Imaging HIV-1 Nuclear Import, Uncoating, and Proviral Transcription.

Live-cell imaging has become a powerful tool for dissecting the behavior of viral complexes during HIV-1 infection with high temporal and spatial resolution. Very few HIV-1 particles in a viral population are infectious and successfully complete replication (~1/50). Single-particle live-cell imaging enables the study of these rare infectious viral particles, which cannot be accomplished in biochemical assays that measure the average property of the entire viral population, most of which are not infectious. The timing and location of many events in the early stage of the HIV-1 life cycle, including nuclear import, uncoating, and integration, have only recently been elucidated. Live-cell imaging also provides a valuable approach to study interactions of viral and host factors in distinct cellular compartments and at specific stages of viral replication. Successful live-cell imaging experiments require careful consideration of the fluorescent labeling method used and avoid or minimize its potential impact on normal viral replication and produce misleading results. Ideally, it is beneficial to utilize multiple virus labeling strategies and compare the results to ensure that the virion labeling did not adversely influence the viral replication step that is under investigation. Another potential benefit of using different labeling strategies is that they can provide information about the state of the viral complexes. Here, we describe our methods that utilize multiple fluorescent protein labeling approaches to visualize and quantify important events in the HIV-1 life cycle, including docking HIV-1 particles with the nuclear envelope (NE) and their nuclear import, uncoating, and proviral transcription.

Single-Virion Analysis: A Method to Visualize HIV-1 Particle Content Using Fluorescence Microscopy.

HIV-1 virions incorporate viral RNA, cellular RNAs, and proteins during the assembly process. Some of these components, such as the viral RNA genome and viral proteins, are essential for viral replication, whereas others, such as host innate immune proteins, can inhibit virus replication. Therefore, analyzing the virion content is an integral part of studying HIV-1 replication. Traditionally, virion contents have been examined using biochemical assays, which can provide information on the presence or absence of the molecule of interest but not its distribution in the virion population. Here, we describe a method, single-virion analysis, that directly examines the presence of molecules of interest in individual viral particles using fluorescence microscopy. Thus, this method can detect both the presence and the distribution of molecules of interest in the virion population. Single-virion analysis was first developed to study HIV-1 RNA genome packaging. In this assay, HIV-1 unspliced RNA is labeled with a fluorescently tagged RNA-binding protein (protein A) and some of the Gag proteins are labeled with a different fluorescent protein (protein B). Using fluorescence microscopy, HIV-1 particles can be identified by the fluorescent protein B signal and the presence of unspliced HIV-1 RNA can be identified by the fluorescent protein A signal. Therefore, the proportions of particles that contain unspliced RNA can be determined by the fraction of Gag particles that also have a colocalized RNA signal. By tagging the molecule of interest with fluorescent proteins, single-virion analysis can be easily adapted to study the incorporation of other viral or host cell molecules into particles. Indeed, this method has been adapted to examine the proportion of HIV-1 particles that contain APOBEC3 proteins and the fraction of particles that contain a modified Gag protein. Therefore, single-virion analysis is a flexible method to study the nucleic acid and protein content of HIV-1 particles.

HIV-1 uncoating requires long double-stranded reverse transcription products.

HIV-1 cores, which contain the viral genome and replication machinery, must disassemble (uncoat) during viral replication. However, the viral and host factors that trigger uncoating remain unidentified. Recent studies show that infectious cores enter the nucleus and uncoat near the site of integration. Here, we show that efficient uncoating of nuclear cores requires synthesis of a double-stranded DNA (dsDNA) genome >3.5 kb and that the efficiency of uncoating correlates with genome size. Core disruption by capsid inhibitors releases viral DNA, some of which integrates. However, most of the viral DNA is degraded, indicating that the intact core safeguards viral DNA. Atomic force microscopy and core content estimation reveal that synthesis of full-length genomic dsDNA induces substantial internal strain on the core to promote uncoating. We conclude that HIV-1 cores protect viral DNA from degradation by host factors and that synthesis of long double-stranded reverse transcription products is required to trigger efficient HIV-1 uncoating.

HIV-1 RNA genome packaging: it's G-rated.

A member of the Retroviridae, human immunodeficiency virus type 1 (HIV-1), uses the RNA genome packaged into nascent virions to transfer genetic information to its progeny. The genome packaging step is a highly regulated and extremely efficient process as a vast majority of virus particles contain two copies of full-length unspliced HIV-1 RNA that form a dimer. Thus, during virus assembly HIV-1 can identify and selectively encapsidate HIV-1 unspliced RNA from an abundant pool of cellular RNAs and various spliced HIV-1 RNAs. Several "G" features facilitate the packaging of a dimeric RNA genome. The viral polyprotein Gag orchestrates virus assembly and mediates RNA genome packaging. During this process, Gag preferentially binds unpaired guanosines within the highly structured 5' untranslated region (UTR) of HIV-1 RNA. In addition, the HIV-1 unspliced RNA provides a scaffold that promotes Gag:Gag interactions and virus assembly, thereby ensuring its packaging. Intriguingly, recent studies have shown that the use of different guanosines at the junction of U3 and R as transcription start sites results in HIV-1 unspliced RNA species with 99.9% identical sequences but dramatically distinct 5' UTR conformations. Consequently, one species of unspliced RNA is preferentially packaged over other nearly identical RNAs. These studies reveal how conformations affect the functions of HIV-1 RNA elements and the complex regulation of HIV-1 replication. In this review, we summarize cis- and trans-acting elements critical for HIV-1 RNA packaging, locations of Gag:RNA interactions that mediate genome encapsidation, and the effects of transcription start sites on the structure and packaging of HIV-1 RNA.

Potent dual block to HIV-1 infection using lentiviral vectors expressing fusion inhibitor peptide mC46- and Vif-resistant APOBEC3G.

Gene therapy strategies that effectively inhibit HIV-1 replication are needed to reduce the requirement for lifelong antiviral therapy and potentially achieve a functional cure. We previously designed self-activating lentiviral vectors that efficiently delivered and expressed a Vif-resistant mutant of APOBEC3G (A3G-D128K) to T cells, which potently inhibited HIV-1 replication and spread with no detectable virus. Here, we developed vectors that express A3G-D128K, membrane-associated fusion inhibitor peptide mC46, and O6-methylguanine-DNA-methyltransferase (MGMT) selectable marker for in vivo selection of transduced CD34+ hematopoietic stem and progenitor cells. MGMT-selected T cell lines MT4, CEM, and PM1 expressing A3G-D128K (with or without mC46) potently inhibited NL4-3 infection up to 45 days post infection with no detectable viral replication. Expression of mC46 was sufficient to block infection >80% in a single-cycle assay. Importantly, expression of mC46 provided a selective advantage to the A3G-D128K-modified T cells in the presence of replication competent virus. This combinational approach to first block HIV-1 entry with mC46, and then block any breakthrough infection with A3G-D128K, could provide an effective gene therapy treatment and a potential functional cure for HIV-1 infection.

HIV-1 usurps transcription start site heterogeneity of host RNA polymerase II to maximize replication fitness.

HIV-1 relies on host RNA polymeraseII (Pol II) to transcribe its genome and uses multiple transcription start sites (TSS), including three consecutive guanosines located near the U3-R junction, to generate transcripts containing three, two, and one guanosine at the 5' end, referred to as 3G, 2G, and 1G RNA, respectively. The 1G RNA is preferentially selected for packaging, indicating that these 99.9% identical RNAs exhibit functional differences and highlighting the importance of TSS selection. Here, we demonstrate that TSS selection is regulated by sequences between the CATA/TATA box and the beginning of R. Furthermore, we have generated two HIV-1 mutants with distinct 2-nucleotide modifications that predominantly express 3G RNA or 1G RNA. Both mutants can generate infectious viruses and undergo multiple rounds of replication in T cells. However, both mutants exhibit replication defects compared to the wild-type virus. The 3G-RNA-expressing mutant displays an RNA genome-packaging defect and delayed replication kinetics, whereas the 1G-RNA-expressing mutant exhibits reduced Gag expression and a replication fitness defect. Additionally, reversion of the latter mutant is frequently observed, consistent with sequence correction by plus-strand DNA transfer during reverse transcription. These findings demonstrate that HIV-1 maximizes its replication fitness by usurping the TSS heterogeneity of host RNA Pol II to generate unspliced RNAs with different specialized roles in viral replication. The three consecutive guanosines at the junction of U3 and R may also maintain HIV-1 genome integrity during reverse transcription. These studies reveal the intricate regulation of HIV-1 RNA and complex replication strategy.

Adaptation of HIV-1/HIV-2 Chimeras with Defects in Genome Packaging and Viral Replication.

Frequent recombination is a hallmark of retrovirus replication. In rare cases, recombination occurs between distantly related retroviruses, generating novel viruses that may significantly impact viral evolution and public health. These recombinants may initially have substantial replication defects due to impaired interactions between proteins and/or nucleic acids from the two parental viruses. However, given the high mutation rates of retroviruses, these recombinants may be able to evolve improved compatibility of these viral elements. To test this hypothesis, we examined the adaptation of chimeras between two distantly related human pathogens: HIV-1 and HIV-2. We constructed HIV-1-based chimeras containing the HIV-2 nucleocapsid (NC) domain of Gag or the two zinc fingers of HIV-2 NC, which are critical for specific recognition of viral RNA. These chimeras exhibited significant defects in RNA genome packaging and replication kinetics in T cells. However, in some experiments, the chimeric viruses replicated with faster kinetics when repassaged, indicating that viral adaptation had occurred. Sequence analysis revealed the acquisition of a single amino acid substitution, S18L, in the first zinc finger of HIV-2 NC. This substitution, which represents a switch from a conserved HIV-2 residue to a conserved HIV-1 residue at this position, partially rescued RNA packaging and replication kinetics. Further analysis revealed that the combination of two substitutions in HIV-2 NC, W10F and S18L, almost completely restored RNA packaging and replication kinetics. Our study demonstrates that chimeras of distantly related retroviruses can adapt and significantly enhance their replication by acquiring a single substitution. IMPORTANCE Novel retroviruses can emerge from recombination between distantly related retroviruses. Most notably, HIV-1 originated from zoonotic transmission of a novel recombinant (SIVcpz) into humans. Newly generated recombinants may initially have significant replication defects due to impaired interactions between viral proteins and/or nucleic acids, such as between cis- and trans-acting elements from the two parental viruses. However, provided that the recombinants retain some ability to replicate, they may be able to adapt and repair the defective interactions. Here, we used HIV-1 and HIV-2 Gag chimeras as a model system for studying the adaptation of recombinant viruses. We found that only two substitutions in the HIV-2 NC domain, W10F and S18L, were required to almost fully restore RNA genome packaging and replication kinetics. These results illustrate the extremely flexible nature of retroviruses and highlight the possible emergence of novel recombinants in the future that could pose a significant threat to public health.

Transcription Start Site Heterogeneity and Preferential Packaging of Specific Full-Length RNA Species Are Conserved Features of Primate Lentiviruses.

HIV-1 must package its RNA genome to generate infectious viruses. Recent studies have revealed that during genome packaging, HIV-1 not only excludes cellular mRNAs, but also distinguishes among full-length viral RNAs. Using NL4-3 and MAL molecular clones, multiple transcription start sites (TSS) were identified, which generate full-length RNAs that differ by only a few nucleotides at the 5' end. However, HIV-1 selectively packages RNAs containing one guanosine (1G RNA) over RNAs with three guanosines (3G RNA) at the 5' end. Thus, the 5' context of HIV-1 full-length RNA can affect its function. To determine whether the regulation of genome packaging by TSS usage is unique to NL4-3 and MAL, we examined 15 primate lentiviruses including transmitted founder viruses of HIV-1, HIV-2, and several simian immunodeficiency viruses (SIVs). We found that all 15 viruses used multiple TSS to some extent. However, the level of TSS heterogeneity in infected cells varied greatly, even among closely related viruses belonging to the same subtype. Most viruses also exhibited selective packaging of specific full-length viral RNA species into particles. These findings demonstrate that TSS heterogeneity and selective packaging of certain full-length viral RNA species are conserved features of primate lentiviruses. In addition, an SIV strain closely related to the progenitor virus that gave rise to HIV-1 group M, the pandemic pathogen, exhibited TSS usage similar to some HIV-1 strains and preferentially packaged 1G RNA. These findings indicate that multiple TSS usage and selective packaging of a particular unspliced RNA species predate the emergence of HIV-1. IMPORTANCE Unspliced HIV-1 RNA serves two important roles during viral replication: as the virion genome and as the template for translation of Gag/Gag-Pol. Previous studies of two HIV-1 molecular clones have concluded that the TSS usage affects unspliced HIV-1 RNA structures and functions. To investigate the evolutionary origin of this replication strategy, we determined TSS of HIV-1 RNA in infected cells and virions for 15 primate lentiviruses. All HIV-1 isolates examined, including several transmitted founder viruses, utilized multiple TSS and selected a particular RNA species for packaging. Furthermore, these features were observed in SIVs related to the progenitors of HIV-1, suggesting that these characteristics originated from the ancestral viruses. HIV-2, SIVs related to HIV-2, and other SIVs also exhibited multiple TSS and preferential packaging of specific unspliced RNA species, demonstrating that this replication strategy is broadly conserved across primate lentiviruses.

Intranuclear Positions of HIV-1 Proviruses Are Dynamic and Do Not Correlate with Transcriptional Activity.

The relationship between spatiotemporal distribution of HIV-1 proviruses and their transcriptional activity is not well understood. To elucidate the intranuclear positions of transcriptionally active HIV-1 proviruses, we utilized an RNA fluorescence in situ hybridization assay and RNA stem loops that bind to fluorescently labeled bacterial protein (Bgl-mCherry) to specifically detect HIV-1 transcription sites. Initially, transcriptionally active wild-type proviruses were located closer to the nuclear envelope (NE) than expected by random chance in HeLa (∼1.4 µm) and CEM-SS T cells (∼0.9 µm). Disrupting interactions between HIV-1 capsid and host cleavage and polyadenylation specificity factor (CPSF6) resulted in localization of proviruses to lamina-associated domains (LADs) adjacent to the NE in HeLa cells (∼0.9 - 1.0 µm); however, in CEM-SS T cells, there was little or no shift toward the NE (∼0.9 µm), indicating cell-type differences in the locations of transcriptionally active proviruses. The distance from the NE was not correlated with transcriptional activity, and transcriptionally active proviruses were randomly distributed throughout the HeLa cell after several cell divisions, indicating that the intranuclear locations of the chromosomal sites of integration are dynamic. After nuclear import HIV-1 cores colocalized with nuclear speckles, nuclear domains enriched in pre-mRNA splicing factors, but transcriptionally active proviruses detected 20 h after infection were mostly located outside but near nuclear speckles, suggesting a dynamic relationship between the speckles and integration sites. Overall, these studies establish that the nuclear distribution of HIV-1 proviruses is dynamic and the distance between HIV-1 proviruses and the NE does not correlate with transcriptional activity. IMPORTANCE HIV-1 integrates its genomic DNA into the chromosomes of the infected cell, but how it selects the site of integration and the impact of their location in the 3-dimensional nuclear space is not well understood. Here, we examined the nuclear locations of proviruses 1 and 5 days after infection and found that integration sites are first located near the nuclear envelope but become randomly distributed throughout the nucleus after a few cell divisions, indicating that the locations of the chromosomal sites of integration that harbor transcriptionally active proviruses are dynamic. We also found that the distance from the nuclear envelope to the integration site is cell-type dependent and does not correlate with proviral transcription activity. Finally, we observed that HIV-1 cores were localized to nuclear speckles shortly after nuclear import, but transcriptionally active proviruses were located adjacent to nuclear speckles. Overall, these studies provide insights into HIV-1 integration site selection and their effect on transcription activities.

Plasma Membrane Anchoring and Gag:Gag Multimerization on Viral RNA Are Critical Properties of HIV-1 Gag Required To Mediate Efficient Genome Packaging.

Human immunodeficiency virus type 1 (HIV-1) Gag selects and packages the HIV RNA genome during virus assembly. However, HIV-1 RNA constitutes only a small fraction of the cellular RNA. Although Gag exhibits a slight preference to viral RNA, most of the cytoplasmic Gag proteins are associated with cellular RNAs. Thus, it is not understood how HIV-1 achieves highly efficient genome packaging. We hypothesize that besides RNA binding, other properties of Gag are important for genome packaging. Many Gag mutants have assembly defects that preclude analysis of their effects on genome packaging. To bypass this challenge, we established complementation systems that separate the particle-assembling and RNA-binding functions of Gag: we used a set of Gag proteins to drive particle assembly and an RNA-binding Gag to package HIV-1 RNA. We have developed two types of RNA-binding Gag in which packaging is mediated by the authentic nucleocapsid (NC) domain or by a nonviral RNA-binding domain. We found that in both cases, mutations that affect the multimerization or plasma membrane anchoring properties of Gag reduce or abolish RNA packaging. These mutant Gag can coassemble into particles but cannot package the RNA genome efficiently. Our findings indicate that HIV-1 RNA packaging occurs at the plasma membrane and RNA-binding Gag needs to multimerize on RNA to encapsidate the viral genome. IMPORTANCE To generate infectious virions, HIV-1 must package its full-length RNA as the genome during particle assembly. HIV-1 Gag:RNA interactions mediate genome packaging, but the mechanism remains unclear. Only a minor portion of the cellular RNA is HIV-1 RNA, and most of the RNAs associated with cytoplasmic Gag are cellular RNAs. However, >94% of the HIV-1 virions contain viral RNA genome. We posited that, besides RNA binding, other properties of Gag contribute to genome packaging. Using two complementation systems, we examined features of Gag that are important for genome packaging. We found that the capacities for Gag to multimerize and to anchor at the plasma membrane are critical for genome packaging. Our results revealed that Gag needs to multimerize on viral RNA at the plasma membrane in order to package RNA genome.

Selective packaging of HIV-1 RNA genome is guided by the stability of 5' untranslated region polyA stem.

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.

HIVIntact: a python-based tool for HIV-1 genome intactness inference.

The characterisation of the HIV-1 reservoir, which consists of replication-competent integrated proviruses that persist on antiretroviral therapy (ART), is made difficult by the rarity of intact proviruses relative to those that are defective. While the only conclusive test for the replication-competence of HIV-1 proviruses is carried out in cell culture, genetic characterization of genomes by near full-length (NFL) PCR and sequencing can be used to determine whether particular proviruses have insertions, deletions, or substitutions that render them defective. Proviruses that are not excluded by having such defects can be classified as genetically intact and, possibly, replication competent. Identifying and quantifying proviruses that are potentially replication-competent is important for the development of strategies towards a functional cure. However, to date, there are no programs that can be incorporated into deep-sequencing pipelines for the automated characterization and annotation of HIV genomes. Existing programs that perform this work require manual intervention, cannot be widely installed, and do not have easily adjustable settings. Here, we present HIVIntact, a python-based software tool that characterises genomic defects in NFL HIV-1 sequences, allowing putative intact genomes to be identified in-silico. Unlike other applications that assess the genetic intactness of HIV genomes, this tool can be incorporated into existing sequence-analysis pipelines and applied to large next-generation sequencing datasets.

HIV-1 cores retain their integrity until minutes before uncoating in the nucleus.

We recently reported that HIV-1 cores that retained >94% of their capsid (CA) protein entered the nucleus and disassembled (uncoated) near their integration site <1.5 h before integration. However, whether the nuclear capsids lost their integrity by rupturing or a small loss of CA before capsid disassembly was unclear. Here, we utilized a previously reported vector in which green fluorescent protein is inserted in HIV-1 Gag (iGFP); proteolytic processing efficiently releases GFP, some of which remains trapped inside capsids and serves as a fluid phase content marker that is released when the capsids lose their integrity. We found that nuclear capsids retained their integrity until shortly before integration and lost their GFP content marker ∼1 to 3 min before loss of capsid-associated mRuby-tagged cleavage and polyadenylation specificity factor 6 (mRuby-CPSF6). In contrast, loss of GFP fused to CA and mRuby-CPSF6 occurred simultaneously, indicating that viral cores retain their integrity until just minutes before uncoating. Our results indicate that HIV-1 evolved to retain its capsid integrity and maintain a separation between macromolecules in the viral core and the nuclear environment until uncoating occurs just before integration. These observations imply that intact HIV-1 capsids are imported through nuclear pores; that reverse transcription occurs in an intact capsid; and that interactions between the preintegration complex and LEDGF/p75, and possibly other host factors that facilitate integration, must occur during the short time period between loss of capsid integrity and integration.

Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CLpro Inhibitors.

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.

Specific Guanosines in the HIV-2 Leader RNA are Essential for Efficient Viral Genome Packaging.

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.

Impact of Nuclear Export Pathway on Cytoplasmic HIV-1 RNA Transport Mechanism and Distribution.

HIV-1 full-length RNA (referred to as HIV-1 RNA here) serves as the viral genome in virions and as a template for Gag/Gag-Pol translation. We previously showed that HIV-1 RNA, which is exported via the CRM1 pathway, travels in the cytoplasm mainly through diffusion. A recent report suggested that the export pathway used by retroviral RNA could affect its cytoplasmic transport mechanism and localization. HIV-1 RNA export is directed by the viral protein Rev and the cis-acting element, Rev response element (RRE). When Rev/RRE is replaced with the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV), HIV-1 RNA is exported through the NXF1 pathway. To determine the effects of the export pathway on HIV-1 RNA, we tracked individual RNAs and found that the vast majority of cytoplasmic HIV-1 RNAs travel by diffusion regardless of the export pathway. However, CTE-containing HIV-1 RNA diffuses at a rate slower than that of RRE-containing HIV-1 RNA. Using in situ hybridization, we analyzed the subcellular localizations of HIV-1 RNAs in cells expressing a CTE-containing and an RRE-containing provirus. We found that these two types of HIV-1 RNAs have similar subcellular distributions. HIV-1 RNA exported through the NXF1 pathway was suggested to cluster near centrosomes. To investigate this possibility, we measured the distances between individual RNAs to the centrosomes and found that HIV-1 RNAs exported through different pathways do not exhibit significantly different distances to centrosomes. Therefore, HIV-1 RNAs exported through CRM1 and NXF1 pathways use the same RNA transport mechanism and exhibit similar cytoplasmic distributions.IMPORTANCE The unspliced HIV-1 full-length RNA (HIV-1 RNA) is packaged into virions as the genome and is translated to generate viral structural proteins and enzymes. To serve these functions, HIV-1 RNA must be exported from the nucleus to the cytoplasm. It was recently suggested that export pathways used by HIV-1 RNA could affect its cytoplasmic transport mechanisms and distribution. In the current report, we examined the HIV-1 RNA transport mechanism by following the movement of individual RNAs and identifying the distribution of RNA using in situ hybridization. Our results showed that whether exported by the CRM1 or NXF1 pathway, HIV-1 RNAs mainly use diffusion for cytoplasmic travel. Furthermore, HIV-1 RNAs exported using the CRM1 or NXF1 pathway are well mixed in the cytoplasm and do not display export pathway-specific clustering near centrosomes. Thus, the export pathways used by HIV-1 RNAs do not alter the cytoplasmic transport mechanisms or distribution.

Unpaired Guanosines in the 5' Untranslated Region of HIV-1 RNA Act Synergistically To Mediate Genome Packaging.

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 <2-fold defects in packaging. However, when mutations at different NC binding sites were combined, severe defects were observed. Furthermore, combining the mutations resulted in synergistic defects in RNA packaging, suggesting redundancy in Gag-RNA interactions and a requirement for multiple Gag binding on viral RNA during HIV-1 genome encapsidation.IMPORTANCE HIV-1 must package its RNA genome during virus assembly to generate infectious viruses. To better understand how HIV-1 packages its RNA genome, we examined the roles of RNA elements identified as binding sites for NC, a Gag-derived RNA-binding protein. Our results demonstrate that binding sites within stem-loop 1 of the 5' untranslated region play important roles in genome packaging. Although mutating one or two NC-binding sites caused only mild defects in packaging, mutating multiple sites resulted in severe defects in genome encapsidation, indicating that unpaired guanosines act synergistically to promote packaging. Our results suggest that Gag-RNA interactions occur at multiple RNA sites during genome packaging; furthermore, there are functionally redundant binding sites in viral RNA.

Visualizing the translation and packaging of HIV-1 full-length RNA.

HIV-1 full-length RNA (HIV-1 RNA) plays a central role in viral replication, serving as a template for Gag/Gag-Pol translation and as a genome for the progeny virion. To gain a better understanding of the regulatory mechanisms of HIV-1 replication, we adapted a recently described system to visualize and track translation from individual HIV-1 RNA molecules in living cells. We found that, on average, half of the cytoplasmic HIV-1 RNAs are being actively translated at a given time. Furthermore, translating and nontranslating RNAs are well mixed in the cytoplasm; thus, Gag biogenesis occurs throughout the cytoplasm without being constrained to particular subcellular locations. Gag is an RNA binding protein that selects and packages HIV-1 RNA during virus assembly. A long-standing question in HIV-1 gene expression is whether Gag modulates HIV-1 RNA translation. We observed that despite its RNA-binding ability, Gag expression does not alter the proportion of translating HIV-1 RNA. Using single-molecule tracking, we found that both translating and nontranslating RNAs exhibit dynamic cytoplasmic movement and can reach the plasma membrane, the major HIV-1 assembly site. However, Gag selectively packages nontranslating RNA into the assembly complex. These studies illustrate that although HIV-1 RNA serves two functions, as a translation template and as a viral genome, individual RNA molecules carry out only one function at a time. These studies shed light on previously unknown aspects of HIV-1 gene expression and regulation.

HIV-1 uncoats in the nucleus near sites of integration.

HIV-1 capsid core disassembly (uncoating) must occur before integration of viral genomic DNA into the host chromosomes, yet remarkably, the timing and cellular location of uncoating is unknown. Previous studies have proposed that intact viral cores are too large to fit through nuclear pores and uncoating occurs in the cytoplasm in coordination with reverse transcription or at the nuclear envelope during nuclear import. The capsid protein (CA) content of the infectious viral cores is not well defined because methods for directly labeling and quantifying the CA in viral cores have been unavailable. In addition, it has been difficult to identify the infectious virions because only one of ∼50 virions in infected cells leads to productive infection. Here, we developed methods to analyze HIV-1 uncoating by direct labeling of CA with GFP and to identify infectious virions by tracking viral cores in living infected cells through viral DNA integration and proviral DNA transcription. Astonishingly, our results show that intact (or nearly intact) viral cores enter the nucleus through a mechanism involving interactions with host protein cleavage and polyadenylation specificity factor 6 (CPSF6), complete reverse transcription in the nucleus before uncoating, and uncoat <1.5 h before integration near (<1.5 µm) their genomic integration sites. These results fundamentally change our current understanding of HIV-1 postentry replication events including mechanisms of nuclear import, uncoating, reverse transcription, integration, and evasion of innate immunity.