For instance, the MTase domain confers stability onto the RdRp elongation complex (EC) and and is required for de novo RNA synthesis and nucleotide incorporation by RdRp. Finally, structure function analyses identify key conserved residues at the MTase-RdRp interface that specifically activate RdRp elongation and are essential for ZIKV replication in Huh-7.5 cells. These data demonstrate the requirement for the MTase-RdRp interface in ZIKV replication and identify a specific site within this region as a potential site for therapeutic development.The spread of Zika virus (ZIKV) has caused an international health emergency due to its ability to cause microcephaly in infants. Yet, our knowledge of how ZIKV replicates at the molecular level is limited. For example, how the non-structural protein 5 (NS5) performs replication, and in particular whether the N-terminal methytransferase (MTase) domain is essential for the function of the C-terminal RNA-dependent RNA polymerase (RdRp) remains unclear. In contrast to previous reports, we find that MTase is absolutely essential for all activities of RdRp  Although ZIKV infections have historically caused mild symptoms and can infrequently lead to Guillain-Barré syndrome, the recent massive spread of the virus to the Americas has caused the World Health Organization to declare an international health emergency3. This is due to the ability of the virus to cause a rare neurological condition in infants called microcephaly which is characterized by a significantly reduced head size and impaired neurological and motor skill development4. Strikingly, ZIKV is not only transmitted from mother to fetus, but also via sexual intercourse1. Recent evidence in mice also indicates that ZIKV infection in males may cause sterility5. Hence, our current knowledge of the detrimental effects of ZIKV infection appears to be limited.Mosquito-borne Zika virus (ZIKV) is a member of the Flaviviridae family first identified in 1947 in the Zika Forest region in Uganda7. Although endeavors are underway to develop a vaccine, this can take several years and is not necessarily guaranteed to be effective. Thus, an alternative strategy to combat ZIKV, such as the development of anti-viral drug inhibitors, is an important area of research. Based on the past successes of using polymerase inhibitors to treat human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections, drug inhibitors of the ZIKV RNA-dependent RNA polymerase (RdRp) are likely to be similarly effective in treating ZIKV infections9. However, our current knowledge of how the ZIKV replication machinery functions is limited.ZIKV has spread to 60 countries including the United States where ZIKV infections have been reported in Florida and in Texas12. RNA capping promotes RNA stability, efficient translation and evasion of the host immune response13. Yet, why the MTase is directly fused to RdRp in the Flavivirus genus remains unclear. A plausible explanation is that these linked enzymes cooperate during RNA synthesis. Studies of related Dengue virus (DENV) report conflicting findings on the effects of the MTase on RdRp RNA synthesis activity. For example, stimulation and suppression of DENV RdRp by the MTase have been reported16, whereas another study showed that MTase had no effect on DENV RdRp activity17. Intriguingly, mutation of key residues involved in DENV MTase-RdRp interactions primarily resulted in an increase in RdRp initiation and elongation activities in vitro, suggesting MTase suppresses DENV RdRp activity16. A recent in vitro study using ZIKV proteins suggests that the MTase only significantly contributes to RdRp RNA elongation activity, which contrasts previous reports on DENV14. Clearly, detailed biochemical studies are required to unequivocally determine whether the effects of MTase on RdRp activity are universal among Flaviviruses or vary between different members such as ZIKV.All known Flavivirus RdRp enzymes are directly fused to a methytransferase (MTase) through a flexible linker within the non-structural 5 (NS5) protein that is essential for replication Fig. . The MTa21. Japanese encephalitis virus (JEV) MTase exhibits the same conformation as ZIKV MTase22. A notable difference between ZIKV/JEV and DENV MTase conformations is their respective interactions with the RdRp fingers subdomain which is important for nucleotide binding and template interaction  which appears to contribute to the conformation of the NTP and template channels , and is also necessary for RNA initiation and nucleotide incorporation by RdRp. Structure function analyses identify key conserved residues in MTase that are important for RdRp elongation but not initiation. Importantly, when we mutated these residues to disrupt the specific MTase-RdRp interface site, ZIKV replication was completely abolished in Huh-7.5 cells. These data demonstrate that MTase acts as an essential auxiliary factor for ZIKV RdRp, and identify a small region within the MTase-RdRp interface as a potential drug binding site.E. coli expression vector containing a Hexa-histidine tag (His-tag) and a SUMO tag at the N-terminus. RdRp was purified by standard nickel affinity chromatography methods including cleavage of the His-SUMO tag  encoded by the Asian ZIKV strain PRVABC59 was cloned into a tag Fig. , right. 23. For example, prior reports demonstrated the ability of DENV and HCV RdRps to extend synthetic RNA primer-templates in the presence of ribonucleoside triphosphates (NTPs)24. A multitude of studies on RNA polymerases, including those performed with HCV and DENV RdRps, show that extension of these types of primer-templates requires the polymerase to undergo a conformational change necessary for elongation of the RNA27. For example, recent structural studies of HCV RdRp demonstrate distinct initiation and elongation conformations of the enzyme, and many studies on DNA-dependent RNA polymerases show similar initiation to elongation conformational changes32. Reports also show the ability of DENV and ZIKV RdRps to readily conform to an EC on synthetic RNA primer templates, similar to DNA-dependent RNA polymerases24. In this report, we primarily focused on biochemical investigation of ZIKV RdRp and NS5 ECs.We used similar RNA replication elongation assays performed previously with DENV and HCV RdRps33. Next, ATP along with 32P-α-ATP were added and reactions were terminated after 30 min by the addition of EDTA and formamide. RNA was resolved in denaturing polyacrylamide gels and visualized by phosphorimager and autoradiography. The results show that NS5 is dramatically more proficient in RNA elongation activity compared to RdRp  with a RNA primer-template at 37 °C for 10 min in order to allow the polymerase to adopt a conformation necessary for elongating the RNA, similar to previous studies Fig. , top33. dRp Fig. , left, a13. We examined whether RdRp is impaired in RNA initiation activity compared to NS5 in Fig. de novo RNA synthesis by the polymerase in the presence of NTPs and 32P-α-ATP  of NS5 and RdRp  with a cassette containing the Renilla luciferase followed by the foot-and-mouth disease virus (FMDV) 2 A protease. The resulting ZIKV replicon (ZIKV/SG-RLuc wt) contains the 5′UTR, the first 25 amino acids of the capsid protein, Renilla luciferase, FMDV 2 A protease, the last 28 amino acids of the envelope protein, viral NS1-NS5 proteins, and the 3′ UTR Fig. . The res8. Thus, understanding how the ZIKV NS5 RdRp domain functions and is regulated at the molecular level is important for developing therapeutics against its enzymatic activity. A previous study has reported that the MTase-RdRp interface in ZIKV NS5 only significantly affects elongation activity18. In contrast to this previous report, our studies demonstrate that the MTase domain is essential for all RNA synthesis activities of RdRp. For example, we demonstrate that RdRp is severely deficient in de novo RNA synthesis, EC stability, and nucleotide incorporation. Although multiple reports have specifically focused on the activity of the isolated RdRp domain38, our findings show that it is severely defective in RNA elongation and initiation.Because viral polymerases, such as those encoded by HIV and HCV, have demonstrated to be highly effective drug targets, the RdRp domain of ZIKV NS5 is similarly considered a promising enzyme for therapeutic developmentin vitro demonstrates that this interface is important for RNA elongation. Hence, our findings indicate that the specific MTase-RdRp interface formed by residues E112, P113 and L115 of the MTase domain with RdRp F466 is essential for ZIKV replication by promoting replication elongation. The possibility exists that these residues potentially interact with other ZIKV proteins during the course of replication and thus could conceivably have additional undefined roles in replication, in addition to their replication elongation function.Our studies identify a small MTase-RdRp interaction site that is essential for ZIKV replication in cells. Biochemical data of the identical NS5 mutant 19. Specifically, MTase residues that stabilize the F motif , the F motif structure is dramatically changed and as a result alters the configuration of the NTP and template binding channels19. This structural alteration likely explains why RdRp incorporates nucleotides at an extremely low rate compared to NS5 during elongation. Consistent with our biochemical observations, we find that identical MTase-F motif contacts in NS5 are essential for ZIKV replication in cellular studies, and these contacts are also needed for JEV replication, demonstrating a conserved function at least between ZIKV and JEV39.Importantly, recent crystallographic studies complement our data and provide further insight into potential mechanisms by which the MTase regulates RdRp. For instance, structural data demonstrate that the MTase contributes to the conformation of the NTP and template binding channels46. Our findings indicate that the MTase-F motif interactions in ZIKV NS5 represent a potential site for therapeutic development. For example, compounds designed to specifically prevent the MTase-F466 interaction by binding key contacts in the 112–126 residue region in MTase  at 37 °C. RNA elongation was then initiated by the addition of 2 µM ATP along with 0.5 µl of 32P-α-ATP (Perkin Elmer) in a final volume of 20 µl. Reactions were terminated after 30 minutes by the addition of 25 mM EDTA and 45% formamide. RNA was then resolved in denaturing urea polyacrylamide gels containing 15% formamide, then visualized by autoradiography or phosphorimager. In Fig. 50 nM of the indicated RNA template (467 R/475 R) was pre-incubated with 1 µM or indicated concentrations of RdRp or NS5 proteins for 10 min in the following buffer  was preincubated for 30 s with 1 µM of NS5 or RdRp in the following buffer  at 37 °C. Then 200 µM of NTPs were added for 1 hour until the reactions were terminated by the addition of 25 mM EDTA and 45% formamide. RNA was resolved in denaturing urea polyacrylamide gels and visualized by phosphorimager and autoradiography.50 nM of the 2). Then 10 µM or 2 µM of NTPs were added along with 32P-α-ATP (Perkin Elmer) for the indicated times. Reactions were terminated by the addition of 25 mM EDTA and 45% formamide and RNA was resolved in denaturing urea polyacrylamide gels and visualized by phosphorimager and autoradiography. Relative extension (R.E.) was determined by comparing the relative intensities of run-off products for each experiment and normalizing the most intense run-off product band to 100.50 nM of the indicated RNA (RP478R) was pre-incubated with 1 µM NS5 or RdRp for 30 s in the following buffer  in the presence of 32P-γ-ATP (Perkin Elmer).RNA primer was mixed with a 1.25 fold excess of template RNA in the presence of 50 mM NaCl, then the mixtures were heated to 70–80 °C for 2–5 min, followed by slow cooling to room temp. In the case of Figs D was determined from protein concentration giving half-maximal template binding.Fluorescence anisotropy was performed by incubating the indicated amounts of NS5 or RdRp with 10 nM of fluorescein (FAM) conjugated RNA template (RP467RFAM/RP475R) at room temp in the following buffer: 20 mM Tris-HCl pH 6.8, 0.1 mg/ml bovine serum albumin (BSA), 0.01% NP-40, 10 mM dithiothreitol (DTT), 10% glycerol. After 30 minutes, florescence polarization of FAM was determined using a Clariostar plate reader. KE. coli vector (LifeSensors) resulting in a N-terminal Hexahistidine-SUMO tag. RdRp was expressed overnight at room temp in Rosetta2(DE3)/pLysS cells (Stratagene) after reaching 0.6 optical density (O.D.) and adding 0.4 mM IPTG. Cells were lysed by sonication in the presence of lysis buffer  and protein extract collected after ultracentrifugation at 35,000 rpm for 45 min. Clarified extract was loaded onto a His-trap crude 5 ml column , washed with 15 column volumes of lysis buffer. Protein was then eluted with lysis buffer containing a 10–250 mM imidazole gradient. Peak fractions containing His-SUMO RdRp were pooled and dialyzed into lysis buffer containing 200 mM NaCl and 0 imidazole, then mixed with SUMOStar protease (LifeSensors) and incubated overnight at 4 °C. Finally, cleaved RdRp was purified over a His-trap 5 ml column with a shallow gradient of imidazole. Pure cleaved RdRp was concentrated to 9.11 mg/ml and stored in aliquots at −80 °C. All steps in the purification process were carried out at 4 °C. RdRp encoded by strain SPH2015 was used in Figs E. coli pellets were stored at −80 °C. Frozen pellets were thawed on ice and resuspended in buffer containing 50 mM TrisHCl pH 8.8, 500 mM NaCl, 10% (v/v) glycerol, 10 mM imidazole pH 8, 1.5% (v/v) Igepal CA-630 (Sigma), 5 mM 2-mercaptoethanol (BME), 10 mM PMSF, and 1 SIGMAFAST™ Protease Inhibitor tablet per every 100 ml at a volume of 10 ml of buffer per gram of cell pellet. The resuspended cells were sonicated on ice and then centrifuged at 40,000 g. The clarified cell lysate was loaded onto a 5 ml HisTrap FF Crude column (GE Lifesciences) and washed with buffer A  glycerol, 10 mM imidazole pH 8.0, 5 mM BME and 0.005% v/v Igepal CA-630). Bound fractions were then eluted with a gradient to buffer B  glycerol, 0.005% (v/v) Igepal CA-630, 5 mM BME and 250 mM imidazole pH 8.0). Fractions containing NS5 were pooled, mixed with 10 units of SUMOStar protease (LifeSensors) and dialyzed against buffer C  glycerol, 5 mM DTT and 0.005% v/v Igepal CA-630) for overnight at 4 °C. The digested fractions were then loaded onto a 5 ml HisTrap HP column and washed with buffer C. Cleaved NS5 was separated from uncleaved protein and the protease by applying a gradient to buffer B. Fractions containing NS5 were concentrated, and loaded onto gel filtration column Superdex 200 Increase 10/300 GL  and run in the buffer 50 mM TrisHCl pH 8.8, 500 mM NaCl, 10% glycerol, 0.001% Igepal, 10 mM DTT. NS5 fractions were concentrated, frozen in 5 μl aliquots and stored at −80 °C. All steps in the purification process were carried out at 4 °C. NS5 triple mutant SUMOstar expression plasmid was generated using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies). The NS5 triple mutant protein was purified in an identical manner to wild-type NS5.ZIKV RdRp purification. ZIKV RdRp amino acid sequence 274–904 (Brazilian strain SPH2015) was cloned into pE-SUMOstar Amp vector 47. It contains the viral genome flanked by a T7 promoter and hepatitis delta virus ribozyme sequence (HDVr) at the 5′ and 3′ ends, respectively. To generate the ZIKV subgenomic replicons, we replaced the region of the viral genome encoding amino acid 26 of the capsid protein through amino acid 476 of the envelope protein with Renilla luciferase (RLuc) followed by the foot-and-mouth disease virus (FMDV) 2 A protease. The resulting replicon, ZIKV/SG-RLuc, contains the 5′UTR, first 25 amino acids of the capsid protein, Renilla luciferase, FMDV 2 A protease, last 28 amino acids of the envelope protein, viral NS1-NS5 proteins, and the 3′ UTR. We generated the replication-defective replicon by mutating the RdRp active site motif GDD (NS5 residues 663–665) to GNN. Mutations in the ZIKV polymerase and methyltransferase domains were introduced by overlapping PCRs.The infectious clone of ZIKV Cambodian strain (FSS13025) is publishedin vitro transcribed RNA from ZIKV replicons using TransIT-mRNA Transfection kit . The cells were harvested at 4, 24, 48, and 72 hours post-transfection using 1X cell culture lysis reagent , and the Renilla luciferase activity was measured using FLUOstar Omega .Huh-7.5 cells were seeded into 24-well plates at the density of 50,000 cells per well. Twelve hours later, the cells were transfected with 125 ng of Supplementary Figures


Drosophila, pelota is essential for the progression through meiosis during spermatogenesis and germline stem cell maintenance. Here we show that homozygous Hbs1 mutant flies are viable, female-fertile, but male-sterile, which is due to defects in meiosis and spermatid individualization, phenotypes that are also observed in pelota hypomorphic mutants. In contrast, Hbs1 mutants have no obvious defects in germline stem cell maintenance. We show that Hbs1 genetically interacts with pelota during spermatid individualization. Furthermore, Pelota with a point mutation on the putative Hbs1-binding site cannot substitute the wild type protein for normal spermatogenesis. These data suggest that Pelota forms a complex with Hbs1 to regulate multiple processes during spermatogenesis. Our results reveal a specific requirement of Hbs1 in male gametogenesis in Drosophila and indicate an essential role for the RNA surveillance complex Pelota-Hbs1 in spermatogenesis, a function that could be conserved in mammals.Hbs1, which is homologous to the GTPase eRF3, is a small G protein implicated in mRNA quality control. It interacts with a translation-release factor 1-like protein Dom34/Pelota to direct decay of mRNAs with ribosomal stalls. Although both proteins are evolutionarily conserved in eukaryotes, the biological function of Hbs1 in multicellular organisms is yet to be characterized. In  Drosophila is initiated by the asymmetric divisions of germline stem cells (GSCs) that give rise to the respective differentiated gonialblasts, each of which then undergoes four rounds of mitotic divisions to produce 16 spermatocytes. The spermatocytes then go through two rounds of meioses to yield a cyst of 64 spermatids. At the late stage of spermatogenesis, spermatids undergo morphological changes, such as elongation and individualization, where 64 syncytial spermatids are separated into 64 individual mature sperms1. During meiosis and spermiogenesis, gene transcription is globally reduced and the transcripts used for late stages are stored in the cytoplasm until translation. Therefore, gene regulation at the translational level plays an important part to ensure proper sperm development.Spermatogenesis in Pelota (pelo), which encodes an evolutionarily conserved eukaryotic release factor 1-like protein, was first identified in a genetic screen for genes involved in male fertility in Drosophila melanogaster2. Mutation in the pelo locus causes cell cycle arrest at G2/M transition before the first meiotic division during spermatogenesis3. Deletion of dom34, the orthologue of pelo in yeast, results in defective sporulation and decreased polyribosomes, suggesting that the molecular function of Pelo is related to translational regulation4. Dom34 was found to interact with Hbs1, originally uncovered in yeast as a suppressor of the growth defect in strains deficient for Hsp 70 proteins5, and Hbs1 was also shown to promotes efficient cell growth and protein synthesis6. Importantly, both Dom34p and Hbs1 are identified as important factors in a mRNA quality control pathway named no-go decay, in which mRNAs with ribosomal stalls during translational elongation or at the 3′ end of mRNA are subjected to degradation by endonucleolytic cleavage10. The role of Pelo in mRNA quality control is also confirmed in Drosophila and human cells13.15. eRF1 shares a three-dimensional structure similar to tRNA and functions to terminate translation by recognition of the stop codon at A site of ribosome and hydrolysis of the peptidyl-tRNA bond for peptide release16. eRF1 binds to eRF3 through its C-terminal domain and this interaction increases the affinity of eRF3 for GTP17. Similarly, Pelo-Hbs1 interaction is able to augment the affinity constant of Hbs1 to GTP14. Structure of the Dom34-Hbs1 complex confirms the analogy of the overall similarity to that of eRF1-eRF3, but also reveals that the N-terminal region of Pelo may function differently20. The binding of Hbs1 to Dom34 leads to a conformational change with a tRNA-like structure formation by the middle domain of Dom34 and positioning a conserved basic region expected for translational termination. Indeed, Dom34-Hbs1 shows maximum kinetic efficiency on the ribosome complexes that contain truncated mRNAs21 and promotes dissociation of stalled elongation complexes and release of peptidyl-tRNA in an A site codon-independent manner10. Additionally, the Dom34/Hbs1/Rli1 complex mediates the dissociation of the stalled ribosomes into 40 S and 60 S subunits. Taken together, the Pelo-Hbs1 complex regulates gene expression at translational level by facilitating the recycle of ribosomes and removal of aberrant mRNAs when elongation stalls occur.Structural analysis of archaeal and yeast homologues of Pelo suggests that it is related to eukaryotic release factor 1 (eRF1) in the central and the C-terminal domains except some differences in the N-terminal region, but does not contain the stop codon recognition motif or GGQ motif that helps to catalyze hydrolysis of peptide23. In mammals, Pelo-Hbs1 also promotes dissociation of elongation complexes, but only with an ABC-type ATPase ABCE1 in the complex25. Also, mammalian Pelo seems to have additional roles outside of mRNA quality control and the pleiotropic function of Pelo might be also reflected by the observation that pelo null mice die as early as embryonic day 7.526. Conditional knock-out of Pelo at postnatal stages revealed that Pelo is essential for the maintenance of spermatogonial stem cells by regulating PI3K/AKT pathway and the downstream FOXO1 activity27. In Drosophila, mutation in pelo also causes loss of ovarian GSCs, in part as a result of compromised BMP signaling pathway activation28. These observations indicate an evolutionarily conserved function of Pelo in GSCs and gonad development. We have previously shown that Pelo-Hbs1 is required for transposon silencing in the female germline29. However, the biological function of Hbs1 and Pelo-Hbs1 complex beyond female germline remains to be explored in multicellular organisms.The Pelo-Hbs1 complex seems to be conserved throughout evolution. Although Hbs1 is absent in archaea, its function is performed by elongation factor aEF1aDrosophila Hbs1 by generating and analyzing Hbs1 mutants. We find that Hbs1 mutants are male sterile due to defects in meiosis and spermatid individualization during spermatogenesis. We show that Pelo is also required for spermatid individualization in addition to its known role in early meiosis during spermatogenesis, revealing that Pelo functions at multiple stages during male germline development. By interaction analysis, we show that Hbs1 and Pelo genetically and physically interact with each other. We thus demonstrate an essential role for the Pelo-Hbs1 complex in spermatogenesis, likely through translational regulation.Here in this study, we investigated the biological function of Hbs1 (CG1898) in Drosophila melanogaster encodes a polypeptide of 670 amino acids, with a predicated molecular weight of approximately 74 kDa. Hbs1 is a highly conserved protein in eukaryotic kingdoms, with the full length of fly counterpart sharing a similarity of 48%, 37%, 48%, 45% and 45% with the homologs in yeast, C. elegans, Xenopus, chicken and human, respectively  that covers the entire coding region of Hbs1 , which consists of 64 actin cones, is required for this process. At the beginning of individualization, the actin cones assemble around the spermatid nuclei towards the basal end of the testis  pelo mutants like peloPB60 and pelo1 showed a total blockage of cell division, and mutant spermatocytes did not enter meiosis. Weak peloPA13 mutants displayed cell division arrest either before or at the first meiotic division , and about 12.6 ICs per testis in peloPB60 heterozygotes (peloPB60−/+), but IC number dropped significantly to an average of 4.2 when one copy of pelo was removed, and both the IC and nuclei were severely disorganized  mutation that is supposed to disrupt the Pelo-Hbs1 complex function possibly by altering the allostatic configuration of the interaction interface29, and tested its ability in rescuing spermatogenesis defects caused by the loss of Pelo using a germline-specific driver nosGAL4 , cell cycle was arrested at early stages of meiosis and there was no elongated spermatid nuclei bundles and therefore no IC formed in these animals . Similar to its role in ovarian GSCs, we found that pelo was also important for the maintenance of testicular GSCs , which excludes the possibility that their function can compensate for each other. Finally, the putative Hbs1-binding motif-mutant version of Pelo cannot substitute the wild type Pelo for spermatogenesis, further supporting the idea that Pelo and Hbs1 form a protein complex to function during spermatogenesis. Our results, together with the previously reported results that yeast as well as mammalian homologues of Hbs1 and Pelo interact with each other24, collectively support that the physical interaction between Hbs1 and Pelo are evolutionary conserved from yeast to mammals.Our results also suggest that Hbs1 may collaborate with Pelo to regulate multiple processes during spermatogenesis in 40. Also, increased expression of Hbs1 in yeast could suppress the growth defect in strains deficient for SSB1/2, which encode molecular chaperones that are associated with nascent polypeptides and function to facilitate transportation of newly synthesized peptides to the cytosol5. In addition, yeast Hbs1 mutants show defective protein synthesis upon limiting translation initiation6. Consistently, in partnership with Dom34/Pelo, Hbs1 has been demonstrated to be involved in no-go and no-stop mRNA decay42. In this study, expression of a tagged Hbs1 in the male gonad reveals that Hbs1 is predominantly localized in the cytoplasm of germline cells, consistent with a role of Hbs1 in translational regulation. Dom34/Pelo and Hbs1 have been shown to have a role in degrading aberrant 18S rRNAs. Nonfunctional rRNAs and mRNAs to be removed by no-go decay are both localized within P bodies, places where mRNA decay is conducted43. Therefore, Pelo-Hbs1 could remove both nonfunctional rRNAs and aberrant mRNAs for proper spermatogenesis. In mammalian cells, the Pelo-Hbs1complex along with ABCE1 is important for recycling vacant 80S ribosomes12, it is therefore possible that Pelo-Hbs1 are important for ribosome recycling to ensure proper spermatogenesis.Hbs1-Pelo complex likely regulates spermatogenesis through translational regulation. Hbs1 belongs to a protein family known to be involved in several steps of translational processes, including translational elongation, release, and decay of nonfunctional mRNAsPelo mutants28. This is similar to the situation in yeast, in which Dom34/Pelo mutants display stronger and more profound phenotypes, such as defective sporulation and decreased polyribosomes, whereas Hbs1 mutants show no obvious phenotype6. Moreover, Hbs1 seems to play a minor role in no-go decay compared to the critical requirement of Dom34/Pelo7. It is possible that there are other redundant G proteins that can functionally substitute Hbs1 or interact with Pelo at different stages together with Hbs1 during spermatogenesis, and Pelo may be a key player in the complex, while Hbs1 may only have a supportive role.Despite our finding to show that Hbs1 and Pelo function together during late spermatogenesis, our results also indicate Hbs1-independent functions of Pelo in maintaining both male and female GSCs and in early spermatogenesis. Hbs1 is dispensable for GSC maintenance and mutants show relatively weaker phenotype in male meiosis compared to that of Drosophila, while a short period of transcription exists postmeiotically in mammals45. So translational regulation is critical in regulating gene expression during the postmeiotic stage, where germ cells undergo dramatic remodeling while transcription is shut down in spermiogensis48. In those circumstances, many mRNAs are produced and translationally repressed in spermatocytes until the encoded proteins are required for later spermatogenesis, such as don juan and don juan like, both of which are initially transcribed in primary spermatocytes but translationally repressed until spermatid elongation49. Translational activation is also required for the sequential gene expression in later development in spermatogenesis. Boule, Drosophila orthologue of the vertebrate Y-linked Deleted in Azoospermia-like (DAZl) promotes the efficient translation of twine for meiotic entry50. The general translation system, which includes translation initiation, elongation, and termination, also contributes to the expression of different genes at different stages of germline development. eIF4G and eIF4G2, paralogues of the translation initiation complex component eIF4G, are required for meiotic progression and spermatid differentiation in Drosophila by controlling temporal expression of core cell cycle regulators, implicating that alternate translation initiation machinery may be responsible for gene expression and germline development in spermatogenesis53. Our results here suggest another level of translational regulation during spermatogenesis mediated by RNA surveillance factors. The Drosophila Pelo-Hbs1 complex, which has been demonstrated to be required for no-go decay in cultured Drosophila cells and transposon silencing in female germline29, could be involved in degrading poisonous/defective mRNAs such as transposon transcripts, rRNAs and/or facilitating ribosome recycling that would otherwise lead to defects in meiosis and spermatid differentiation. Conditional depletion of Pelo in postnatal mice causes progressive spermatogonial stem cell loss and sterility, although later spermatogenesis appears normal before germline exhaustion27. It remains possible that the perdurance of gene product follow gene knock-out in stem cells may be sufficient to allow later spermatogenesis. Therefore it would be worthwhile to re-evaluate the potential function of Pelo-Hbs1 complex in mammalian gametogenesis by germline depletion of Hbs1.Translational regulation plays crucial roles in biological processes in which transcription is largely absent and developmental events are obligatory to be carried out. It is known that transcription is nearly absent at postmeiotic stage during spermatogenesis in pelo1, peloPB60, peloPA13; Hbs11, Hbs148 and Hbs1172 ; Pelo (P210A)29and nosGal4VP1638. BamGAL4VP16 (a gift from Dahua Chen)54. Vasa-GAL4 (a gift from Zhaohui Wang)55, and pelo RNAi lines were from VDRC 56.All flies were raised at 25 °C and cultured on standard media, unless otherwise stated. The following fly stocks were used in this study include: peloPB60 was caused by a 107 bp deletion in the second exon, and the predicted protein product is a peptide with 77 amino acids (compared to the full length with 395 amino acids). Flies homozygous for peloPB60 showed similar GSC loss phenotype during oogenesis and meiosis defect during spermatogenesis to transheterozyotes (peloPB60 in trans to a deficiency Df(2 L)Exel6024 in which the entire pelo locus is deleted). peloPB60 homozygous testes also displayed similar severity of meiosis defects to that of pelo1 homozygous mutants, suggesting that both peloPB60 and pelo1 alleles can be considered as strong loss of function or genetic null alleles. peloPA13 was resulted from a 557 bp insertion left by the original KG6646 P element, and homozygous mutant cysts from testes squash displayed meiosis arrest either before the first or the second meiotic cell division. Based on the severity of defects in female GSC maintenance and male meiosis, we considered peloPA13as a hypomorphic allele.KG6646 chromosome was brought together with a transposase source by crossing peloKG6646/Cyo flies to CyO, PBac{w[+mC] = Delta2-3.Exel}2/amos[Tft] flies which carried transposase on the second chromosome (Bloomington stock number:8201). Then the flies with peloKG6646/CyO, PBac{w[+mC] = Delta2-3.Exel}2 were crossed to those with CyO/Sco. In the next generation, pelo/CyO flies without CyO, PBac{w[+mC] = Delta2-3.Exel}2 chromosome were selected to establish excised lines. Hbs1 alleles were established in a similar way using Hbs1EY09557.The peloHbs1 together with 1X Flag coding region at the C-terminal end was amplified by PCR using KOD polymerase  and cloned into Spe I and Not I sites of pCasPeR 4. The following primers were used: 5′ GGACTAGTCC TGAAGCAAAT CAGAACAGTC 3′ and 5′ TTGCGGCCGCAA CTACTTGTCATCGTCATCCTTGTAATC GCG GAT CTT GGT GAC CAT T 3′. To generate epitope-tagged transgenes, ORFs of both pelo and Hbs1 were first cloned into pENTRY/D-TOPO vector and then into pPWF and pPWM vectors respectively through the Gateway reaction to make UASP-Pelo-FLAG and UASP-Hbs1-Myc (The Drosophila gateway vector collection developed by Terrence Murphy: http://emb.carnegiescience.edu/labs/murphy/Gateway%20vectors.html). To generate pelo carrying Pro210 to Ala (P210A) mutation, pelo was cloned from Canton-S ovarian cDNA library and ligated into pEasy-blunt Simple vector (TransGen Biotech). Single amino acid substitution was generated by Easy Mutagenesis System (TransGen Biotech) according to the manufacturer’s instruction. Full length pelo coding sequence was amplified by PCR with primers 5′-GGTACCATGAAGCTGCTGGGCAAATA-3′ and 5′-TCTAGACTAGTCGCTATCGCTATCTGCC-3′. The PCR fragment was restriction digested with KpnI and XbaI, and subsequently cloned into pUASP. All the plasmid DNA was injected for transformation via standard procedures.For the genomic rescue experiment, the genomic region of Drosophila S2 cells, ORFs of Pelo and Hbs1 were cloned into pAWG and pAWR vectors using the Gateway kit. Constructs were transfected into Drosophila S2 cells using the standard calcium phosphate transfection protocol, and the expression pattern of Pelo-RFP and Hbs1-GFP was examined 4 days after the initial transfection.To generate Pelo-RFP and Hbs1-GFP constructs for expression in Actin5c was used for normalization. Fold changes for mRNA levels were calculated using ΔΔCt method57. The primers used are listed in Table Total RNA from 50–60 testis was extracted by RNAiso Plus reagent and Direct-zol RNA Miniprep kit with DNase treatment. Complementary DNA(cDNA) was synthesized by 5X All-In-One RT MasterMix kit. RT-qPCR was performed in three duplicates using SYBR Select Master Mix on ABI PRISM 7500 fast Real-time PCR System(Applied Biosystems). Endogenous 58. Each male was put together with two w1118 virgin females in one food vail in twenty duplicates. The flies were raised at 25 °C and separated from each other after 5 days. The offspring were examined within 2 weeks.Fertility assay was performed as previously describedNewly enclosed males were collected and manually dissected for testes in Testis Buffer . Testes were subsequently transferred onto a coverslip containing a 2-ul drop of Testis buffer. Testes were then opened using forceps, squashed and examined by phase contrast using a Zeiss Axio microscope.Fly testes were dissected in PBS medium, fixed in 4% paraformaldehyde for 10 min at room temperature, and then washed with PBT (PBS containing 0.1% Triton X-100). For DAPI  staining, samples were incubated with 1 mg/mL DAPI in PBT for 6 min, and the reaction was stopped by washing samples with PBT. For phalloidin staining, samples were incubated with rodamine-phalloidin  at 37 °C for 2 hours and subsequently washed with PBT twice. For antibody staining, samples were fixed first and washed three times with PBST. After blocking with 5% normal goat serum in PBT, primary and secondary antibodies were then added according to standard procedures. Antibodies used in this study include anti-α-spectrin antibody , 1:50 dilution), anti-DE-Cadherin , anti-Vasa , anti-FLAG , anti-Myc , anti-α-tublin , Alexa568-conjugated goat anti-mouse and Alexa 488-conjugated goat anti-rabbit secondary antibodies . Samples were then mounted in 70% glycerol in 1 × PBS. Images were captured by either a Zeiss Imager Z1 equipped with an ApoTome system or a Zeiss Meta 510 confocal microscope. All images were processed in Adobe Photoshop and Illustrator.ORF of Pelota was cloned into NdeI and EcoRI sites of pGADT7 AD Vector. The primers used were 5′ CCCATATGAAGCTGCTGGGCAAATACGT 3′, and 5′ CCGAATTCTAGTCGCTATCGCTATCT 3′. ORF of Hbs1 was cloned into Nde I and BamH I sites of pGBKT7 DNA-BD vector, and primers were 5′ CCCATATGTCGCGGCACAGGATAGT 3′ and 5′ CCGGATCCTAGCGGATCTTGGTGACCATT 3′. The yeast two hybrid assay was based on the MATCHMAKER kit (Clontech). pGBKT7-p53 with the Gal4 DNA-BD fused with murine p53 and pGADT7-T with the Gal4 AD fused with SV40 large T-antigen were used as positive control.Supplementary info


The model allowed for docking studies aimed at effective pre-screening of potential inhibitors of ZIKV RdRp. Applicability of the structural model for docking studies was illustrated with the NITD008 artificial nucleotide that is known to effectively inhibit the function of the ZIKV RdRp. The ZIKV RdRp – RNA structural model is provided for all possible variations of the nascent RNA bases pairs to enhance its general utility in docking and modelling experiments. The developed model makes the rational design of novel nucleosides and nucleotide analogues feasible and thus provides a solid platform for the development of advanced antiviral therapy.Zika virus is a global health threat due to significantly elevated risk of fetus malformations in infected pregnant women. Currently, neither an effective therapy nor a prophylactic vaccination is available for clinical use, desperately necessitating novel therapeutics and approaches to obtain them. Here, we present a structural model of the Zika virus RNA-dependent RNA polymerase (ZIKV RdRp) in complex with template and nascent RNAs, Mg Flaviviridae, genus Flavivirus. This genus includes several major human pathogens like the dengue virus, Japanese encephalitis virus, West Nile virus, and tick-borne encephalitis virus. Virtually the entire human population lives where at least one flavivirus species is endemic1. The ZIKV was first identified in 1947 in Uganda2. Until its sudden emergence in Brazil in 2015 and explosive spread through South America, Central America, and the Caribbean, the virus had been thought to produce a rare and mild, self-limiting disease in humans, with common symptoms that include fever, rash, joint pain, and conjunctivitis3. However, during the recent outbreak it was discovered that ZIKV infection is linked to adverse pregnancy and birth defects, most notably microcephaly and other serious brain anomalies4. In adults, ZIKV can cause rare but severe neurological complications, including Guillain-Barré syndrome , acute disseminated encephalomyelitis , acute myelitis, meningitis or encephalitis9 with fatal consequences in isolated cases10. During the most recent outbreak in Brazil, an estimated 440 000–1 300 000 cases of ZIKV infection have been reported11.Zika virus (ZIKV) is an emerging mosquito-borne member of the family 12. Viral RNA bears a type-1 cap structure (m7GpppAm) at the 5′-end and has no poly(A) tail at the 3′-end. The ORF encodes a single polyprotein that is subsequently processed into three structural  and seven non-structural  proteins12. Central enzymatic activities related to virus replication are encoded by NS3 and NS5 proteins. NS3 is the second largest viral protein and possesses both the protease and helicase activities, with NS2B serving as a cofactor for the protease13. The NS5 protein is the largest virus-encoded protein, comprised of about 900 amino acids (AAs), and is also the most highly conserved protein among flaviviruses14. Approximately 270 AAs at the N-terminus form the methyltransferase (MTase) domain, and approximately 620 AAs at the C-terminus form the RNA-dependent RNA polymerase (RdRp)15. The MTase is involved in type 1 cap synthesis at the 5′-end of the viral RNA. At least two activities of the MTase were recognized; i.e., guanine N7 methyltransferase and nucleoside 2′-O ribose methyltransferase16. No MTase domain was found in the other genera of the Flaviviridae family, i.e., hepaciviruses and pestiviruses, which contain an internal ribosome entry site at its 5′-end of viral genome instead of the type 1 cap17.Flaviviruses are small, enveloped viruses carrying genomes which consist of non-segmented single-stranded positive-sense RNA (+ RNA). The genome contains one large open reading frame (ORF), which is flanked with untranslated regionsde novo RNA synthesis to first generate −RNA (negative-sense RNA) using the viral +RNA as a template. In the next round, the −RNA serves as a template for the synthesis of more +RNA strands which are used as mRNA for viral polyprotein synthesis or are packaged into arising viral particles12. NS5 is considered to be one of the most promising targets for antiviral drugs because both domains (MTase and RdRp) are essential for virus replication and are not present in non-infected cells22.The polymerase domain of NS5′ catalyses 24 in addition to the previously solved structure by AbbVie Inc15. Additionally, several crystal structures of other flavivirus polymerases were solved in complex with small molecules26, however, neither of these structures contained an RNA molecule nor any nucleotide or nucleotide analog, which hinders their use in structure based design of novel inhibitors. For these reasons we endeavoured to build a sophisticated model of RNA and nucleotide bound ZIKV RdRp based on the published crystal structures of ZIKV RdRp and its most similar RdRp bound to RNA that can be found in the PDB database which is the Hepatitis C virus (HCV) RdRp. Such a functional model of ZIKV RdRp containing all the necessary components is indispensable for successful docking experiments, which are an essential part of the rational design of inhibitors that might serve as drugs against ZIKV and other flaviviruses. Correctness of the ZIKV RdRp:RNA structural model developed in this work is demonstrated biochemically using point mutants predicted from the structural model and the usefulness of our model is documented using docking experiments.Recently, the structural biology of ZIKV RdRp significantly advanced as several crystal structures of the RdRp were analysed15 and HCV (PDB ID 4WTD)27 crystal structures. The protein without substrate RNA was captured in 5TFR whereas the template RNA, nascent RNA and accessing ADP nucleotide within catalytic site were captured in 4WTD. The S-adenosylhomocysteine cofactor, bound to the MTase domain, was removed from the ZIKV structure. The structural alignment of ZIKV and HCV (chain B) performed using the Measures of Structural Similarity algorithm28 implemented in the Chimera29 yielded our starting structural model. The alignment of HCV and ZIKV RdRp involved 174 amino acid pairs and the RMSD between 174 pruned atom pairs was 1.123 Å  were included in the structural model but were replaced by Mg2+ atoms because magnesium is the physiological ligand. The starting structural model thus corresponded to ZIKV 5TFR structure where the RNA molecules plus the x-ray resolved solvent within the catalytic core were adopted from HCV 4WTD structure. The ADP, which is the accessing RNA nucleotide in HCV, was in our structural model modified to ATP by adding a protonated γ-phosphate. The geometry of added γ-phosphate was adopted from the geometry of ATP in the x-ray structure of human norovirus polymerase (PDB ID 3H5Y)30. One of the two x-ray-resolved water molecules was removed because it occupied the position of the added γ-phosphate. The template RNA and nascent RNA strands were shortened to include only the last A3-U8 and consecutive nascent U2…A602 base pairs, where the A602 was accessing ATP  Figures  and S2. 8 that terminated the nascent RNA strand. One model included O3′-protonated ribose while the other included O3′-deprotonated ribose of U8. Geometries of the two structural models were preliminarily QM/MM-optimized and the complexes were solvated with the SPC explicit water33 using the Maestro program. A water layer with a thickness ~10 Å surrounding the ZIKV-RNA complex included 32 354 water molecules. A neutral charge of the solvated complexes was achieved by adding Cl− anions using the Maestro program. The geometries of the protein, RNAs, two Mg2+ ions and the x-ray-resolved water within catalytic core were constrained during MD simulation by imposing the force constant 100.0 kcal mol−1 Å−1. Hence, only dynamics of the added water molecules and counter-ions was MD-calculated freely, without restraints to encompass the ZIKV-RNA complex with the explicit solvent coherently. The MD simulations were carried out by employing the Desmod program34. The OPLS-AA(2005) force field was used in all MD simulation35. First, the system was equilibrated employing the constraints and periodic boundary conditions, T = 300 K and P = 101325 Pa. The equilibration was followed by 120 ps of NVT simulation. The solvation of ZIKV-RNA complex was obtained upon 120 ps NPT MD simulation. The solvated structural model used in QM/MM calculations included only the water molecules within the 5 Å layer surrounding the ZIKV RdRp:RNA complex  force field35. Only the x-ray geometry of the sugar-backbone part of the template RNA was fixed in all QM/MM geometry optimizations, the rest of the geometric parameters was optimized. All the QM/MM-calculated energy minima were validated with the vibration analysis employing the latest Hessian as implemented within the QSite program40. Note, actual accuracy of the calculated structures de facto corresponds to the accuracies of original x-ray structures employed in preparation of initial Zika-RNA model. Somewhat higher precision of geometrical parameters throughout the text, however, corresponds to the accuracy assumed for the QM/MM method.The QM/MM method was used for optimization of geometry of the ZIKV-RNA complex. The QM and MM part of structural model was described employing the QSite 6.1 program. The QM part included nucleotide-triphosphate N Mg Fig. . The numE. coli Rosetta Gami B (DE3) cells and purified as described in detail previously20 using our using our standard protocols42. Briefly, the ZIKV RdRp  was expressed with a His8SUMO purification and solubilisation tag. Bacterial lysate was prepared using French pressure cell press (Thermo). Proteins were purified by combination of Ni-NTA chelation chromatography followed by the cleavage of the purification tag by the Ulp1 protease and finally purified using size exclusion chromatography (SEC). The purified protein was concentrated to 10 μM concentration and stored at −80 °C in SEC buffer  until needed.Wild type ZIKV RdRp and all the six mutants were expressed in 20. Briefly, it was measured as a frequency of radioactivity labeled ATP*  incorporation into prolonging chain. We used 0.2 ng/μl poly-U  as a template, 10 nM 15-mer poly-A primer and 1 μM ZIKV RdRp in buffer . The reaction was started by addition of 2.5 μM ATP and 3000 Ci/mmol ATP*  and was running for 40 minutes at 30 °C. The RNA product was caught on anion exchange paper . Unbound radioactivity was washed out 10x with 125 mM Na2HPO4 (pH 9), twice with water and once with an ethanol flush. The radiation was measured by a Phosphorimager (Typhoon 9410) and the signal was quantified using ImageJ software43. Reactions without RdRp were used as a negative control and its signal was subtracted from all testing signals. The normalized activity was calculated as a ratio of wild type RdRp (100% activity).The activity of ZIKV RdRp was measured as described previously44 using the default methodology (the polar hydrogen atoms were maintained in the model). The charges of Mg2+ ions were calculated for the QM part of QM/MM-optimized protein-RNA complex that included ATP602 and U8 nascent RNA base pair excluding the A3 and U2 nucleobases. The four hydrogen caps bridging the QM part with the MM part were replaced by hydrogen atoms that were geometry optimized with the PBE method and lapcv* basis using the Jaguar 8.2 program45. The RESP46 charges of the magnesium atoms were calculated using the B3LYP48 method and 6–31 G50 basis set with the Gaussian09.D0151 quantum chemistry package and Amber tool antechamber52. The charge +1.1 e− was calculated for the two magnesium atoms by using both alternatives of the structural models including normal and O3′-deprotonated ribose of U8.The QM/MM-optimized structural model including ATP (au_o3.pdb) was used for all the docking experiments. Initially, the ATP as well as water molecules were removed. The pdbqt file was prepared with the AutoDock Tools 1.5.6The QM-calculated charges of the magnesium ions were manually inserted into the pdbqt file. We also extracted several water molecules , which play an essential role in the binding site, from the original pdb file into a separate pdb file, prepared pdbqt file containing these water molecules in AutoDock Tool 1.5.6 and manually inserted them back into the pdbqt file with protein and ions.de novo preparation of the ligand NIT008 and converted the 2D structure to a 3D structure using standard function with the force field based on CHARMM parametrization. The 2′-endo (South) and 3′-endo (North) conformations of this compound were prepared and optimized using B3LYP-D353 method and 6–31 basis set with inclusion of the implicit water environment modelled by Polarizable Continuum Model (PCM)54. The necessary conversion of the format was performed using AutoDock Tools 1.5.6.ATP was extracted from the original model into a separate pdb file and converted to pdbqt by AutoDock Tools 1.5.6. We used ACD/ChemSketch 12.01 for 55 with the default docking method at larger exhaustiveness = 200 and greater binding space  in comparison with the subsequent docking experiments. The docking experiments were also performed with the Autodock Vina 1.1 using the default docking method with exhaustiveness = 100 under similar conditions as in our previous work57. The docking was performed into the smaller binding pocket of 20 × 20 × 20 Å centred at −2.55, −7.53 and 1.67 Å.We have performed several validation experiments, which show the importance of all the components  for successful docking experiments. All these experiments were performed using Autodock Vina 1.128 as implemented in the Chimera58. The alignment of HCV and ZIKV RdRp involved 174 amino acid pairs and the RMSD between 174 pruned atom pairs was 1.123 Å . The protein-RNA complex was protonated assuming pH = 7.0 and solvated by explicit water molecules keeping all geometry parameters derived from the two crystal structures fixed as is summarized in Fig. 8  calculations was derived from the ZIKV RdRp (PDB ID 5TFR) and HCV (PDB ID 4WTD) crystal structures because no flaviviral RdRp was so far crystallized in complex with RNA. The two structures were superimposed using the Measures of Structural Similarity algorithm Figures  and S2. 602-A2 pair it was even 41°  cation and to the Pα atom of the NTP  distance depended critically on whether the terminal nucleotide 8 of nascent RNA was or was not O3′-deprotonated  and O3′-Mn(A) distance was 2.194 Å and 2.280 Å, respectively. The O3′-Mn(A) distance in the HCV RdRp crystal structure was 2.129 Å. For O3′-deprotonated nucleotide 8, the O3′-Mg(A) distance ranged from 1.970 Å to 1.977 Å  that are thus predicted essential  had significantly lower activity compared to the wild type protein (decrease of 65%). Total loss of enzymatic activity was measured with the mutants involving AAs that interact with Mg2+ . The enzymatic activity of RdRp with mutation Trp501Ala was also reduced (40% reduction). Tryptophan is a large aromatic amino acid and although Trp505 does not directly participate in RNA polymerization (based on the model) it might be important for the stability of ZIKV RdRp. As was assumed, the mutation Val572Ala in ZIKV RdRp had negligible effect on the polymerase activity.The activity of all these recombinant RdRp was measured as the ability to incorporate ATP*  into elongated polyA RNA and compared with the wild type ZIKV RdRp Fig. . The ZIK55. Water molecules were omitted for the docking experiments except for the water molecules within the active site.The docking experiments and validation were performed by the open-source software AutoDock Vina 1.1 using the default methodology2+ and waters) resulted in completely incorrect pose of ATP in the binding site  and 3′-endo (North) because the docking algorithm employing AutoDock Vina does not alter the conformation of the ribose ring. The two conformers of NITD008 employed in the docking studies were geometry optimized using the B3LYP-D353 method, 6–31 basis set and by including the effect of implicit water modelled by the Polarizable Continuum Model (PCM)54.Subsequently, we used this validated model for docking of the well-known flavivirus RdRp inhibitor NITD008  conformation Fig.  fits nic2+ ions, accessing NTP, template, and nascent RNAs and the protein. First, we will focus on the local geometric parameters to illustrate the structural basis for rationalizing the docking studies and the mutation experiments.The QM/MM-optimized model of the ZIKV RdRp:RNA complex highlighted many important interactions among all the partners within the catalytic core that involved Mg2+ ions (Tables 2+ ion (A) clearly provides the principal linkage of the nascent RNA with the accessing NTP that is activated only upon O3′-deprotonation of the terminal nucleotide within the nascent RNA. This assumption is coherent with recent benchmark calculations of the P-O bond formation operated by DNA Polymerases β and λ61. Our QM/MM calculations furthermore revealed that the interactions between the NTP phosphate and other RNA molecules remained preserved, only respective bond distances varied upon altered RNA sequence  was critical with respect to RNA activation as it provides principal chemical linkage of the nascent RNA strand with the accessing NTP. Highly variable nucleobase-protein interaction network demonstrated versatility of the amino acid residues within catalytic core that is indispensable for proper deposition and capturing of RNA molecules irrespectively of their sequence. The contacts among RNAs, the two Mg2+ cations and the ZIKV RdRp, which were geometrically characterized in Tables The QM/MM calculations also demonstrated a rather conservative pattern of the RNA phosphate-metal-protein interactions within ZIKV RdRp catalytic core as compared to somewhat more versatile nucleobase-protein interactions that were obviously adjusted according to the particular RNA sequence. Hence, the homologous parts of the RNA molecules involving particularly phosphate and ribose units are deposited within the ZIKV RdRp core in a uniform way regardless of RNA sequence namely due to the effect of the two catalytically important Mg64. Motifs A – E are included in the palm domain and motifs F – G in the fingers domains  are responsible for interaction with Mg2+ ions and mediate the two metal ion catalysis65 that enhances substrate recognition and catalytic specificity66. Mutation of either Asp535 or Asp665 results in a total loss of ZIKV RdRp polymerization activity  is highly evolutionary conserved in tick-borne encephalitis virus (TBEV), Dengue virus (DENV) and ZIKV RdRps. Our model predicts that residue Lys403 interacts with ribose 3′-hydroxyl group of the template RNA  in the Ala mutant. We therefore suggest that the large aromatic Trp559 residue is required for proper folding and/or stability of ZIKV RdRp. Alternatively, Trp559 could contribute to membrane binding as often aromatic residues do70 and viral RdRps are known to be membrane associated72, however, such an aromatic residue needs to be surface exposed and Trp559 is rather buried within close proximity of the active site.Motif B is located in the palm domain and contains Ser - Gly residues responsible for RNA trans-location. However, polymerases with mutated Ser residues retained their ability to incorporate multiple nucleotides73, can be effectively docked into the binding site despite the bulky alkyne substituent at 2′-position.The docking experiments, which we performed using our model with ATP as the accessing NTP, clearly showed that using simple docking software such as AutoDock Vina can lead to satisfactory results. Our experiments with the redocking of ATP resulted in a very nice overlay of the original ATP pose optimized by QM/MM calculations Fig. . The val74. The most favorable results were obtained when the formal charges of magnesium atoms were set to +1.1 e−. . Secondly, the MD calculated and QM/MM geometry optimized solvation of the active site of the RdRp seems to be crucial for formation of appropriate interaction(s) of the triphosphate with the magnesium ions and subsequent position of the nucleobase. However, omission of the water molecules adjacent to the nucleobase might be necessary for appropriate docking of the base modified nucleoside/tide derivatives. Finally, the experiment performed with NITD008 clearly shows that conformation of the ribose ring plays a crucial role in successful docking and it can significantly affect the results. Our data showed that NITD008 in 3′-endo (North) conformation can accommodate the binding site of ZIKV RdRp in an efficient way with minimum distortion of both the nucleobase and the sugar moieties in comparison to ATP in the original model (Fig. Nonetheless, several parameters appear to be essential for successful docking. Firstly, the appropriate charge of the magnesium ions seems to be critical for the optimum positioning of the docked ligand, which is in agreement with the docking studies performed by Chen and co-workersdel Fig. . NITD0082+ ions was employed in rationalization of measured catalytic function of the six mutants as compared to the activity of the wild type enzyme. The structural model highlighted the importance of particular amino acid residues in direct contact either with RNA molecules and/or magnesium ions. By contrast, mutation of the adjacent residues, which are not involved in the direct contact with these essential components of the reaction, exerted significantly lower effect on polymerase activity. The ZIKV RdRp:RNA models provide solid basis for molecular modelling of RdRp behaviour in relation with its biochemical function. Therefore, it can be utilized by medicinal chemists for the design of novel nucleoside and nucleotide derivatives as direct-acting antiviral agents against the ZIKV and other flaviviral pathogens.In conclusion, the comprehensive structural model of ZIKV RdRp:RNA including the protein, template and nascent RNAs, approaching nucleoside triphosphate and magnesium ions was derived from available crystal structures. The QM/MM-optimized ZIKV RdRp:RNA models including all possible nascent RNA base pairs are designated for future docking studies aimed at the design of novel nucleotide analogues against the ZIKV RdRp. These models will be also very useful for the rationalization of the biochemical experiments focused on the catalytic function of ZIKV RdRp because no structural model that includes both the protein and the RNAs is available at the moment. Exploitation of the ZIKV RdRp:RNA model was successfully demonstrated with the docking of NITD008, a nucleoside triphosphate analog. The protein-mutation study assuming the picture of catalytic core was rationalized by using our RdRp:RNA model. In particular, the classification of contacts among amino acids within the core, RNA and MgSupplementary Information


FolE enzymes catalyze the first enzyme in the de novo folate biosynthesis pathway. Herein, we demonstrate that folE motif RNAs selectively bind the enzyme cofactor tetrahydrofolate (THF) and several of its close derivatives. These aptamers, commonly found in Gram-negative bacteria, are distinct from aptamers of the previous validated THF riboswitch class found in Gram-positive bacteria. Our findings indicate that folE motif RNAs are aptamer domains for a second THF riboswitch class, named THF-II. The biochemical validation of THF-II riboswitches further highlights the ability of bacteria to utilize diverse RNA structures to sense universal enzyme cofactors that are predicted to be of ancient origin.We previously reported a large collection of structured noncoding RNAs (ncRNAs) that includes many riboswitch candidates identified through comparative sequence analysis of bacterial intergenic regions. One of these candidates, initially named the “ S-adenosylmethionine (SAM)  , and forne (SAM) . Given tfolE motif” RNAs, and were reported , called THF-II. Members of this riboswitch class were initially named “reported  among a folE motif RNAs form a somewhat more complex secondary structure than was originally proposed. In addition, we use biochemical assays to demonstrate that folE motif RNA constructs, designed based on the revised consensus model, function as selective aptamers for THF and certain other folate derivatives, including dihydrofolate. These and other characteristics indicate that folE motif RNAs are components of riboswitches for THF and some of its related natural derivatives. Compared to the structural and functional characteristics of the previously reported THF riboswitch class  of a gene (folE) encoding GTP cyclohydrolase I. This enzyme catalyzes the first committed step in the de novo biosynthesis pathway for the enzyme cofactor folate or related compounds such as biopterin . This effort yielded an additional 35 representatives, to reach a total of 86 unique RNA sequences , all present from species in the order Rhizobiales or in metagenomics DNA sequences. A revised and expanded consensus sequence and a secondary structure model were then generated based on this entire collection , TFold (cd00651), and GTP_cyclohydrol (cd00642) by the DNA database RefSeq version 63. On reinspection, these genes are now all annotated as folE in RefSeq version 80, which codes for GTP cyclohydrolase I. This observation strongly suggests that the folE motif is involved in sensing a molecule that incorporates a pterin-like moiety  for THF is ∼19 µM. Similarly, we examined the ability of another folE motif representative to bind THF. In-line probing assays using a 62-nt RNA from Ochrobactrum intermedium also yield data consistent with a 1:1 stoichiometry and an apparent KD of ∼4 µM .To determine the binding affinity of THF-II aptamers, band intensities at sites 1–4 B were qufolE motif consensus model was assessed by examining the effects of single mutations , dihydrofolate (DHF) and 7,8-dihydroneopterin (DHN) are bound by the aptamer with near equal KD values . In contrast, no structural modulation was observed when the RNA was incubated with folic acid, 6-biopterin, 5-methyl THF or folinic acid, even at concentrations as high as 300 µM . This substantial ligand-dependent increase in RNA strand scission, coupled with the fact that the U nucleotide at this position is highly conserved, suggests that this position is involved in forming a tertiary structure that holds the phosphodiester linkage in an in-line geometry , 80 mM HEPES-KOH (pH 7.5 at 23°C), 24 mM MgCl2, 2 mM spermidine, 40 mM DTT, and 2 mM of each of the four NTPs. The desired RNA product was isolated via denaturing (8 M urea) 10% polyacrylamide gel electrophoresis . The gel band containing the desired RNA product was excised after UV shadowing using a hand-held UV lamp and a fluorescent TLC plate. RNA was extracted by crush-soaking for 30 min at 23°C in a 450 µL solution containing 200 mM NaCl, 10 mM Tris-HCl (pH 7.5 at 23°C), and 1 mM EDTA. RNA was precipitated from the resulting supernatant by the addition of 1 mL of ice-cold 100% ethanol, then pelleting via centrifugation at 13,000 rpm for 20 min. The resulting RNA pellet was dried under vacuum centrifugation, then resuspended in 20 µL deionized water (dH2O).Synthetic DNA oligonucleotides of the desired template sequences that contain a T7 RNA polymerase (T7 RNAP) promoter on the 5′ terminus were transcribed in vitro for 4 h at 37°C in a 100 µL reaction containing laboratory-prepared T7 RNAP  according to the manufacturer's protocol. Half of this reaction was then 32P-radiolabeled at the 5′ terminus using T4 polynucleotide kinase in a 20 µL reaction mixture containing 25 mM CHES (pH 9.0 at 23°C), 5 mM MgCl2, 3 mM DTT and 20 µCi[γ-32P]ATP via incubation for 1 h at 37°C. The resulting radiolabeled RNA was purified by denaturing 10% PAGE and the desired RNA was extracted as described above.To generate 5′ 32P-labeled RNA was incubated at 25°C in a 10 µL reaction volume for ∼40 h in the presence of 100 mM KCl, 50 mM Tris-HCl (pH 8.3 at 23°C), 20 mM MgCl2 and the presence or absence of the desired ligand. The RNA spontaneous cleavage products were resolved by denaturing 10% PAGE, and the resulting gel was dried and imaged with a PhosphorImager . Fraction bound values were established by varying the concentration of ligands in individual in-line probing reactions, quantifying regions that exhibited structural modulation using ImageQuant 5.1 , then normalized to a nonmodulating band, and finally scaled between 0 and 1. Apparent dissociation constant (KD) values were estimated by plotting fraction bound to the logarithm of the molar concentration of the ligand then overlaying a theoretical 1:1 binding curve (Hill coefficient of 1) on the data points using GraphPad Prism 7.In-line probing experiments were conducted as previously described . BrieflySupplemental material is available for this article.


Furthermore, catalytic antisense RNAs inhibited viral production up to 90% measured as p24 antigen levels in ex vivo assays. The use of chimeric RNA molecules targeting different domains represents an attractive antiviral strategy to be explored for the prevention of side effects from current drugs and of the rapid emergence of escape variants of HIV-1.The 5’-untranslated region (5’UTR) of the HIV-1 RNA is an attractive target for engineered ribozymes due to its high sequence and structural conservation. This region encodes several conserved structural RNA domains essential in key processes of the viral replication and infection cycles. This paper reports the inhibitory effects of catalytic antisense RNAs composed of two inhibitory RNA domains: an engineered ribozyme targeting the 5’ UTR and a decoy or antisense domain of the dimerization initiation site (DIS). These chimeric molecules are able to cleave the HIV-1 5’UTR efficiently and prevent viral genome dimerization  The quasi-species structure of the viral population and its high mutation rate enable the virus to rapidly evade the immune response and promote the appearance of variants resistant to therapeutic drugs. Current therapies are based on a combination of different viral protein inhibitors that achieve a high reduction of the viral load, allowing a partial restoration of the immune function. Patients must maintain an aggressive lifelong treatment which produces serious side effects, so there is a real need to pursue the development of new therapies. Besides carrying the genetic information the HIV genomic RNA contains several conserved structural domains which play essential roles in viral replication and infection cycles. These functional RNA domains exhibit the highest sequence and structural conservations of the viral genome, making them very attractive therapeutic targets. The direct targeting of the viral RNA genome to interfere with the function of genomic RNA domains is a strategy to be explored, with nucleic acids being strong candidates as specific inhibitors for the development of this potential therapeutic technology. The human immunodeficiency virus HIV-1) is the etiological agent of AIDS [ is the eIn silico studies based on biochemical data support the notion that the LDI is the most abundant conformer [ex vivo structure probing assays indicate the prevalence of the BMH conformer, i.e. the dimerizing-competent form [The HIV is a lentivirus which contains two long directly repeated regions at both ends of the genome. The 5’-untranslated region (5’UTR) of primary RNA transcripts comprises several functional RNA domains involved in key processes of the viral cycle like the trans-activation of transcription, polyadenylation, reverse transcription, dimerization, splicing and packaging . The HIVonformer , while eent form . Both coent form . The minimal catalytic domains of the natural hammerhead and hairpin ribozymes have been engineered to generate trans-cleaving catalytic RNAs A 8,9,10,109,10, In vitro analysis demonstrated that both sense and antisense-DIS domains interfere with viral dimerization. The antiviral activity was evaluated by transfecting cells with either pre-synthesized RNA molecules or plasmid DNAs encoding the chimeric inhibitor RNAs. We report here the characterization of a series of chimeric molecules composed of a trans-cleaving ribozyme, either a hairpin or a hammerhead, targeting two different sites within the HIV-5’UTR and an anti-DIS specific molecule, either a DIS sense molecule that may act as a decoy element and an antisense DIS molecule B. In vitHairpin (HP) and hammerhead (HH) ribozymes were designed to target sequences 107-120 and 153-166 of the NL4.3 HIV-1 strain, which we had previously shown as being sequences accessible to both kinds of ribozyme . EngineeThe DIS region is a 34 nt-long RNA domain folded into a stem-loop structure that expands from nucleotide position 244 to 278 in the NL4.3 HIV-1 strain. A molecule containing the 23 most apical nucleotides of the DIS stem-loop, positions 249 to 271, was used as the decoy domain (DIS). A molecule having the complementary sequence of the same 23 nt was used as the antisense domain αDIS; B. The deIn vitro cleavage activity of each chimera RNA was assayed and compared to that of the corresponding trans-cleaving ribozyme lacking any additional domain  to the same HP and HH ribozymes used in this work, resulted in an improvement of the cleavage activity . The TARin vitro in a high salt concentration and can be resolved by native gel electrophoresis [The HIV-1 virion particles contain two genome RNA copies. The process by which two genome copies get non-covalently bond is called dimerization and it is initiated at the DIS domain located at the 5’-end of the genomic RNA . The dimphoresis .2O, using a 20-fold molar excess of non-radiolabelled inhibitory RNA. The dimerization reaction was started by the addition of a reaction buffer. The dimer fraction was detected as the molecular species showing a slower mobility in a native polyacrylamide gel electrophoresis. The RNA chimeras based on HH159 and HP159 clearly interfered with dimer formation whereas ribozymes HH159 and HP159 did not have any effect. These chimeras led to an almost complete suppression of the dimer in favour of the appearance of a new species which migrates between the dimer and the monomer. This new species might be a heterodimer formed between the inhibitory RNA and the 5’UTR, although we cannot rule out other possibilities  were mixed together in Hbilities .Similarly, the HH113 and HP113 chimeras also suppressed dimer formation almost completely and yielded a new species, supposedly a heterodimer, which was detected between the dimer and the monomer. Surprisingly, ribozymes HP113 and HH113 induced the appearance of a new molecular species that migrates between the heterodimer and the 5’UTR-5’UTR homodimer and a second species that migrated faster in the gel. Interestingly, catalytic RNAs targeting position 113 were the only inhibitors able to cleave the substrate under dimerization conditions (data not shown). It is feasible that the new species detected might correspond to a partially or completely cleaved dimer, which would explain the disappearance of the dimer species in the presence of HH113 . LikewisA HEK293T cell system was used for assaying the effect of inhibitor RNAs on HIV viral production. The HEK293T cells do not express the surface receptors required for HIV-1 infection; the virus can replicate in these cells but viral particles cannot infect them. Transfection with pro-viral DNA mimics the post-integration stage of the viral cycle and viral particles in the supernatant after transfection correspond to first generation virions.Inhibitor RNAs were cloned in a pcDNA3-derived plasmid, pU6, in which both CMV promoter and BGH polyadenylation signals were substituted by those belonging to human U6 snRNA . The pU6in vitro synthesized inhibitory RNAs were transfected. These RNAs were transcribed in vitro by T7 RNA polymerase from PCR templates obtained by amplification from the pU6 constructs. The templates were designed to obtain RNAs containing both U6 snRNA flanking hairpins in order to be identical to those produced by the U6-driven expression cassettes  is a gene induced by double-stranded RNA and virus infection ,26. BasaAmplification of the ISG56 gene was not observed in any RT-PCR from total RNA extracted from cells co-transfected with pNL4.3 and any of the following pU6 construct derivatives: empty vector, HP113DIS, HH113αDIS, HP113αDIS or HP159αDIS . Only inin vitro. The TAR is present within both LDI and BMH conformers, while the DIS structure is only present in a very limited percentage of the 5’UTR molecules. The monomeric conformers LDI and BMH could not be resolved under the dimerization conditions tested A. Only aex vivo probing assays [To further characterize the anti-HIV dimerization effect of the inhibitory RNAs, a DIS decoy RNA was used as a competitor. The DIS decoy might stabilize BMH within a heterodimer and could be a tool for the identification of different isoforms and for understanding their equilibrium relationships. Dimerization reactions were performed in the presence of a 60-fold molar excess of a cold 23 nt-long DIS decoy, which was added to the mix just before the reaction began. The DIS decoy completely abolished dimer formation, while a new RNA complex appeared which migrated slightly slower than the BMH monomers B. This iin vitro transcription from appropriate DNA templates using bacteriophage T7 RNA polymerase. The DNA templates were generated from pG3HH and pG3HP anti-LTR HIV-1 plasmid series as described by Puerta-Fernández and co-workers [BglII and BamHI restriction enzymes to eliminate the antisense TAR domain coding sequence and ligated with the DIS inserts, yielding the new pG3HH and pG3HP series encoding anti-DIS sequences. In vitro transcription was performed as previously described [BglII or BamHI linearized pG3HH and pG3HP plasmid templates to synthesize ribozymes or inhibitor chimera RNAs, respectively.Inhibitor RNAs were obtained by -workers  by replaescribed  from BglEx vivo expressing plasmid constructs were generated from the pU6-Rzs plasmid series as described previously [KpnI and ApaI and cloned in the KpnI and ApaI unique restriction sites within the U6 promoter cassette of the pU6-Rzs plasmid series. eviously . Inhibitex vivo HIV-1 inhibition assays were generated by in vitro transcription with T7 RNA polymerase from PCR amplicons. Templates were generated from the pU6 constructs using 5’T7U6 (5’TAATACGACTCACTATAGGGGTGCTCGCTTCGGCAGCACAT3’) and 3’ApaIU6 (5’AGCGGGCCCAAAAAGCGGACCGAAGTCCGC3’) oligonucleotides which contained the T7 RNA polymerase promoter at the 5’ end. Transcription from these templates yielded chimera RNAs containing U6 snRNA flanking hairpins at both ends, as they would be generated by ex vivo transcription. Pre-synthesized RNAs for in vitro by T7 RNA polymerase from a PCR template obtained from the pNL43 plasmid (GeneBank no. AF324493) with primers 5’T7NL43 (5’TAATACGACTCA CTATAGGGTCTCTCTGGTTAC3’) and 3’T7NL43 (5’AATTTTTGGCGTA CTCACCAGT3’). The amplicon incorporated the T7 RNA polymerase promoter at the 5’-end of HIV-1 genomic region from the +1 to +308 nucleotide coding sequence.The 5’UTR RNA was transcribed 2O and the reaction was initiated by the addition of 2 µL of 5X cleavage buffer  and incubated at 37 ºC. Aliquots were taken at different times  and the reaction stopped by adding an equal volume of 2X denaturing loading buffer . The results were resolved in 8% denaturing polyacrylamide gels in 1X TBE using 0.5X TBE as the running buffer. The cleavage reactions were performed in a 10 µL final volume in the presence of 0.2 nmol of internally radiolabelled 5’UTR RNA and 20 nmol of non-radiolabelled catalytic inhibitor RNAs. The RNA mix was denatured (10 min 65 ºC) and renatured (10 min 37 ºC) in H2O. The RNA mix was denatured (10 min 65 ºC) and renatured (10 min 37 ºC) and the reactions were initiated by the addition of 2 µL of 5X dimerization buffer . The reactions were performed for 20 min at 37 ºC and then stopped by the addition of a same volume of 2X native gel loading buffer . The reaction products were resolved by 4% native polyacrylamide gel electrophoresis in 1X TBE using 1X TBE as the running buffer. Electrophoresis was performed at 4 ºC and no more than 15 mA per gel. Monomerization control was performed by the addition of 5X monomerization buffer . For detection of the 5’UTR conformers BMH and LDI, the dimerization mix contained 0.1 µmol of 5’UTR and 6 µmol of DIS decoy, and the reactions were resolved in 4% native polyacrylamide gels in 0.25X TBE using 0.25X TBE as the running buffer.Dimerization competition reactions were performed in a final volume of 10 µL. Internally radiolabelled 5’UTR RNA (157 nmol) was mixed with non-radiolabelled inhibitory RNA (3.14 pmol) in 8 µL HL-glutamine 2 mM the day before transfection. Then, 1 µL of Lipofectamine 2000 reagent  was mixed with 25 µL of Opti-MEM®  and incubated for 5 min at room temperature. Following this, 100 ng of pNL4.3 were mixed with either 300 ng of a pU6 DNA plasmid construct coding for a specific RNA inhibitor or 500 ng of a pre-synthesized inhibitory RNA in 25 µL of Opti-MEM®, and incubated for 5 min. Lipofectamine and nucleic acid solutions were mixed and incubated at room temperature for 20 min. Then, 450 µL of DMEM, 10% FBS and 2 mM L-glutamine were added. The supernatant was carefully removed from each well by aspiration and 500 µL transfecting-mix was added. Two days after transfection, the supernatants were removed and processed using the Genscreen HIV-1 Ag assay  for viral protein p24 antigen quantification, following the manufacturer’s instructions. The DNA for transfection was prepared using the Plasmid Mini Kit , following manufacturer’s instructions. Each treatment was assayed in triplicate and statistical analysis was performed by Kruskal-Wallis non-parametric test followed by multiple Dunnet comparison tests to check differences against the control.For these assays 250,000 HEK293T cells per well were plated in a 24-well plate using 500 µL/well of DMEM, 10% fetal bovine serum (FBS) and ® reagent (Invitrogen), following manufacturer’s recommendations but adding an extra incubation of the aqueous phase after chloroform extraction with 1 U of RQ1DNase  at 37 ºC for 15 min to prevent genomic DNA contamination. This was followed by an additional chloroform extraction. Reverse transcription was performed using the Reverse-Transcriptor kit (Roche), where 100 ng of total RNA was incubated at 37 ºC for 20 min with 0.5 U of RQ1 DNase (Promega) and 20 U of the RNase inhibitor (Promega). The RNA was incubated at 65 ºC for 10 min, then the temperature was brought down quickly to 4 ºC and 3.2 µg of random hexamer primers were added. The reaction mixes contained 1 mM dNTPs and 10 U of reverse transcriptase (RT) in reaction buffer in a final volume of 20 µL. Annealing was performed at 25 ºC for 10 min and the reactions were run at 60 ºC for one hour. Finally, the RT was inactivated at 85 ºC for 5 min. Semi-quantitative PCR was performed by the Reddy Mix PCR Master Mix (Thermo Scientific). The reactions were performed in a total volume of 10 µL containing 2.5 µL of cDNA, 3 pmol of each GAPDH-specific primer and 10 pmol of ISG56-specific primers. Total RNA was extracted from the cells by the TrizolNowadays, there are multiple RNA-based approaches with the therapeutic potential to interfere with viral functions. Functional RNA moieties have been involved in many regulatory processes. Understanding these functionalities, like RNA catalysis or HIV genomic RNA dimerization, may enable the design of therapeutic RNAs. Here we have proven that the combination of functional inhibitor RNA moieties directed against different HIV-1 5’UTR RNA targets in a single chimeric molecule results in bi-functional RNA molecules that fully conserve both inhibitory activities. Both hammerhead and hairpin ribozyme domains were explored as 5’UTR RNA trans-cleaving elements targeting two available cleavage sites surrounding the +113 and +159 positions. The DIS domain within the 5’UTR RNA, responsible for triggering the HIV-1 RNA dimerization, was used as target for a decoy and an antisense RNA.ex vivo HIV-1 inhibition assays demonstrated up to 90% effect measured as viral protein p24 levels in the supernatant of cells transfected with the HIV pro-viral DNA in a post-integrative model system. Inhibitory chimera RNAs achieved a complete in vitro inhibition of the essential viral dimerization process. A model of the implication of both 5’UTR folding isoforms LDI and BMH in the dimerization process is proposed. The results summarized here indicate that in the absence of ionic strength, the 5’UTR folds in vitro into the BMH monomeric conformer, which is competent for dimerization. This conformer quickly switches to the LDI form after the addition of ions. Under these conditions, a low proportion of folded BMH molecules can dimerize into a stable complex, ensuring the existence of enough HIV genome dimers for packaging and the generation of infective viral particles.The