD1 was designed to target the conserved TRS-L region in the SARS-CoV 5’ UTR , and theThe RNA-degrading chimeras follow a well-established precedent from the protein field, namely, the proteolysis targeting chimera or PROTAC. PROTACs bind to their target protein using a guide arm as “bait”. The effector arm of PROTACs recruits an endogenous E3 ubiquitin ligase resulting in polyubiquitination and subsequent proteasomal degradation of the target protein . The DisKd = 18 µM to RNase L monomer), presenting a modest antiviral effect as a single agent against human parainfluenza virus in cells -RNase L pathway. In a viral infection, OAS senses dsRNA and synthesizes 2',5'-linked oligoadenylates (2–5A) that activate RNase L by dimerization . RNase Lin cells . The strin cells . Recentlin cells . One of in cells  (Haniff in cells .4) as an RNase L recruiter (Following the first small-molecule RIBOTAC report, another type of nucleic acid-based RIBOTAC targeting SARS-CoV-2 demonstrating efficacy in virus-infected cells was also disclosed . This tyecruiter . These Recruiter .Molecules targeting conserved viral RNA sequences and structures are a newly emerged pharmacological modality that can significantly expand our antiviral arsenal. ASOs that recognize primary viral RNA sequences can be rapidly designed and optimized in early drug discovery. The major obstacles to the clinical use of ASOs are the unfavorable cellular uptake and distribution . Recentlin vivo activity and toxicity profile of RNA-targeting small molecules as antivirals are still obscure. Major efforts are required to address these issues before RNA-targeting molecules can be used as antiviral drugs in clinics.Targeting RNA structures will broaden the spectrum of the small-molecule “druggability”. Compared to traditional protein targets in viruses, such as RdRP and proteases, a completely different target specificity will be obtained for RNA ligands. As illustrated in the SARS-CoV-2 5’ UTR, the RNA structures but not the exact sequences are conserved across betacoronavirus strains . Such stFueled by the current advances in RNA-binding small molecules, ASOs, and RNA-degrading chimeras, RNA-targeting strategies have already been demonstrated the use in inhibiting SARS-CoV-2. With further advances in structure modeling for RNAs and understanding of the RNA-ligand interactions, the RNA-targeting drug discovery platforms have the potential to quickly generate antiviral candidates to address future viral outbreaks.


Chironomus riparius has been involved in thermal stress response. One of the telomeric transcripts, the so-called CriTER-A variant, is highly overexpressed upon heat shock. On the other hand, its homologous variant CriTER-B, which is the most frequently encoded noncoding RNA in the telomeres of C. riparius, is only slightly affected by thermal stress. Interestingly, both transcripts show high sequence homology, but less is known about their folding and how this could influence their differential behaviour. Our study suggests that CriTER-A folds as two different conformers, whose relative proportion is influenced by temperature conditions. Meanwhile, the CriTER-B variant shows only one dominant conformer. Thus, a temperature-dependent conformational equilibrium can be established for CriTER-A, suggesting a putative functional role of the telomeric transcriptome in relation to thermal stress that could rely on the structure–function relationship of the CriTER-A transcripts.The telomeric transcriptome of  Telomeres are complex and dynamic structures consisting of DNA repeats, proteins, and telomeric transcripts. The transcriptional activity of telomeres received great attention after the discovery of the telomeric repeat-containing RNA (TERRA) molecules in mammals. They are regulated nuclear noncoding RNAs (ncRNAs) transcribed from telomeres that overturned the longstanding belief that telomeres were transcriptionally silent ,2. TheseDrosophila melanogaster, shows transposons in the telomeres [Although all telomeres perform similar functions, telomeric organisation is diverse. Besides the widely conserved telomeric model consisting of tandem arrays of short G/C-rich repeats maintained by specific enzymes, named telomerases, there are alternative telomere structures with different types of telomeric DNA. This is the case with telomeres of dipterans. One of the best known, elomeres ,13. Anotelomeres ,16,17,18Chironomus riparius, also known as Chironomus thummi, possesses one of these noncanonical telomeres consisting of 176-bp-long tandemly repeats at their chromosome ends. Early analysis of its telomeres showed the existence of transcriptional activity that could be detected after thermal stress as a giant telomeric puff remarkably observed in the right telomere of chromosome 3 (hereinafter ‘III-R’). Activation of transcription under heat shock was also detected in other telomeres but to a lesser extent [r extent . Later, r extent ,21,22. Tr extent . The seqr extent . Three dr extent . TsA is C. riparius that are transcribed from these telomeric sequences: the CriTER-A (Chironomus riparius TElomeric RNA from TsA), the CriTER-B (Chironomus riparius TElomeric RNA from TsB), and the antisense transcripts produced from a complementary strand of TsB, the so-called αCriTER-B [Additionally, three different ncRNAs were identified in CriTER-B . FurtherCriTER-B . ExperimCriTER-B ,25. OverC. riparius is the sole biological system in which two homologous telomeric ncRNAs with substantial and characterized differences in the sequence, CriTER-A and CriTER-B, has been reported, exhibiting each differential transcriptional activity under different environmental conditions. Despite the sequence variation, secondary structure elements are highly conserved in evolution for most functional RNAs. Thus, the potential structural differences between CriTER-A and CriTER-B could result in different functionalities according to environmental conditions, such as thermal stress. To investigate the putative differential structure and function of both these transcripts, a comparative structural analysis was carried out. We hypothesised that despite the sequence homology between these two telomeric transcripts, they exhibit conformational variations that could rely on their different behaviour and relative abundance upon heat shock conditions. Thus, this manuscript provides a structural analysis of the transcripts corresponding to the monomeric units (176 bp), CriTER-A and its homologue CriTER-B. We obtained comprehensive results proving the existence of distinctive folding and conformational behaviour for these two ncRNAs and demonstrating the existence of a conformational switch in CriTER-A, which is associated with temperature changes. These findings support the functional role of CriTER-A in relation to thermal stress and offer novel evidence of the existence of thermosensing structural elements in a stress-inducible telomeric RNA.Revealing the structure and function of telomeric RNA transcripts is crucial for understanding telomere biology and the fundamental biological processes in which they are involved, such as stress response and senescence ,26. As fC. riparius cells; and 35 °C, which entails a thermal stress situation in this biological model  and 35 °C . Interestingly, different conformers could be detected at 20 °C for both telomeric transcripts CriTER-A  and CriTER-B , with the relative proportion between them remaining constant even at high RNA concentrations  were carried out. For this purpose, RNA transcripts encoding the natural monomeric CriTER-A and CriTER-B RNA molecules were trations . The preions  in a Mg2+-dependent way whereas no differences in the CriTER-B conformers were detected. Thus, CriTER-B shows high conformational homogeneity with a compact and likely static structure and a well-defined architecture with three major stem-loops separated by single-stranded regions  could be detected for both telomeric transcripts, one of them (A1 and B1) being much more prevalent (~80%). Their relative abundance does not depend on the RNA concentration or the presence of Mg regions  in contr regions , seems tTaken together, these results suggest that the structural tuning observed for the CriTER-A variants could be a key piece in the molecular mechanism underlying the CriTER-A function as a thermal sensor and point to A2 as the main functionally important conformer under heat stress conditions. As far as we know, this is the first time that distinguished structural features correlated to temperature changes of two homologue telomeric transcripts have been reported. Thus, these observations are an important step towards enhancing our understanding of telomeric ncRNA function in relation to cellular stress response.EcoRI sites were added in both extremes to facilitate later work.The TsA and TsB sequences are available in GenBank, Accession nos. M33211 and AJ295633, respectively. The sequences for the sense transcripts CriTER-A and CriTER-B correspond to the reverse-complementary ones of these both telomeric sequences. The plasmids with one monomeric unit of each transcript were ordered from GenScript (Netherlands). The receipt vector was pUC57. DNA templates encompassing the sequence coding for CriTER-A and CriTER-B transcripts were obtained by PCR from their respective plasmids with the oligonucleotides see  T7pCriTEFor SHAPE assays and 2′-hydroxyl molecular interference (HMX) analyses, DNA templates coding for the transcripts CriTER-A+cas and CriTER-B+cas were also obtained by amplification. In this case, the RNA transcript design required slight modifications in order to optimise the recovery of the SHAPE reactivity data. Hence, two different cassettes at the 5′ and the 3′ ends were included, flanking the central sequence encoding CriTER-A or CriTER-B. These cassettes do not interfere with the structural dynamic of the transcripts (data not shown). The cassette located at the 5′ end is 48 nts long and shows an autonomous folding. It allows for the precise monitoring of SHAPE reactivities at the 5′ end of the RNA molecule under study since it moves away from the fluorescent peak corresponding to the full-length product. At the 3′ end, the 45 nts 3′ cassette provides an annealing site for efficient primer extension. Briefly, molecules T7pCriTER-A+cas and T7pCriTER-B+cas bearing these cassettes were obtained by amplification from their corresponding plasmids. Oligonucleotides T7p_CriTERA_5cas and asCriTERAB_3cas generated the T7pCriTER-A+cas template, while oligonucleotides T7p_CriTERB_5cas and asCriTERAB_3cas allowed for the production of the T7pCriTER-Bcas template see .RNA synthesis was accomplished with the generated DNA templates using the TranscriptAid T7 High Yield Transcription Kit (ThermoFisher Scientific), following the manufacturer’s instructions. The resulting RNA products were purified, and their quality was tested as previously described .32P-labeled RNA were denatured for 2 min at 95 °C and subsequently transferred to ice for 15 min. RNA molecules were then incubated at 20 or 35 °C in folding buffer , either in the presence or absence of 1 mM MgCl2, for 30 min. RNA isoforms were immediately resolved in 6% nondenaturing polyacrylamide gels (acrylamide:bisacrylamide 19:1). Electrophoresis was performed at 4 °C and 12 V/cm, either in TBM buffer  for analyses performed in the presence of Mg2+; or in TBE buffer  for those studies carried out in the absence of Mg2+. Gels were dried, scanned, and analysed as previously described [The analysis of structural heterogeneity for CriTER-A and CriTER-B monomers was performed in a dose-dependent manner. Briefly, increasing concentrations (1–100 nM) of the Competition assays were performed with 10 nM of the labelled CriTER-A or CriTER-B constructs in the presence of a molar excess of the corresponding unlabelled transcripts . Briefly, RNA molecules were denatured and renatured as noted above and mixed at the indicated concentrations in the presence of folding buffer. Reactions proceeded at 20 °C for 30 min. Complexes were resolved in TBE buffer as described above. Gels were dried and scanned as reported in .SHAPE analysis of CriTER-A and CriTER-B folding was performed essentially as previously described ,33. For 2 for 5 min. Probing reactions were initiated by the addition of 15 mM of freshly diluted NMIA in DMSO. Chemical probing proceeded for 30 min at 35 °C and was stopped by snap cooling and ethanol precipitation. Control reactions were carried out in parallel in the absence of NMIA, with an equivalent volume of DMSO. The RNA was washed once with 80% ethanol, and different CriTER-A conformers were resolved by nondenaturing polyacrylamide gels as described above. Specific conformers were visualised by UV shadowing, eluted, and purified as previously described [For CriTER-A, 10 pmol of purified RNA were denatured for 2 min at 95 °C and then cooled on ice for 15 min. The RNA was then incubated at 35 °C in folding buffer with 1 mM MgClescribed . One pmoescribed , which rShape-dif methodology for the CriTER-B variant was carried out as reported ,37. Fivehttp://www.unafold.org/, accessed on: 25 August 2021), RNAFold, included in Vienna RNA package [http://rna.tbi.univie.ac.at/, accessed on: 25 August 2021), and RNAStructure [http://rna.urmc.rochester.edu/, accessed on: 25 August 2021). Preliminary RNA secondary structure predictions for this study were performed using RNAStructure web server  [There are different implementations, which enable us to estimate the RNA secondary structure from a single sequence. The most popular are Mfold/Unafold  (http:// package  (http://tructure  (http://st 2021) ,42. MFE st 2021) .The prediction of the theoretical RNA secondary structure models taking the SHAPE mapping date was generated using ShapeKnots , includi


The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti‐virals. We combine in vitro RNA analysis with molecular dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5′ UTR of the SARS‐CoV‐2 genome. Furthermore, we determine the binding of metallo‐supramolecular helicates (cylinders) to this RNA structure. These nano‐size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3‐base bulge and the central cross 4‐way junction located in stem loop 5. Finally, we show these RNA‐binding cylinders suppress SARS‐CoV‐2 replication, highlighting their potential as novel anti‐viral agents. Viral untranslated regulatory regions (UTRs) contain a variety of conserved yet dynamic RNA structures crucial for viral replication. Combining in vitro RNA analysis with molecular dynamics simulations 3D models of the structure and dynamics of key regions of the 5’ UTR of SARS‐CoV‐2 are created. Metallo‐supramolecular helicates are shown to bind at a bulge and junction, and in cell assays confirm that they inhibit viral infection. These structures play a role in RNA‐RNA interactions  and in protein binding for the initiation of mRNA production, translation, and viral replication. Moreover, these RNA structures may act as trans acting elements or mediate translational frameshifting, a common feature in viruses with plus‐strand RNA genomes.Nucleic acid sensors mediate the early detection and host response to virus infections, and recognise either viral nucleic acids or “unusual” cellular nucleic acids present upon infection.Compared to protein‐ and DNA‐recognition, RNA‐recognition by drugs has been much less explored. Nucleic acid recognition often focuses on sequence recognition but for RNA, which folds into complex shapes, its structure provides an opportunity for specific targeting; indeed, it is the structure of the UTR that is conserved for function, rather than sequence. Small molecule libraries have been screened for RNA binding 23, 24We have explored nano‐size metallo‐supramolecular cylinders Figure  as RNA‐bGiven this anti‐viral activity against HIV‐1, we were interested to assess whether these cylinders would bind structures in the UTR of SARS‐CoV‐2. We report now combined modelling and biophysical approaches to define the 3D structures of the SARS‐CoV‐2 5′ UTR, and demonstrate cylinder binding to specific bulge structures in the 5′ UTR. Furthermore, we show that cylinders inhibit SARS‐CoV‐2 viral replication in cells.To create a 3D dynamic model of the 5′ UTR from the published genome sequenceTo experimentally probe the UTR, we used SHAPE,  analysis where the 5′ UTR RNA sequence was first folded in vitro and the open strand (non‐duplex) RNA sites  acylated with 1‐methyl‐7‐nitroisatoic anhydride (1M7). These sites were then identified through a reverse transcription reaction that generates DNA fragments which end at the 1M7 tagged sites and were readily analysed by gel electrophoresis Figure . Two priThe results  mutations in the 5′ UTR. We examined the available sequences in the gisaidAfter identifying the distinct stems loops (SLn) that were conserved throughout the results from the secondary structure prediction, we attempted the more challenging step of creating a 3‐dimensional representation of the structure. We focused on SL3 and SL5 as they have a variety of different structural features including bulges and loops. Although the exact structure/function of SL5 is not yet determined (to our knowledge), it contains the initiation codon and it is similar to the SL5 of SARS‐CoV‐112, 13We employed the recent RNA‐force field developed by Mathews,48, 49Considering the SL5 RNA in absence of cylinder, molecular dynamics reveal the following features of the stem: a) There is a bulge at G138‐U140 which is highly flexible with a lot of transient stacking between its bases   cylinder (M=Fe), we also compared the analogous nickel(II) and ruthenium(II) cylinders , and the frequency of cells expressing the viral encoded spike glycoprotein quantified . MTT cell metabolic activity/viability assays confirmed that the cylinder is not cytotoxic to Vero cells in the timeframe of these experiments (See Supplementary Information).Having established that the cylinder can bind to and modify the structure and reactivity of the SARS‐CoV‐2 5′ UTR in vitro, we explored their potential to inhibit viral replication in cellulo. Simian Vero cells were infected with SARS‐CoV‐2 virus England 2  in the presence and absence of the Ru and Ni cylinders, [Md Figure . Both cyWe have shown that by combining experimental SHAPE results with molecular dynamics simulations we can create 3D models of the structure and dynamics of key individual stems that make up the 5′ UTR of SARS‐CoV‐2. These stems contain a number of intriguing structural motifs also found in the UTRs of other viruses, and which offer the possibility of developing new anti‐viral agents that act against a broad spectrum of diseases. The unique nucleic acid binding activity of the supramolecular cylinders is ideally suited to target these types of structures and we show that the cylinders can bind non‐covalently to an RNA bulge in stem loop 5, as well as the central cross (4‐way junction) of that loop. The ability to bind at different crucial RNA structural sites that are essential for virus replication limits the opportunity for the virus to mutate and to evade drug action. In line with their RNA binding, these nanosized supramolecular helicates inhibit infection at concentrations where they have negligible cellular toxicity.These helicate cylinders are currently the only metallo‐supramolecular architectures that have been demonstrated to thread through RNA bulge and junction structures, but there is a growing interest in metallo‐supramolecular designs to bind nucleic acid structures.53, 54The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


As transcriptional co-activator of AP-1/Jun, estrogen receptors and NF-κB, nuclear protein RBM39 also involves precursor mRNA (pre-mRNA) splicing. Porcine reproductive and respiratory syndrome virus (PRRSV) causes sow reproductive disorders and piglet respiratory diseases, which resulted in serious economic losses worldwide. In this study, the up-regulated expression of RBM39 and down-regulated of inflammatory cytokines  were determined in PRRSV-infected 3D4/21 cells, and accompanied with the PRRSV proliferation. The roles of RBM39 altering phosphorylation of c-Jun to inhibit the AP-1 pathway to promote PRRSV proliferation were further verified. In addition, the nucleocytoplasmic translocation of RBM39 and c-Jun from the nucleus to cytoplasm was enhanced in PRRSV-infected cells. The three RRM domain of RBM39 are crucial to support the proliferation of PRRSV. Several PRRSV RNA  binding with RBM39 were determined, which may also contribute to the PRRSV proliferation. Our results revealed a complex mechanism of RBM39 by altering c-Jun phosphorylation and nucleocytoplasmic translocation, and regulating binding of RBM39 with viral RNA to prompt PRRSV proliferation. The results provide new viewpoints to understand the immune escape mechanism of PRRSV infection. RNA-binding proteins (RBPs) have been found in all living organisms . ThroughRBM39 (RNA binding motif protein 39), also called CAPER, HCC1.3/1.4,Caperα,FSAP59,RNPC2 , is a nuThe activation protein-1 (AP-1) transcription factors are immediate early response genes involved in a diverse set of transcriptional regulatory processes , 20. ThePorcine reproductive and respiratory syndrome virus (PRRSV) is a critical pathogen in pig and can trigger a serious negative impact on the economic development of pigs , 27, whi2. All media were obtained from Biological Industries and supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. The PRRSV was preserved in our laboratory and produced by transfection of 3D4/21 cells with the PRRSV-JXwn06 infectious clone plasmid. In this study, the PRRSV was used with a titer of 104 PFU/ml. Sendai virus (SeV) and vesicular stomatitis virus (VSV) was preserved in our laboratory.Human embryonic kidney 293 T cells (HEK293 T) and HeLa cells were cultured in Dulbecco’s modified essential medium (DMEM), porcine alveolar macrophages (PAM) cell and PAM cell line 3D4/21 in RPMI-1640 medium  in incubator at 37°C with 5% COMurine RBM39 pAb was prepared by immunizing mice with the purified protein. Anti-PRRSV nsp2 antibody was a gift from Prof. Jun Han of China Agricultural University and Rabbit anti-PRRSV-N protein pAb was purchased from YBio Technology (YB-23941R). Mouse anti-Flag/β-actin/GADPH mAb and Peroxidase-Conjugated Goat anti-Rabbit/Mouse IgG (H+L) were purchased from Yeasen Technology. Rabbit anti-phospho-JNK1+2+3 (Thr183+Tyr185) pAb was purchased from Bioss (bs-1640R); Rabbit anti-c-Jun mAb was purchased from Abways Technology (CY5290); Rabbit anti-phospho-c-Jun (Ser73) mAb was purchased from Cell Signaling Technology (3270T); Rabbit anti-HA pAb, Goat anti-Mouse IgG (H+L) Alexa Fluor 555 and Goat anti-Mouse/Rabbit IgG (H+L) FITC was purchased from Invitrogen; Mouse anti-Flag/HA-tags beads was purchased from Abmart. Phosphatase inhibitors were purchased from APE×BIO. Quick CIP were purchased from Biolabs.RBM39 and c-Jun gene was cloned from 3D4/21 cells cDNA as template. Flag/HA-RBM39 and Flag/HA-c-Jun plasmids were constructed by seamless cloning technology. Flag-RRM1, RRM2, RRM3, ΔRRM1, ΔRRM2, ΔRRM3, ΔRRM, ΔNLS plasmids were constructed by reverse amplification by Flag-RBM39 plasmid as template. Plasmid pET-28a-RBM39-X7 that delete the complete 5’-terminal NLS sequence was also constructed by seamless cloning method, to purify the RBM39 protein and make its antibodies. AP-1/IFN-β/Renilla luciferase reporter plasmids were constructed as previously described . The pri2. After 4-6 hours, discard the mixed solution and replace to complete medium, and continue to grow cells for 24-48 h.Previous to transient transfection, adherent HEK293T, HeLa or 3D4/21 cells were seeded in a 6/12/24-well plate . Cells were transfected at the appropriate confluence with eukaryotic expression plasmid. Briefly, 10 µM polyethyleneimine (PEI) was preheated at 85°C and replace the old medium with Reduced-Serum Medium  before transfection 1 h in advance. Add a suitable equal volume of medium to the two centrifuge tubes, then add the appropriate plasmid and PEI, shake and mix, and place at room temperature for 5 minutes. Then, mix and place them at room temperature for 20-30 minutes. Afterwards, add the mixture to the cells and incubate the cells in 37°C incubator containing 5% COCell samples were lysed by radioimmunoprecipitation assay (RIPA) (Solarbio) added with protease inhibitor cocktail phenylmethanesulfonyl fluoride . Cells lysis supernatants were added to 5×loading buffer, following boiled for 10 min and separated by SDS-PAGE. The separated proteins were transferred onto a polyvinylidene fluoride (PVDF) blotting membrane  and blocked for 1h with 5% skimmed milk in 1×TBST  at room temperature, followed by incubation with the primary antibodies diluted in 1×TBST at 4°C overnight and secondary HRP-conjugated antibodies diluted in 1×TBST for 1h at room temperature. Finally, the membrane was detected through Pierce ECL Western Blotting Substrate  and exposed through Chemi Doc XRS Imaging System .T) method (2−ΔΔCt). The thermal cycler program consisted of 95°C for 10 min and then 45 cycles at 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s. The β-actin was used as an internal control for normalization. All the primers used for quantitative real-time PCR are shown in Quantification of the relative levels of gene expression was performed using qRT-PCR. Total RNAs were extracted by RNAiso plus (Takara). The cDNA was synthesized with EasyScript First-Strand cDNA Synthesis SuperMix according to the manufacturer’s instructions (TransGene). The relative levels of gene expression were analyzed by QTOWER3G IVD Fluorescent Quantitative PCR System  using TransStart Top Green qPCR SuperMix (TransGene) with three-step amplification and the data were calculated by the comparative cycle threshold  and the Renilla luciferase control reporter plasmid was used as a reference. The cells were collected and lysed at 24 h post-transfection, and the cell supernatant was used to detect luciferase activity through Dual Luciferase Reporter Gene Assay Kit (Yeasen Technology) according to the manufacturer’s instructions.via SDS-PAGE, transferred to PVDF membrane, and detected with the proper antibodies.HEK293 T Cells cultivated in six-well plates were transfected or co-transfected with appropriate eukaryotic expression plasmid. The cells were harvested and resuspended by PBS 24 h post-transfection, and lysed on ice for 15 min in 400 µl RIPA lysis buffer. Then the samples were centrifuged at 12,000 rpm for 5 min. We took out the 50 μL supernatant in new tube, added 10 μL loading buffer and boiled them to make input samples. Subsequently, anti-Flag-labeled beads (Sigma) were activated by RIPA and added the rest of the cell lysis supernatant. The reaction mixture was incubated at 4°C overnight. The beads were washed three times with lysis buffer containing PMSF for 5 min each time and added 50 μL lysates and 10 μL loading buffer, boiled for 10 min to make IP samples. Finally, the proteins bound to the beads were separated Adherent HEK293T, HeLa or 3D4/21 cells grown on glass coverslips in 12-well plates at 30% to 50% confluence were transfected with plasmids expressing RBM39 and/or c-Jun. The empty vector plasmid was used as a negative control (NC). Subsequently, 3D4/21 cells were infected with PRRSV at a multiplicity of infection (MOI) of 0.4. At 24 h post-transfection or post-infection, the cells washed with 1×phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.5% Triton X-100 for 15 min and blocked with 5% bovine serum albumin (BSA) dissolved in 1×PBS containing 0.05% of Tween-20 (PBST) for 2h at room temperature. Anti-HA (hemagglutinin), anti-Flag and anti-PRRSV-N primary antibodies were diluted in 2% BSA and incubated with slides overnight at 4°C. Following a wash performed with PBS 3 times for 5 min each time, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse/rabbit IgG or IF555-conjugated goat anti-mouse IgG secondary antibody diluted in 2% BSA at 37°C for 1 h in a humidified chamber. The slides were washed with PBST and nuclei were counterstained with Hoechst 33258 dye (Solarbio) for 5 min. Confocal images were collected with confocal laser scanning microscope  and taken at 40 × or 100 × magnification (Olympus).A small interfering RNA (siRNA) assay, which was synthesized from Sigma Table 3,via corresponding primers.3D4/21 cells plated in 6-well plates and grown to 70%-80% confluence were transfected with 2μg HA-RBM39 or Flag-RBM39 plasmids. Then the cells were infected with 0.4 MOI PRRSV 12 h after transfection. After another 24 hours, the cells were collected and lysed with 400 μl RIPA supplemented with murine RNase inhibitor (1:100). Part of the supernatant was used for immunoblotting analysis, and the remaining supernatant was incubated with HA/Flag-tags beads overnight at 4°C. The co-precipitated RNAs were extracted, reverse transcribed and detected through PCR Data were presented as the mean of three independent replicates. Statistical significance was analyzed by the unpaired t-test using Origin and GraphPad Prism software. Statistical differences were considered to be statistically significant at P-value < 0.05 .PRRSV infection can result to upregulation or downregulation of many genes in cells. To find out genes that displayed significant changes in transcription after PRRSV infection, a transcriptome sequencing analysis derived from PAM cell was performed. RBM39 was found to be significantly upregulated after PRRSV infection by statistical analysis S2A and The effect of RBM39 on the virus has not been reported before. To explore the effect of RBM39 on PRRSV proliferation, the 3D4/21 and PAM cells were transfected with Flag-RBM39 expression plasmids or empty vector, followed by infected with 0.4 MOI PRRSV 12 h post-transfection. Total RNA was extracted from the cells at 24 h post-transfection, and subsequently, transcription levels of PRRSV nsp2 and N gene were analyzed by qRT-PCR. Compared with the control groups, the overexpression of RBM39 promoted the mRNA level of the PRRSV nsp2 and N gene S2D. Meavia the reverse amplification method, namely Flag-RRM1, RRM2, RRM3, ΔRRM1, ΔRRM2, ΔRRM3, ΔRRM, ΔNLS . The results showed that overexpression of RBM39 drastically decreased the transcription and secretion levels of these genes in 3D4/21 cells  results found that RBM39 binds and interact with the AP-1 component c-Jun . In addiThe c-Jun is an important part of AP-1 signaling pathway and a significant immune-related protein. To investigate whether RBM39 impacts AP-1 signaling pathway, HEK293 T cells were transfected or co-transfected with HA-RBM39 and AP-1 promoter reporter plasmid. The results display that the activity of AP-1 promoter reporter gene was reduced by RBM39 overexpression in a dose-dependent manner . After PRRSV infection, RBM39 is up-regulated, which promotes the formation of RBM39 and c-Jun complex in the cytoplasm and cytoplasmic retention. (2) PRRSV infection makes JNK phosphorylation incorporated into the nucleus, and promotes the phosphorylation of nuclear transcription factor c-Jun; RBM39 and the phosphorylated c-Jun in the nucleus form a complex that is prone to translocation from the nucleus to the cytoplasm, which further reduces the retention of c-Jun in the nucleus; Decreased phosphorylated c-Jun in the nucleus, down-regulating the transcription complex of c-Jun and c-Fos, resulting in down-regulation of the transcription level of IFN-β,TNF-α,IL-1β,IL-6 and other genes controlled by the AP-1 promoter. (3) The enhancement of the binding capacity of RBM39 with PRRSV RNA hints that the improvement of viral RNA stability also contributes to the PRRSV proliferation Figure 9The RNA-binding protein family can promote or inhibit the proliferation of viruses, and the mechanism of action is different. For example, Pseudouridine Synthase 4 (Pus4) could compete with the capsid protein (CP) to bind to plus-strand Brome Mosaic Virus (BMV) RNAs and prevent virion formation . EAP EB–42. FátyIn addition, the virus itself also encodes RNA-binding protein for its pivotal role in replication, transcription, etc. The NP protein (nucleoprotein) of influenza virus is a structural single-stranded RNA binding protein, which is a key adaptor molecule between the virus and the host cell process , 49. NP http://www.cbs.dtu.dk/services/NetNES/, neither RBM39 nor c-Jun has NES  sequences. Therefore, after PRRSV infection, the translocation of them to the cytoplasm may involve other proteins containing the NES sequence, which leading to the translocation of the two.After PRRSV infection, both RBM39 and c-Jun showed the phenomenon of translocation from the nucleus to the cytoplasm. Quantities of previous studies have shown that RNA-binding proteins have been transferred from the inside to the outside of the nucleus. Another RBP protein in the RBM family, RBM14, was relocated to the nucleolus after influenza A infection . CIRP isvia siRNA changes the splice form of vascular endothelial growth factor A (VEGF-A)  , 60, 61.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Conceived and designed the experiments: JH. Performed the experiments: YS, XL, YG, MZ, RS, JS, ZT, and LZ. Analyzed the data: YS and JH. Contributed reagents/materials: JH. Wrote the paper: YS and JH. All authors contributed to the article and approved the submitted version.This work was supported by the National Key Research and Development Program of China (2018YFD0500500) and the key underprop project of Tianjin Science and Technology Bureau in China (20YFZCSN00340).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.