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Strategies to control HIV-1 replication without antiviral therapy are needed to achieve a functional cure. To exploit the innate antiviral function of restriction factor cytidine deaminase APOBEC3G (A3G), we developed self-activating lentiviral vectors that efficiently deliver HIV-1 Vif-resistant mutant A3G-D128K to target cells. To circumvent APOBEC3 expression in virus-producing cells, which diminishes virus infectivity, a vector containing two overlapping fragments of A3G-D128K was designed that maintained the gene in an inactive form in the virus-producer cells. However, during transduction of target cells, retroviral recombination between the direct repeats reconstituted an active A3G-D128K in 89%–98% of transduced cells. Lentiviral vectors that expressed A3G-D128K transduced CD34+ hematopoietic stem and progenitor cells with a high efficiency (>30%). A3G-D128K expression in T cell lines CEM, CEMSS, and PM1 potently inhibited spreading infection of several HIV-1 subtypes by C-to-U deamination leading to lethal G-to-A hypermutation and inhibition of reverse transcription. SIVmac239 and HIV-2 were not inhibited, since their Vifs degraded A3G-D128K. A3G-D128K expression in CEM cells potently suppressed HIV-1 replication for >3.5 months without detectable resistant virus, suggesting a high genetic barrier for the emergence of A3G-D128K resistance. Because of this, A3G-D128K expression in HIV-1 target cells is a potential anti-HIV gene therapy approach that could be combined with other therapies for the treatment and functional cure of HIV-1 infection., Graphical Abstract

To generate infectious virus, HIV-1 must package two copies of its full-length RNA into particles. HIV-1 transcription initiates from multiple, neighboring sites, generating RNA species that only differ by a few nucleotides at the 5' end, including those with one (1G) or three (3G) 5' guanosines. Strikingly, 1G RNA is preferentially packaged into virions over 3G RNA. We investigated how HIV-1 distinguishes between these nearly identical RNAs using in-gel chemical probing combined with recently developed computational tools for determining RNA conformational ensembles, as well as cell-based assays to quantify the efficiency of RNA packaging into viral particles. We found that 1G and 3G RNAs fold into distinct structural ensembles. The 1G RNA, but not the 3G RNA, primarily adopts conformations with an intact polyA stem, exposed dimerization initiation site, and multiple, unpaired guanosines known to mediate Gag binding. Furthermore, we identified mutants that exhibited altered genome selectivity and packaged 3G RNA efficiently. In these mutants, both 1G and 3G RNAs fold into similar conformational ensembles, such that they can no longer be distinguished. Our findings demonstrate that polyA stem stability guides RNA-packaging selectivity. These studies also uncover the mechanism by which HIV-1 selects its genome for packaging: 1G RNA is preferentially packaged because it exposes structural elements that promote RNA dimerization and Gag binding.

The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.

HIV-2, a human pathogen that causes acquired immunodeficiency syndrome, is distinct from the more prevalent HIV-1 in several features including its evolutionary history and certain aspects of viral replication. Like other retroviruses, HIV-2 packages two copies of full-length viral RNA during virus assembly and efficient genome encapsidation is mediated by the viral protein Gag. We sought to define cis-acting elements in the HIV-2 genome that are important for the encapsidation of full-length RNA into viral particles. Based on previous studies of murine leukemia virus and HIV-1, we hypothesized that unpaired guanosines in the 5' untranslated region (UTR) play an important role in Gag:RNA interactions leading to genome packaging. To test our hypothesis, we targeted 18 guanosines located in 9 sites within the HIV-2 5' UTR and performed substitution analyses. We found that mutating as few as three guanosines significantly reduce RNA packaging efficiency. However, not all guanosines examined have the same effect; instead, a hierarchical order exists wherein a primary site, a secondary site, and three tertiary sites are identified. Additionally, there are functional overlaps in these sites and mutations of more than one site can act synergistically to cause genome packaging defects. These studies demonstrate the importance of specific guanosines in HIV-2 5'UTR in mediating genome packaging. Our results also demonstrate an interchangeable and hierarchical nature of guanosine-containing sites, which was not previously established, thereby revealing key insights into the replication mechanisms of HIV-2.

The viral protein Gag selects full-length HIV-1 RNA from a large pool of mRNAs as virion genome during virus assembly. Currently, the precise mechanism that mediates the genome selection is not understood. Previous studies have identified several sites in the 5′ untranslated region (5′ UTR) of HIV-1 RNA that are bound by nucleocapsid (NC) protein, which is derived from Gag during virus maturation. However, whether these NC binding sites direct HIV-1 RNA genome packaging has not been fully investigated. In this report, we examined the roles of single-stranded exposed guanosines at NC binding sites in RNA genome packaging using stable cell lines expressing competing wild-type and mutant HIV-1 RNAs. Mutant RNA packaging efficiencies were determined by comparing their prevalences in cytoplasmic RNA and in virion RNA. We observed that multiple NC binding sites affected RNA packaging; of the sites tested, those located within stem-loop 1 of the 5′ UTR had the most significant effects. These sites were previously reported as the primary NC binding sites by using a chemical probe reverse-footprinting assay and as the major Gag binding sites by using an in vitro assay. Of the mutants tested in this report, substituting 3 to 4 guanosines resulted in

The HIV-1 RNA genome contains complex structures with many structural elements playing regulatory roles during viral replication. A recent study has identified multiple RNA structures with unknown functions that are conserved among HIV-1 and two simian immunodeficiency viruses. To explore the roles of these conserved RNA structures, we introduced synonymous mutations into the HIV-1 genome to disrupt each structure. These mutants exhibited similar particle production, viral infectivity, and replication kinetics relative to the parent NL4-3 virus. However, when replicating in direct competition with the wild-type NL4-3 virus, mutations of RNA structures at inter-protein domain junctions can cause fitness defects. These findings reveal the ability of HIV-1 to tolerate changes in its sequences, even in apparently highly conserved structures, which permits high genetic diversity in HIV-1 population. Our results also suggest that some conserved RNA structures may function to fine-tune viral replication.

Genome packaging is an essential step to generate infectious HIV-1 virions and is mediated by interactions between the viral protein Gag and cis-acting elements in the full-length RNA. The sequence necessary and sufficient to allow RNA genome packaging into an HIV-1 particle has not been defined. Here, we used two distinct reporter systems to determine the HIV-1 sequence required for heterologous, non-viral RNAs to be packaged into viral particles. Although the 5’ untranslated region (UTR) of the HIV-1 RNA is known to be important for RNA packaging, we found that its ability to mediate packaging relies heavily on the context of the downstream sequences. Insertion of the 5’ UTR and the first 32-nt of gag into two different reporter RNAs is not sufficient to mediate the packaging of these RNA into HIV-1 particles. However, adding the 5’ half of the gag gene to the 5’ UTR strongly facilitates the packaging of two reporter RNAs; such RNAs can be packaged at >50% of the efficiencies of an HIV-1 near full-length vector. To further examine the role of the gag sequence in RNA packaging, we replaced the 5’ gag sequence in the HIV-1 genome with two codon-optimized gag sequences and found that such substitutions only resulted in a moderate decrease of RNA packaging efficiencies. Taken together, these results indicated that both HIV-1 5’ UTR and the 5’ gag sequence are required for efficient packaging of non-viral RNA into HIV-1 particles, although the gag sequence likely plays an indirect role in genome packaging.

To increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we observed that only a portion, ∼13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions. IMPORTANCE Nascent HIV-1 particles assemble on plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we labeled and monitored the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stayed near the plasma membrane for only a few minutes in the absence of Gag, whereas most HIV-1 RNAs stayed at the plasma membrane for 15 to 60 min in the presence of Gag. Our results also demonstrated that only a small proportion of the HIV-1 RNAs, approximately 1/10 to 1/3 of the RNAs that reached the plasma membrane, was incorporated into viral protein complexes. These studies determined the dynamics of HIV-1 RNA on the plasma membrane and obtained temporal information on RNA-Gag interactions that lead to RNA encapsidation.

Cell-to-cell transmission is an efficient mechanism to disseminate human immunodeficiency virus type 1 (HIV-1) and human T cell lymphotropic virus type 1 (HTLV-1). However, it has been challenging to quantify the level of cell-to-cell transmission because the virus-producing cells cannot be easily distinguished from infected target cells. We have previously described replication-dependent vectors that can quantify infection events in cocultured cells. These vectors contain an antisense-oriented promoter and reporter gene interrupted by a sense-oriented intron from the human gamma-globin gene. This strategy prevents expression of the reporter gene in the transfected cells but permits its expression in target cells after infection. However, the gamma-globin intron is not efficiently removed by splicing in the aforementioned vectors, thereby reducing the level of reporter gene expression after transduction into target cells. Here, we used two approaches to improve the replication-dependent vectors. First, we improved the splicing events that remove the gamma-globin intron by optimizing the intron insertion site within the reporter gene. Second, we improved the packaging of the spliced RNA without the gamma-globin intron by targeting the intron-containing RNA via microRNA 30 (miR30)-based short hairpin RNAs. Using two optimized fluorescent reporter vectors and flow cytometry, we determined that multiply HIV-1-infected cells were generated at a higher frequency in coculture than in cell-free infection; furthermore, this increase was dependent upon viruses bearing HIV-1 Env. Compared with previously described vectors, these improved vectors can quantify the infection in lymphocytes and in primary cells with a higher sensitivity and allow the detection and quantitation of multiply infected cells, providing better tools to study retroviral cell-mediated infection. IMPORTANCE The human-pathogenic retroviruses HTLV-1 and HIV-1 can be transmitted more efficiently in vivo via direct contact of infected cells with healthy target cells than through cell-free virion-mediated infection. Despite its importance, cell-to-cell transmission has been difficult to quantify because the previously infected cells and the newly infected cells are mixed together in the same culture. In the current study, we generated vectors that are significantly improved over the previously described replication-dependent vectors. As a result, these improved vectors can efficiently detect and quantify cell-to-cell transmission or new infection events in cells in mixed culture. These luciferase- or fluorescence protein-based reporter vectors can be used to quantify and study HIV-1 or HTLV-1 cell-mediated infection in a simple one-step transfection/infection assay.

Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly. IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly. Understanding the mechanism used by HIV-1 to ensure genome packaging provides significant insights into viral assembly and replication.

A key step of retroviral replication is packaging of the viral RNA genome during virus assembly. Specific packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA genome. In HIV-1, similar to most retroviruses, the packaging signal is located within the 5′ untranslated region and extends into the gag -coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10- to 50-fold) in viral RNA packaging and virus titer. We examined here the role of the ribosomal frameshift signal in HIV-1 RNA packaging by studying the RNA packaging and virus titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently, and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation. IMPORTANCE To generate infectious viruses, HIV-1 must package viral RNA genome during virus assembly. The specific HIV-1 genome packaging is mediated by interactions between the structural protein Gag and elements near the 5′ end of the viral RNA known as packaging signal. In this study, we examined whether the Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans , none of the tested ribosomal frameshift signal mutants has defects in RNA packaging or virus titer. These studies provide important information on how HIV-1 regulates its genome packaging and generate infectious viruses necessary for transmission to new hosts.

Although the predominant effect of host restriction APOBEC3 proteins on HIV-1 infection is to block viral replication, they might inadvertently increase retroviral genetic variation by inducing G-to-A hypermutation. Numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. Confounding factors contributing to the debate include the extent of lethal (stop codon) and sublethal hypermutation induced by different APOBEC3 proteins, the inability to distinguish between G-to-A mutations induced by APOBEC3 proteins and error-prone viral replication, the potential impact of hypermutation on the frequency of retroviral recombination, and the extent to which viral recombination occurs in vivo, which can reassort mutations in hypermutated genomes. Here, we determined the effects of hypermutation on the HIV-1 recombination rate and its contribution to genetic variation through recombination to generate progeny genomes containing portions of hypermutated genomes without lethal mutations. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 × 10−5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (4 × 10−21 and1 × 10−11, respectively) and its contribution to viral mutations is far below mutations generated during error-prone reverse transcription. Taken together, we conclude that the contribution of APOBEC3-induced hypermutation to HIV-1 genetic variation is substantially lower than that from mutations during error-prone replication., Author Summary HIV-1 populations exhibit high levels of genetic variation, allowing the virus to escape immunological and pharmacological selection pressures, and thwarting efforts to develop an effective vaccine; thus, it is critical to understand all sources of HIV-1 genetic diversity. In addition to error-prone reverse transcription and frequent recombination, APOBEC3 proteins, expressed as part of the host intracellular antiviral defense mechanism, could potentially contribute to viral genetic variation through sublethal hypermutation or rescue of hypermutated portions of the genomes without lethal mutations through recombination. Here, we show that hypermutation by APOBEC3 proteins does not affect the rate of recombination; in addition, even in proviruses generated from heterozygous virions containing wild-type and hypermutated genomes, hypermutation only modestly increased the viral mutation rate by twofold. Since the frequency of copackaged wild-type and hypermutated genomes in patients is extremely low, we conclude that hypermutation does not significantly contribute to HIV-1 genetic variation. Analysis of hypermutated sequences from infected patients indicates that the frequency of sublethal mutagenesis is negligible and its contribution to viral variation is substantially lower than that of error-prone viral replication.

Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA-Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA-Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms.

HIV-1 and HIV-2 are derived from two distinct primate viruses and share only limited sequence identity. Despite this, HIV-1 and HIV-2 Gag polyproteins can coassemble into the same particle and their genomes can undergo recombination, albeit at an extremely low frequency, implying that HIV-1 and HIV-2 RNA can be copackaged into the same particle. To determine the frequency of HIV-1 and HIV-2 RNA copackaging and to dissect the mechanisms that allow the heterologous RNA copackaging, we directly visualized the RNA content of each particle by using RNA-binding proteins tagged with fluorescent proteins to label the viral genomes. We found that when HIV-1 and HIV-2 RNA are present in viral particles at similar ratios, ∼10% of the viral particles encapsidate both HIV-1 and HIV-2 RNAs. Furthermore, heterologous RNA copackaging can be promoted by mutating the 6-nucleotide (6-nt) dimer initiation signal (DIS) to discourage RNA homodimerization or to encourage RNA heterodimerization, indicating that HIV-1 and HIV-2 RNA can heterodimerize prior to packaging using the DIS sequences. We also observed that the coassembly of HIV-1 and HIV-2 Gag proteins is not required for the heterologous RNA copackaging; HIV-1 Gag proteins are capable of mediating HIV-1 and HIV-2 RNA copackaging. These results define the cis- and trans -acting elements required for and affecting the heterologous RNA copackaging, a prerequisite for the generation of chimeric viruses by recombination, and also shed light on the mechanisms of RNA-Gag recognition essential for RNA encapsidation.

With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment, and vaccine development.

Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2 proviruses, one with a wild-type gag / gag-pol and the other with a mutant gag that cannot express functional Gag/Gag-Pol. Viral titers and RNA analyses revealed that mutant viral RNAs can be packaged at efficiencies comparable to that of viral RNA from which wild-type Gag/Gag-Pol is translated. These results do not support the cis -packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins that specifically recognize stem-loop motifs in the viral genomes, an assay termed single virion analysis. These studies revealed that >90% of the HIV-2 particles contained viral RNAs and that RNAs derived from different viruses were copackaged frequently. Furthermore, the frequencies of heterozygous particles in the viral population could be altered by changing a 6-nucleotide palindromic sequence at the 5′-untranslated region of the HIV-2 genome. This finding indicates that selection of copackaging RNA partners occurs prior to encapsidation and that HIV-2 Gag proteins primarily package one dimeric RNA rather than two monomeric RNAs. Additionally, single virion analyses demonstrated a similar RNA distribution in viral particles regardless of whether both viruses had a functional gag or one of the viruses had a nonfunctional gag , providing further support for the trans- packaging hypothesis. Together, these results revealed mechanisms of HIV-2 RNA packaging that are, contrary to previous studies, in many respects surprisingly similar to those of HIV-1.

Recombination is a major force for generating human immunodeficiency virus type 1 (HIV-1) diversity and produces numerous recombinants circulating in the human population. We previously established a cell-based system using green fluorescent protein gene (gfp) as a reporter to study the mechanisms of HIV-1 recombination. We now report an improved system capable of detecting recombination using authentic viral sequences. Frameshift mutations were introduced into the gag gene so that parental viruses do not express full-length Gag; however, recombination can generate a progeny virus that expresses a functional Gag. We demonstrate that this Gag reconstitution assay can be used to detect recombination between two group M HIV-1 variants of the same or of different subtypes. Using both gfp and gag assays, we found that, similar to group M viruses, group O viruses also recombine frequently. When recombination between a group M virus and a group O virus was examined, we found three distinct barriers for intergroup recombination. First, similar to recombination within group M viruses, intergroup recombination is affected by the identity of the dimerization initiation signal (DIS); variants with the same DIS recombined at a higher rate than those with different DIS. Second, using the gfp recombination assay, we showed that intergroup recombination occurs much less frequently than intragroup recombination, even though the gfp target sequence is identical in all viruses. Finally, Gag reconstitution between variants from different groups is further reduced compared with green fluorescent protein, indicating that sequence divergence interferes with recombination efficiency in the gag gene. Compared with identical sequences, we estimate that recombination rates are reduced by 3-fold and by 10- to 13-fold when the target regions in gag contain 91% and 72-73% sequence identities, respectively. These results show that there are at least three distinct mechanisms preventing exchange of genetic information between divergent HIV-1 variants from different groups.

A long-standing question in retrovirus biology is how RNA genomes are distributed among virions. In the studies presented in this report, we addressed this issue by directly examining HIV-1 RNAs in virions using a modified HIV-1 genome that contained recognition sites for BglG, an antitermination protein in the Escherichia coli bgl operon, which was coexpressed with a fragment of BglG RNA binding protein fused to a fluorescent protein. Our results demonstrate that the majority of virions (>90%) contain viral RNAs. We also coexpressed HIV-1 genomes containing binding sites for BglG or the bacteriophage MS2 coat protein along with 2 fluorescent protein-tagged RNA binding proteins. This method allows simultaneously labeling and discrimination of 2 different RNAs at single-RNA-detection sensitivity. Using this strategy, we obtained physical evidence that virions contain RNAs derived from different parental viruses (heterozygous virion) at ratios expected from a random distribution, and we found that this ratio can be altered by changing the dimerization sequences. Our studies of heterozygous virions also support a generally accepted but unproven assumption that most particles contain 1 dimer. This study provides answers to long-standing questions in HIV-1 biology and illustrates the power and sensitivity of the 2-RNA labeling method, which can also be adapted to analyze various issues of RNA biogenesis including the detection of different RNAs in live cell imaging.

Primate lentiviruses are composed of several distinct lineages, including human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus SIVagm. HIV-1 and HIV-2 have significant differences in the mechanisms of viral RNA encapsidation. Therefore, the RNA packaging mechanisms of SIVagm cannot be predicted from the studies of HIV-1 and HIV-2. We examined the roles of the nucleocapsid (NC) zinc finger motifs on RNA packaging by mutating the conserved zinc finger (CCHC) motifs, and whether SIVagm has a preference to package RNA in cis by comparing the RNA packaging efficiencies of gag mutants in the presence of a wild-type vector. Our results indicate that the SIVagm NC domain plays an important role in Gag–RNA recognition; furthermore SIVagm is distinct from the other currently known primate lentiviruses as destroying either zinc finger motif in the NC causes very drastic RNA packaging defects. Additionally, trans -packaging is a major mechanism for SIVagm RNA encapsidation.

Full-length HIV-1 RNA plays a central role in viral replication by serving as the mRNA for essential viral proteins and as the genome packaged into infectious virions. Proper RNA trafficking is required for the functions of RNA and its encoded proteins; however, the mechanism by which HIV-1 RNA is transported within the cytoplasm remains undefined. Full-length HIV-1 RNA transport is further complicated when group-specific antigen (Gag) protein is expressed, because a significant portion of HIV-1 RNA may be transported as Gag–RNA complexes, whose properties could differ greatly from Gag-free RNA. In this report, we visualized HIV-1 RNA and monitored its movement in the cytoplasm by using single-molecule tracking. We observed that most of the HIV-1 RNA molecules move in a nondirectional, random-walk manner, which does not require an intact cytoskeletal structure, and that the mean-squared distance traveled by the RNA increases linearly with time, indicative of diffusive movement. We also observed that a single HIV-1 RNA molecule can move at various speeds when traveling through the cytoplasm, indicating that its movement is strongly affected by the immediate environment. To examine the effect of Gag protein on HIV-1 RNA transport, we analyzed the cytoplasmic HIV-1 RNA movement in the presence of sufficient Gag for virion assembly and found that HIV-1 RNA is still transported by diffusion with mobility similar to the mobility of RNAs unable to express functional Gag. These studies define a major mechanism of HIV-1 gene expression and resolve the long-standing question of how the RNA genome is transported to the assembly site.

Recombination is a major mechanism that generates variation in populations of human immunodeficiency virus type 1 (HIV-1). Mutations that confer replication advantages, such as drug resistance, often cluster within regions of the HIV-1 genome. To explore how efficiently HIV-1 can assort markers separated by short distances, we developed a flow cytometry-based system to study recombination. Two HIV-1-based vectors were generated, one encoding the mouse heat-stable antigen gene and green fluorescent protein gene ( GFP ), and the other encoding the mouse Thy-1 gene and GFP . We generated derivatives of both vectors that contained nonfunctional GFP inactivated by different mutations. Recombination in the region between the two inactivating mutations during reverse transcription could yield a functional GFP . With this system, we determined that the recombination rates of markers separated by 588, 300, 288, and 103 bp in one round of viral replication are 56, 38, 31, and 12%, respectively, of the theoretical maximum measurable recombination rate. Statistical analyses revealed that at these intervals, recombination rates and marker distances have a near-linear relationship that is part of an overall quadratic fit. Additionally, we examined the segregation of three markers within 600 bp and concluded that HIV-1 crossover events do not exhibit high negative interference. We also examined the effects of target cells and viral accessory proteins on recombination rate. Similar recombination rates were observed when human primary CD4 + T cells and a human T-cell line were used as target cells. We also found equivalent recombination rates in the presence and absence of accessory genes vif , vpr , vpu , and nef . These results illustrate the power of recombination in generating viral population variation and predict the rapid assortment of mutations in the HIV-1 genome in infected individuals.

Encapsidating the viral genome into virions is an essential step in generating infectious viral particles. Most HIV-1 particles contain two copies of full-length viral RNA indicating genome encapsidation is an efficient and regulated process. Interactions between the HIV-1 structural protein Gag and cis-acting elements in the viral RNA mediate the packaging of viral RNA. The HIV-1 genome selects its copackaged RNA partner, or dimerizes, prior to encapsidation. Several aspects of virus biology and host–virus interactions important for the packaging of HIV-1 viral genomes are discussed in this review.

High-frequency recombination is a hallmark of HIV-1 replication. Recombination can occur between two members of the same subtype or between viruses from two different subtypes, generating intra- or intersubtype recombinants, respectively. Many intersubtype recombinants have been shown to circulate in human populations. We hypothesize that sequence diversity affects the emergence of viable recombinants by decreasing recombination events and reducing the ability of the recombinants to replicate. To test our hypothesis, we compared recombination between two viruses containing subtype B pol genes (B/B) and between viruses with pol genes from subtype B or F (B/F). Recombination events generated during a single cycle of infection without selection pressure on pol gene function were analyzed by single-genome sequencing. We found that recombination occurred slightly (∼30%) less frequently in B/F than in B/B viruses, and the overall distribution of crossover junctions in pol was similar for the two classes of recombinants. We then examined the emergence of recombinants in a multiple cycle assay, so that functional pol gene products were selected. We found that the emerging B/B recombinants had complex patterns, and the crossover junctions were distributed throughout the pol gene. In contrast, selected B/F recombinants had limited recombination patterns and restricted crossover junction distribution. These results provide evidence for the evolved coadapted sites in variants from different subtypes; these sites may be segregated by recombination events, causing the newly generated intersubtype recombinants to undergo purifying selection. Therefore, the ability of the recombinants to replicate is the major barrier for many of these viruses.

Once transcribed, the nascent full-length RNA of HIV-1 must travel to the appropriate host cell sites to be translated or to find a partner RNA for copackaging to form newly generated viruses. In this report, we sought to delineate the location where HIV-1 RNA initiates dimerization and the influence of the RNA transport pathway used by the virus on downstream events essential to viral replication. Using a cell-fusion-dependent recombination assay, we demonstrate that the two RNAs destined for copackaging into the same virion select each other mostly within the cytoplasm. Moreover, by manipulating the RNA export element in the viral genome, we show that the export pathway taken is important for the ability of RNA molecules derived from two viruses to interact and be copackaged. These results further illustrate that at the point of dimerization the two main cellular export pathways are partially distinct. Lastly, by providing Gag in trans, we have demonstrated that Gag is able to package RNA from either export pathway, irrespective of the transport pathway used by the gag mRNA. These findings provide unique insights into the process of RNA export in general, and more specifically, of HIV-1 genomic RNA trafficking., Author Summary High genetic diversity of HIV-1 presents a difficult barrier for drug treatment and vaccine development. HIV-1 efficiently reassorts mutations in its genome by packaging two copies of RNA into one virion and using portions of each RNA as the template for DNA synthesis. Thus, a key to HIV-1's genetic diversity is how it packages two RNAs into one virion. We have previously shown that HIV-1 packages RNA as a dimer, rather than two monomers. In this report, we determined that HIV-1 RNA finds its copackaged dimer partner mostly in the cytoplasm, not the nucleus, of the infected cells. We also found that the host cell machinery used to transport the HIV-1 RNA out of the nucleus affects the ability of two RNAs to copackage into one virion: HIV-1 RNAs transported using the CRM-1 pathway does not copackage efficiently with RNA transported by the NXF1 pathway. This is also the first demonstration that RNAs transported by the two major cellular pathways, CRM-1 and NXF1, are partially segregated at some point in the cytoplasm. Therefore, this work provides novel insights to both HIV replication mechanisms and cellular RNA transport pathways.

During human immunodeficiency virus type 1 (HIV-1) assembly, the nucleocapsid (NC) and the PTAP motif in p6 of Gag play important roles in RNA encapsidation and virus release, respectively. We have previously demonstrated that functional complementation occurs between an NC mutant and a PTAP mutant to rescue viral replication. In this report, we examined the amounts of functional NC and PTAP motif that are required during virus replication. When NC and PTAP mutants were coexpressed at 5:1, 5:5, and 1:5 ratios, virus titers were rescued at 5%, 51%, and 86% of the wild-type level, respectively. These results indicate that HIV-1 requires a small amount of functional PTAP motif but far more functional NC to complete efficient replication. Further analyses reveal that RNA packaging can be significantly rescued in viruses containing a small amount of functional NC. However, most of the NC proteins must be functional to generate the wild-type level of R-U5 DNA product. Once the R-U5 product is generated, viruses containing half of the functional NC can complete reverse transcription and DNA integration at near-wild-type efficiency. These results define the quantitative requirements of NC and p6 during HIV-1 replication and provide insights into the requirement for the development of anti-HIV strategies using NC and p6 as targets.

HIV-1 intersubtype recombinants have an increasingly important role in shaping the AIDS pandemic. We sought to understand the molecular mechanisms that generate intersubtype HIV-1 recombinants. To approach this, we analyzed recombinants of HIV-1 subtypes B and C and identified their crossover junctions in the viral genome from the 5′ LTR to the end of pol. We identified 56 recombination events in 56 proviruses; the distribution of these events indicated an apparent recombination gradient: there were significantly more crossover junctions in the 3′ half than in the 5′ half of the analyzed region. HIV-1 subtypes B and C have different dimerization initiation signal (DIS). We hypothesized that the inability of subtype B and C RNAs to form perfect base-pairing of the DIS affects the dimeric RNA structure and causes a decrease in recombination events at the 5′ end of the viral genome. To test this hypothesis, we examined recombinants generated from a subtype C virus and a modified subtype B virus containing a subtype C DIS. In the 56 proviruses analyzed, we identified 96 recombination events, which are significantly more frequent than in the B/C recombinants. Furthermore, these crossover junctions were distributed evenly throughout the region analyzed, indicating that the recombination gradient was corrected by matching the DIS. Therefore, base-pairing at the DIS has an important function during HIV-1 reverse transcription, most likely in maintaining nucleic-acid structure in the complex. These findings not only reveal elements important to retroviral recombination but also provide insights into the generation of HIV-1 intersubtype recombinants that are important to AIDS epidemic.

Frequent human immunodeficiency virus type 1 (HIV-1) recombination occurs during DNA synthesis when portions of the two copackaged RNAs are used as templates to generate a hybrid DNA copy. Therefore, the frequency of copackaging of genomic RNAs from two different viruses (heterozygous virion formation) affects the generation of genotypically different recombinants. We hypothesized that the selection of copackaged RNA partners is largely determined by Watson-Crick pairing at the dimer initiation signal (DIS), a 6-nucleotide palindromic sequence at the terminal loop of stem-loop 1 (SL1). To test our hypothesis, we examined whether heterozygous virion formation could be encouraged by manipulation of the DIS. Three pairs of viruses were generated with compensatory DIS mutations, designed so that perfect DIS base pairing could only occur between RNAs derived from different viruses, not between RNAs from the same virus. We observed that vector pairs with compensatory DIS mutations had an almost twofold increase in recombination rates compared with wild-type viruses. These data suggest that heterozygous virion formation was enhanced in viruses with compensatory DIS mutations (from 50% to more than 90% in some viral pairings). The role of the SL1 stem in heterozygous virion formation was also tested; our results indicated that the intermolecular base pairing of the stem sequences does not affect RNA partner selection. In summary, our results demonstrate that the Watson-Crick pairing of the DIS is a major determinant in the selection of the copackaged RNA partner, and altering the base pairing of the DIS can change the proportion of heterozygous viruses in a viral population. These results also strongly support the hypothesis that HIV-1 RNA dimers are formed prior to encapsidation.

Sequence differences in the dimerization initiation signal (DIS) affect the rate of recombination between subtype B and subtype C HIV-1. To test the hypothesis that DIS sequences can be used to predict intersubtype recombination potentials, we measured the recombination rate between CRF01_A/E (AE) and B, which contain mismatches in the DIS, and between AE and C, which have an identical DIS. Compared with the intrasubtype recombination rate, the recombination rate between AE and subtype B virus was 9-fold lower, and the rate between AE and subtype C virus was 2-fold lower. Thus, DIS sequences can be used to predict the recombination potential between HIV-1 subtypes. Further analyses revealed that the 2-fold lower recombination rate between AE and C viruses can be restored to the intrasubtype recombination rate by matching a part of the LTR and a portion of the viral genome. Therefore, the lower intersubtype recombination rate between AE and C is not caused by a given region but is a cumulative effect by more than one region.

A long-standing question in retrovirus biology is how RNA genomes are spatially organized within virions. Here, we explore the relative 3D spatial distribution of RNA dimers (1) within ∼130-nm diameter virus-like particles (VLPs) with single-RNA sensitivity (2) using the double-helix microscope. The double-helix microscope, a modification of the standard epifluorescence microscope, is capable of precise and accurate three-dimensional (3D) localization of fixed fluorescent single molecules throughout a 2 μm-thick focal volume (3). Furthermore, the use of a sophisticated registration algorithm, based upon a locally weighted quadratic transformation function, enables accurate co-localization of yellow and red fluorescent emitters with

The tightly linked pcbABC genes encode the chlorophyll a/b-binding apoproteins in the oxygenic photosynthetic prokaryote Prochlorothrix hollandica. Northern blotting experiments employing gene-specific DNA probes have identified a complex pattern of transcription from the pcb region. A large 4.4-kb transcript detected in cultures maintained in high light, low light and in darkness results from the cotranscription of all three genes, whereas pcbAB, pcbBC and individual pcbA, B, and C mRNAs are similarly detected in all light regimes. The half lives of the RNAs vary from 15 min for the pcbABC transcript, to over 60 min for the pcbA and pcbC mRNAs. The lack of identifiable promoter sequences other than the region upstream from pcbA, plus the enhanced stability of the individual single gene transcripts, suggest that the smaller RNA species arise from processing of larger transcripts. Transcription and mRNA turnover occurs largely independent of light intensity, in contrast to what is seen in most other phototrophs, in which light influences the accumulation of antenna apoprotein gene mRNAs.

How retroviruses regulate the amount of RNA genome packaged into each virion has remained a long-standing question. Our previous study showed that most HIV-1 particles contain two copies of viral RNA, indicating that the number of genomes packaged is tightly regulated. In this report, we examine the mechanism that controls the number of RNA genomes encapsidated into HIV-1 particles. We hypothesize that HIV-1 regulates genome packaging by either the mass or copy number of the viral RNA. These two distinct mechanisms predict different outcomes when the genome size deviates significantly from that of wild type. Regulation by RNA mass would result in multiple copies of a small genome or one copy of a large genome being packaged, whereas regulation by copy number would result in two copies of a genome being packaged independent of size. To distinguish between these two hypotheses, we examined the packaging of viral RNA that was larger (≈17 kb) or smaller (≈3 kb) than that of wild-type HIV-1 (≈9 kb) and found that most particles packaged two copies of the viral genome regardless of whether they were 17 kb or 3 kb. Therefore, HIV-1 regulates RNA genome encapsidation not by the mass of RNA but by packaging two copies of RNA. To further explore the mechanism that governs this regulation, we examined the packaging of viral RNAs containing two packaging signals that can form intermolecular dimers or intramolecular dimers (self-dimers) and found that one self-dimer is packaged. Therefore, HIV-1 recognizes one dimeric RNA instead of two copies of RNA. Our findings reveal that dimeric RNA recognition is the key mechanism that regulates HIV-1 genome encapsidation and provide insights into a critical step in the generation of infectious viruses., Author Summary Viruses must package their genomes in particles to pass their genetic information to the next generation. Although many aspects of RNA packaging are well-studied, how retroviruses regulate the number of genomes in the particle is currently unknown. Based on the dimeric nature of retroviral genomes in particles, it was often assumed that two copies of RNA were packaged into one particle. This assumption was validated recently when we demonstrated that most HIV-1 particles contain two copies of viral RNA, which revealed that the number of genomes packaged is tightly controlled. In this report, we examined the mechanism that regulates the amount of RNAs encapsidated into HIV-1 particles. Our results showed that RNA packaging is not regulated by the mass of the viral RNA as two copies of small or large genomes are packaged. However, packaging of two copies of RNA can be perturbed; HIV-1 can encapsidate one copy of its genome when the RNA contains two packaging/dimerization signals that allow for intramolecular dimer (self-dimer) formation. These studies revealed that HIV-1 regulates genome packaging by recognizing the dimeric RNA structure, and suggest that the interaction of viral protein Gag and dimeric RNA serves as the nucleation point of virus assembly.

Recombination can assort polymorphic alleles to increase diversity in the HIV-1 population. To better understand the recombination potential of subtype A HIV-1, we generated viruses containing sequences from two variants circulating in Russia and analyzed the polymerase gene (pol) of the recombinants after one round of HIV-1 replication using single-genome sequencing. We observed that recombination occurred throughout pol and could easily assort alleles containing mutations that conferred resistance to currently approved antivirals. We measured the recombination rate in various regions of pol including a G-rich region that has been previously proposed to be a recombination hot spot. Our study does not support a recombination hot spot in this G-rich region. Importantly, of the 58 proviral sequences containing crossover event(s) in pol, we found that each sequence was a unique genotype indicating that recombination is a powerful genetic mechanism in assorting the genomes of subtype A HIV-1 variants.