HIV-1 Assembly Research Articles

Synchronized HIV assembly by tunable PIP2 changes reveals PIP2 requirement for stable Gag anchoring.

HIV-1 assembles at the plasma membrane (PM) of infected cells. PM association of the main structural protein Gag depends on its myristoylated MA domain and PM PI(4,5)P2. Using a novel chemical biology tool that allows rapidly tunable manipulation of PI(4,5)P2 levels in living cells, we show that depletion of PI(4,5)P2 completely prevents Gag PM targeting and assembly site formation. Unexpectedly, PI(4,5)P2 depletion also caused loss of pre-assembled Gag lattices from the PM. Subsequent restoration of PM PI(4,5)P2 reinduced assembly site formation even in the absence of new protein synthesis, indicating that the dissociated Gag molecules remained assembly competent. These results reveal an important role of PI(4,5)P2 for HIV-1 morphogenesis beyond Gag recruitment to the PM and suggest a dynamic equilibrium of Gag-lipid interactions. Furthermore, they establish an experimental system that permits synchronized induction of HIV-1 assembly leading to induced production of infectious virions by targeted modulation of Gag PM targeting.

The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV.

The interactions that occur during HIV Pr55Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC) studies, and we have revealed the thermodynamic properties of HIV assembly for the first time. Thermodynamic analysis showed that the binding between RNA and HIV Pr55Gag is an energetically favourable reaction (ΔG<0) that is further enhanced by the oligomerization of Pr55Gag. The change in enthalpy (ΔH) widens sequentially from: (1) Pr55Gag-Psi RNA binding during HIV genome selection; to (2) Pr55Gag-Guanosine Uridine (GU)-containing RNA binding in cytoplasm/plasma membrane; and then to (3) Pr55Gag-Adenosine(A)-containing RNA binding in immature HIV. These data imply the stepwise increments of heat being released during HIV biogenesis may help to facilitate the process of viral assembly. By mimicking the interactions between A-containing RNA and oligomeric Pr55Gag in immature HIV, it was noted that a p6 domain truncated Pr50Gag Δp6 is less efficient than full-length Pr55Gag in this thermodynamic process. These data suggest a potential unknown role of p6 in Pr55Gag-Pr55Gag oligomerization and/or Pr55Gag-RNA interaction during HIV assembly. Our data provide direct evidence on how nucleic acid sequences and the oligomeric state of Pr55Gag regulate HIV assembly.

HIV-1 Matrix-31 Membrane Binding Peptide Interacts Differently with Membranes Containing PS vs. PI(4,5)P2

Efficient assembly of HIV-1 at the plasma membrane (PM) of the T-cell specifically requires PI(4,5)P2. It was previously shown that a highly basic region (HBR) of the matrix protein (MA) on the Gag precursor polyprotein Pr55Gag is required for membrane association. MA is N-terminally myristoylated, which enhances its affinity to membranes. In this work we used X-ray scattering and neutron reflectivity to determine how the physical properties and structure of lipid bilayers respond to the addition of binding domain peptides, either in the myristoylated form (MA31myr) or without the myristoyl group (MA31). Neutron reflectivity measurements showed the peptides predominantly located in the hydrocarbon interior. Diffuse X-ray scattering showed differences in membrane properties upon addition of peptides and the direction of the changes depended on lipid composition. The PI(4,5)P2-containing bilayers softened, thinned and became less ordered as peptide concentration increased. In contrast, POPS-containing bilayers with equivalent net charge first stiffened, thickened and became more ordered with increasing peptide concentration. As softening the host cell's PM upon contact with the protein lowers the free energy for membrane restructuring, thereby potentially facilitating budding of viral particles, our results suggest that the role of PI(4,5)P2 in viral assembly goes beyond specific stereochemical membrane binding. These studies reinforce the importance of lipids in virology.

The tumour suppressor APC promotes HIV-1 assembly via interaction with Gag precursor protein

Diverse cellular proteins and RNAs are tightly regulated in their subcellular localization to exert their local function. Here we report that the tumour suppressor adenomatous polyposis coli protein (APC) directs the localization and assembly of human immunodeficiency virus (HIV)-1 Gag polyprotein at distinct membrane components to enable the efficient production and spread of infectious viral particles. A proteomic analysis and subsequent biomolecular interaction assay reveals that the carboxyl terminus of APC interacts with the matrix region of Gag. Ectopic expression of APC, but not its familial adenomatous polyposis-related truncation mutant, prominently enhances HIV-1 production. Conversely, the depletion of APC leads to a significant decrease in membrane targeting of viral components, resulting in the severe loss of production of infectious virions. Furthermore, APC promotes the directional assembly of viral components at virological synapses, thereby facilitating cell-to-cell viral transmission. These findings reveal an unexpected role of APC in the directional spread of HIV-1.

Fully Automated Targeted Confocal and Single-Molecule Localization Microscopy.

Single-molecule localization microscopy (SMLM) enables imaging of biological structures in the nanometre range. Long measurement times are the consequence of this kind of microscopy due to the need of acquiring thousands of images. We built a setup that automatically detects target structures using confocal microscopy and images them with SMLM. Utilizing the Konstanz Information Miner (KNIME), we were able to connect a confocal microscope with an SMLM unit for targeted screening. In this process, we developed KNIME plugins to communicate with the microscope components and combined them to a workflow. Thus, measuring biological nanometre-sized structures in a sufficient number to get statistical significance becomes feasible. For proof of principle HIV-1 assembly complexes in HeLa cells derived from transfection of replication deficient viral construct were imaged by a fully automated screen.

The Anti-HIV-1 Cytidine Deaminase APOBEC3G Is a Cellular Site-Specific RNA Editing Enzyme

Background:The APOBEC3 (A3) family of cytidine deaminases in primates is comprised of seven homologous enzymes that are structurally related to the RNA editing enzyme APOBEC1. APOBEC3G (A3G) is a restriction factor for HIV-1 and endogenous retroviruses, and is highly expressed in T lymphocytes. Encapsidation of A3G into HIV-1 particles is essential for its antiviral activity which leads to hypermutation of its cDNA in target cells, and requires RNA binding by A3G to form a ribonucleoprotein complex with viral proteins. A3G can also reduce HIV-1 production in producer cells independently of its DNA deaminating activity. A3G has homologous N-and C-terminal catalytic domains (NTD and CTD) but only the CTD is active for deamination of ssDNAs. The zinc-coordinating catalytic residues as well as non-catalytic residues in A3G-NTD are known to bind RNA and this interaction is required for A3G's binding to the HIV-1 nucleocapsid for recruitment into nascent virions as well as for A3G dimerization. A3G binds to DNA and RNA substrates with similar affinity. Thus far, studies have demonstrated DNA deamination by A3G whereas deamination has not been observed in HIV-1 RNA or synthetic RNA oligonucleotides, thereby, ruling out the RNA editing function of A3G. We recently described that the structurally related enzyme A3A induces widespread site-specific C-to-U RNA editing of cellular transcripts in pro-inflammatory macrophages and in monocytes exposed to hypoxia and/or interferons. We hypothesized that A3G may also have RNA editing function, which may play a role in HIV-1 restriction.Methods:To determine if A3G is capable of RNA editing, we transiently overexpressed the protein in 293T cells, a model routinely used by various labs to study A3G function and its mode of HIV-1 restriction, and then performed transcriptome-wide RNA sequencing (RNA-Seq), Sanger sequencing and site-directed mutagenesis.Results: RNA sequencing analysis showed site-specific RNA editing in hundreds of genes' transcripts, including approximately 200 genes that acquire protein recoding. The transcripts edited by A3G are largely distinct from those edited by A3A. We find that several host genes including NMT1, CHMP4B, MAPK1, ACIN1, MED1, NFAT5, RBM14 which areinvolved in HIV-1 infection acquire pathogenic recoding RNA mutations by A3G-mediated RNA editing. By performing Sanger sequencing of PCR-amplified cDNA, we validated site-specific, non-synonymous C-to-U RNA editing for 21 of 21 (100%) tested sites in 20 genes that we had selected for experimental confirmation. As expected no genomic mutations were seen in the DNA sequences corresponding to the RNA-edited sites in 11 tested genes. The discovery of A3G's RNA editing function prompted us to study the role of the N-terminal domain in RNA editing. We made mutations in the zinc-coordinating and non-catalytic residues in both N-terminal and C-terminal domains of A3G. We demonstrate that mutating zinc-coordinating residues in either N- and C-terminal domains of A3G in 293T cells greatly reduce or abolish editing in its target transcripts.Conclusions: We demonstrate a novel RNA editing function for the A3G cytidine deaminase. Our study shows that the RNAs of genes involved in HIV-1 replication, assembly, transcription and infectivity are targets of A3G-mediated RNA editing. This result raises the possibility that the editing of host transcripts may be a novel mechanism by which HIV-1 infection is inhibited by A3G. Our findings suggest a previously unrecognized role for the N-terminal domain of A3G in RNA editing. A3G is the second of the seven members of the APOBEC3 family of cytidine deaminases and the first two-domain cytidine deaminase for which a previously unrecognized RNA editing activity has been discovered. It suggests that other APOBEC3 proteins may also possess hitherto unknown RNA editing activity that may underpin some of their biological roles. Our findings have the potential to significantly expand on the role of C-to-U RNA editing in epitranscriptomic regulation in T lymphocytes, define specific gene targets of A3G-mediated RNA editing and open new avenues of inquiry on the functions of APOBEC3 genes in HIV-1 restriction. DisclosuresNo relevant conflicts of interest to declare.

Mapping out the intricate relationship of the HIV envelope protein and the membrane environment

The HIV gp160 envelope fusion protein is situated in the viral membrane and mediates virus entry into its host cell. Increasing evidence suggests that virtually all parts of the HIV envelope are structurally and functionally dependent on membranes. Protein-lipid interactions and membrane properties influence the dynamics of a manifold of gp160 biological activities such as membrane fusion, immune suppression and gp160 incorporation into virions during HIV budding and assembly. In the following we will summarize our current understanding of this interdependence between membrane interaction, structural conformation and functionality of the different gp160 domains. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

HIV-1 matrix-31 membrane binding peptide interacts differently with membranes containing PS vs. PI(4,5)P2

Efficient assembly of HIV-1 at the plasma membrane (PM) of the T-cell specifically requires PI(4,5)P2. It was previously shown that a highly basic region (HBR) of the matrix protein (MA) on the Gag precursor polyprotein Pr55Gag is required for membrane association. MA is N-terminally myristoylated, which enhances its affinity to membranes. In this work we used X-ray scattering and neutron reflectivity to determine how the physical properties and structure of lipid bilayers respond to the addition of binding domain peptides, either in the myristoylated form (MA31myr) or without the myristoyl group (MA31). Neutron reflectivity measurements showed the peptides predominantly located in the hydrocarbon interior. Diffuse X-ray scattering showed differences in membrane properties upon addition of peptides and the direction of the changes depended on lipid composition. The PI(4,5)P2-containing bilayers softened, thinned and became less ordered as peptide concentration increased. In contrast, POPS-containing bilayers with equivalent net charge first stiffened, thickened and became more ordered with increasing peptide concentration. As softening the host cell's PM upon contact with the protein lowers the free energy for membrane restructuring, thereby potentially facilitating budding of viral particles, our results suggest that the role of PI(4,5)P2 in viral assembly goes beyond specific stereochemical membrane binding. These studies reinforce the importance of lipids in virology.

An Essential Role of INI1/hSNF5 Chromatin Remodeling Protein in HIV-1 Posttranscriptional Events and Gag/Gag-Pol Stability.

ABSTRACTINI1/hSNF5/SMARCB1/BAF47 is an HIV-specific integrase (IN)-binding protein that influences HIV-1 transcription and particle production. INI1 binds to SAP18 (Sin3a-associated protein, 18 kDa), and both INI1 and SAP18 are incorporated into HIV-1 virions. To determine the significance of INI1 and the INI1-SAP18 interaction during HIV-1 replication, we isolated a panel of SAP18-interaction-defective (SID)-INI1 mutants using a yeast reverse two-hybrid screen. The SID-INI1 mutants, which retained the ability to bind to IN, cMYC, and INI1 but were impaired for binding to SAP18, were tested for their effects on HIV-1 particle production. SID-INI1 dramatically reduced the intracellular Gag/Gag-Pol protein levels and, in addition, decreased viral particle production. The SID-INI1-mediated effects were less dramatic in trans complementation assays using IN deletion mutant viruses with Vpr-reverse transcriptase (RT)-IN. SID-INI1 did not inhibit long-terminal-repeat (LTR)-mediated transcription, but it marginally decreased the steady-state gag RNA levels, suggesting a posttranscriptional effect. Pulse-chase analysis indicated that in SID-INI1-expressing cells, the pr55Gag levels decreased rapidly. RNA interference analysis indicated that small hairpin RNA (shRNA)-mediated knockdown of INI1 reduced the intracellular Gag/Gag-Pol levels and further inhibited HIV-1 particle production. These results suggest that SID-INI1 mutants inhibit multiple stages of posttranscriptional events of HIV-1 replication, including intracellular Gag/Gag-Pol RNA and protein levels, which in turn inhibits assembly and particle production. Interfering INI1 leads to a decrease in particle production and Gag/Gag-Pol protein levels. Understanding the role of INI1 and SAP18 in HIV-1 replication is likely to provide novel insight into the stability of Gag/Gag-Pol, which may lead to the development of novel therapeutic strategies to inhibit HIV-1 late events. IMPORTANCE Significant gaps exist in our current understanding of the mechanisms and host factors that influence HIV-1 posttranscriptional events, including gag RNA levels, Gag/Gag-Pol protein levels, assembly, and particle production. Our previous studies suggested that the IN-binding host factor INI1 plays a role in HIV-1 assembly. An ectopically expressed minimal IN-binding domain of INI1, S6, potently and selectively inhibited HIV-1 Gag/Gag-Pol trafficking and particle production. However, whether or not endogenous INI1 and its interacting partners, such as SAP18, are required for late events was unknown. Here, we report that endogenous INI1 and its interaction with SAP18 are necessary to maintain intracellular levels of Gag/Gag-Pol and for particle production. Interfering INI1 or the INI1-SAP18 interaction leads to the impairment of these processes, suggesting a novel strategy for inhibiting posttranscriptional events of HIV-1 replication.

Molecular Determinants Directing HIV-1 Gag Assembly to Virus-Containing Compartments in Primary Macrophages.

The subcellular sites of HIV-1 assembly, determined by the localization of the structural protein Gag, vary in a cell-type-dependent manner. In T cells and transformed cell lines used as model systems, HIV-1 assembles at the plasma membrane (PM). The binding and localization of HIV-1 Gag to the PM are mediated by the interaction between the matrix (MA) domain, specifically the highly basic region, and a PM-specific acidic phospholipid, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. In primary macrophages, prominent accumulation of assembling or assembled particles is found in the virus-containing compartments (VCCs), which largely consist of convoluted invaginations of the PM. To elucidate the molecular mechanism of HIV-1 Gag targeting to the VCCs, we examined the impact of overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P2, in primary macrophages. We found that the VCC localization and virus release of HIV-1 are severely impaired upon 5ptaseIV overexpression, suggesting an important role for the MA-PI(4,5)P2 interaction in HIV-1 assembly in primary macrophages. However, our analysis of HIV-1 Gag derivatives with MA changes showed that this interaction contributes to Gag membrane binding but is dispensable for specific targeting of Gag to the VCCs per se We further determined that deletion of the NC domain abolishes VCC-specific localization of HIV-1 Gag. Notably, HIV-1 Gag localized efficiently to the VCCs when the NC domain was replaced with a leucine zipper dimerization motif that promotes Gag multimerization. Altogether, our data revealed that targeting of HIV-1 Gag to the VCCs requires NC-dependent multimerization. In T cells and model cell lines, HIV-1 Gag localizes to the PM in a manner dependent on the MA-PI(4,5)P2 interaction. On the other hand, in primary macrophages, HIV-1 Gag localizes to convoluted intracellular membrane structures termed virus-containing compartments (VCCs). Although these compartments have been known for decades, and despite the implication of viruses in VCCs being involved in virus reservoir maintenance and spread, the viral determinant(s) that promotes Gag targeting to VCCs is unknown. In this study, we found that the MA-PI(4,5)P2 interaction facilitates efficient Gag membrane binding in macrophages but is not essential for Gag targeting to VCCs. Rather, our results revealed that NC-dependent multimerization promotes VCC targeting. Our findings highlight the differential roles played by MA and NC in HIV-1 Gag membrane binding and targeting and suggest a multimerization-dependent mechanism for Gag trafficking in primary macrophages similar to that for Gag localization to uropods in polarized T cells.

An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation.

Immature HIV-1 assembles at and buds from the plasma membrane before proteolytic cleavage of the viral Gag polyprotein induces structural maturation. Maturation can be blocked by maturation inhibitors (MIs), thereby abolishing infectivity. The CA (capsid) and SP1 (spacer peptide 1) region of Gag is the key regulator of assembly and maturation and is the target of MIs. We applied optimized cryo-electron tomography and subtomogram averaging to resolve this region within assembled immature HIV-1 particles at 3.9 angstrom resolution and built an atomic model. The structure reveals a network of intra- and intermolecular interactions mediating immature HIV-1 assembly. The proteolytic cleavage site between CA and SP1 is inaccessible to protease. We suggest that MIs prevent CA-SP1 cleavage by stabilizing the structure, and MI resistance develops by destabilizing CA-SP1.

Crystal structure of an HIV assembly and maturation switch.

Virus assembly and maturation proceed through the programmed operation of molecular switches, which trigger both local and global structural rearrangements to produce infectious particles. HIV-1 contains an assembly and maturation switch that spans the C-terminal domain (CTD) of the capsid (CA) region and the first spacer peptide (SP1) of the precursor structural protein, Gag. The crystal structure of the CTD-SP1 Gag fragment is a goblet-shaped hexamer in which the cup comprises the CTD and an ensuing type II β-turn, and the stem comprises a 6-helix bundle. The β-turn is critical for immature virus assembly and the 6-helix bundle regulates proteolysis during maturation. This bipartite character explains why the SP1 spacer is a critical element of HIV-1 Gag but is not a universal property of retroviruses. Our results also indicate that HIV-1 maturation inhibitors suppress unfolding of the CA-SP1 junction and thereby delay access of the viral protease to its substrate.

Specific Destruction of HIV Proviral p17 Gene in T Lymphoid Cells Achieved by the Genome Editing Technology.

Recent development in genome editing technologies has enabled site-directed deprivation of a nucleotide sequence in the chromosome in mammalian cells. Human immunodeficiency (HIV) infection causes integration of proviral DNA into the chromosome, which potentially leads to re-emergence of the virus, but conventional treatment cannot delete the proviral DNA sequence from the cells infected with HIV. In the present study, the transcription activator-like effector nucleases (TALENs) specific for the HIV p17 gene were constructed, and their activities to destroy the target sequence were evaluated. SSA assay showed a high activity of a pair of p17-specific TALENs. A human T lymphoid cell line, Jurkat, was infected with a lentivirus vector followed by transfection with the TALEN–HIV by electroporation. The target sequence was destructed in approximately 10–95% of the p17 polymerase chain reaction clones, and the efficiencies depended on the Jurkat–HIV clones. Because p17 plays essential roles for assembly and budding of HIV, and this gene has relatively low nucleotide sequence diversity, genome editing procedures targeting p17 may provide a therapeutic benefit for HIV infection.

The intracellular plasma membrane-connected compartment in the assembly of HIV-1 in human macrophages.

BackgroundIn HIV-infected macrophages, newly formed progeny virus particles accumulate in intracellular plasma membrane-connected compartments (IPMCs). Although the virus is usually seen in these compartments, it is unclear whether HIV assembly is specifically targeted to IPMCs or whether some viruses may also form at the cell surface but are not detected, as particles budding from the latter site will be released into the medium.ResultsTo investigate the fidelity of HIV-1 targeting to IPMCs compared to the cell surface directly, we generated mutants defective in recruitment of the Endosomal Sorting Complexes Required for Transport (ESCRT) proteins required for virus scission. For mutants unable to bind the ESCRT-I component Tsg101, HIV release was inhibited and light and electron microscopy revealed that budding was arrested. When expressed in human monocyte-derived macrophages (MDM), these mutants formed budding-arrested, immature particles at their assembly sites, allowing us to capture virtually all of the virus budding events. A detailed morphological analysis of the distribution of the arrested viruses by immunofluorescence staining and confocal microscopy, and by electron microscopy, demonstrated that HIV assembly in MDMs is targeted primarily to IPMCs, with fewer than 5 % of budding events seen at the cell surface. Morphometric analysis of the relative membrane areas at the cell surface and IPMCs confirmed a large enrichment of virus assembly events in IPMCs.Serial block-face scanning electron microscopy of macrophages infected with a budding-defective HIV mutant revealed high-resolution 3D views of the complex organisation of IPMCs, with in excess of 15,000 associated HIV budding sites, and multiple connections between IPMCs and the cell surface.ConclusionsUsing detailed quantitative analysis, we demonstrate that HIV assembly in MDMs is specifically targeted to IPMCs. Furthermore, 3D analysis shows, for the first time, the detailed ultrastructure of an IPMC within a large cell volume, at a resolution that allowed identification of individual virus assembly events, and potential portals through which virus may be released during cell-cell transfer. These studies provide new insights to the organisation of the HIV assembly compartments in macrophages, and show how HIV particles accumulating in these protected sites may function as a virus reservoir.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0272-3) contains supplementary material, which is available to authorized users.

Requirements for nucleocapsid-mediated regulation of reverse transcription during the late steps of HIV-1 assembly.

HIV-1 is a retrovirus replicating within cells by reverse transcribing its genomic RNA (gRNA) into DNA. Within cells, virus assembly requires the structural Gag proteins with few accessory proteins, notably the viral infectivity factor (Vif) and two copies of gRNA as well as cellular factors to converge to the plasma membrane. In this process, the nucleocapsid (NC) domain of Gag binds to the packaging signal of gRNA which consists of a series of stem-loops (SL1-SL3) ensuring gRNA selection and packaging into virions. Interestingly, mutating NC activates a late-occurring reverse transcription (RT) step in producer cells, leading to the release of DNA-containing HIV-1 particles. In order to decipher the molecular mechanism regulating this late RT, we explored the role of several key partners of NC, such as Vif, gRNA and the cellular cytidine deaminase APOBEC3G that restricts HIV-1 infection by targeting the RT. By studying combinations of deletions of these putative players, we revealed that NC, SL1-SL3 and in lesser extent Vif, but not APOBEC3G, interplay regulates the late RT.

Analysis of the human immunodeficiency virus-1 RNA packageome.

All retroviruses package cellular RNAs into virions. Studies of murine leukemia virus (MLV) revealed that the major host cell RNAs encapsidated by this simple retrovirus were LTR retrotransposons and noncoding RNAs (ncRNAs). Several classes of ncRNAs appeared to be packaged by MLV shortly after synthesis, as precursors to tRNAs, small nuclear RNAs, and small nucleolar RNAs were all enriched in virions. To determine the extent to which the human immunodeficiency virus (HIV-1) packages similar RNAs, we used high-throughput sequencing to characterize the RNAs within infectious HIV-1 virions produced in CEM-SS T lymphoblastoid cells. We report that the most abundant cellular RNAs in HIV-1 virions are 7SL RNA and transcripts from numerous divergent and truncated members of the long interspersed element (LINE) and short interspersed element (SINE) families of retrotransposons. We also detected precursors to several tRNAs and small nuclear RNAs as well as transcripts derived from the ribosomal DNA (rDNA) intergenic spacers. We show that packaging of a pre-tRNA requires the nuclear export receptor Exportin 5, indicating that HIV-1 recruits at least some newly made ncRNAs in the cytoplasm. Together, our work identifies the set of RNAs packaged by HIV-1 and reveals that early steps in HIV-1 assembly intersect with host cell ncRNA biogenesis pathways.

333. Intracellular Ankyrin Specific to p24 and zfp Specific to 2-LTR Are Alternatives to CCR5 Knockdown for Stem Cell Gene Therapy

HIV-1 genome integration and capsid assembly are some of the most important steps to viral life cycle. Designed scaffold proteins, zinc finger and ankyrin repeat protein, were characterized as intracellular antiviral agents that can interfere in these steps. Zinc finger protein, 2LTRZFP, plays a vital role in blocking HIV integration. This protein targets the 2-LTR-circle junction of the HIV-1 DNA but it would have no effect on the HIV-infected cells. Another protein, AnkGAG1D4, will then specifically bind to the viral capsid resulting in inhibition of HIV-1 assembly. Since only one particular scaffold protein cannot confer full protection against the virus. Combination of these two proteins became the key to enhance inhibitory function to block HIV-1 replication at early and late infection stage and also help eliminate latent viral reservoirs. The human T cell line modified by using a lentiviral vector bearing 2LTRZFP alone or combination with N-myristoylatedMyr(+)AnkGAG1D4 were protected from viral infection in HIV-1 challenge. Interestingly, the Myr(+)AnkGAG1D4 showed significantly antiviral effect in HIV-1-preinfected SupT1 cells and also possessed a broad antiviral activity against SIV, and SHIV. These results demonstrate that 2LTRZFP and Myr(+)AnkGAG1D4 proteins can be the novel anti-HIV-1 therapeutic agents for human applications.

Mutations of Conserved Residues in the Major Homology Region Arrest Assembling HIV-1 Gag as a Membrane-Targeted Intermediate Containing Genomic RNA and Cellular Proteins.

ABSTRACTThe major homology region (MHR) is a highly conserved motif that is found within the Gag protein of all orthoretroviruses and some retrotransposons. While it is widely accepted that the MHR is critical for assembly of HIV-1 and other retroviruses, how the MHR functions and why it is so highly conserved are not understood. Moreover, consensus is lacking on when HIV-1 MHR residues function during assembly. Here, we first addressed previous conflicting reports by confirming that MHR deletion, like conserved MHR residue substitution, leads to a dramatic reduction in particle production in human and nonhuman primate cells expressing HIV-1 proviruses. Next, we used biochemical analyses and immunoelectron microscopy to demonstrate that conserved residues in the MHR are required after assembling Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane. The exact point of inhibition at the plasma membrane differed depending on the specific mutation, with one MHR mutant arrested as a membrane-associated intermediate that is stable upon high-salt treatment and other MHR mutants arrested as labile, membrane-associated intermediates. Finally, we observed the same assembly-defective phenotypes when the MHR deletion or conserved MHR residue substitutions were engineered into Gag from a subtype B, lab-adapted provirus or Gag from a subtype C primary isolate that was codon optimized. Together, our data support a model in which MHR residues act just after membrane targeting, with some MHR residues promoting stability and another promoting multimerization of the membrane-targeted assembling Gag oligomer.IMPORTANCE The retroviral Gag protein exhibits extensive amino acid sequence variation overall; however, one region of Gag, termed the major homology region, is conserved among all retroviruses and even some yeast retrotransposons, although the reason for this conservation remains poorly understood. Highly conserved residues in the major homology region are required for assembly of retroviruses; however, when these residues are required during assembly is not clear. Here, we used biochemical and electron microscopic analyses to demonstrate that these conserved residues function after assembling HIV-1 Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane but before Gag has completed the assembly process. By revealing precisely when conserved residues in the major homology region are required during assembly, these studies resolve existing controversies and set the stage for future experiments aimed at a more complete understanding of how the major homology region functions.

Quantification of HIV-1 Gag Localization Within Virus Producer Cells.

Trafficking of newly synthesized Gag protein to the plasma membrane is one of the important steps during HIV-1 assembly. It requires participation of both viral and cellular determinants. Several techniques have been used to measure the amount of Gag that is associated with plasma membrane. Here we describe a microscopy-based method to estimate the distribution of Gag protein within the producer cell. This method can be used in conjunction with other biochemical techniques to quantify the distribution of Gag within a virus-producing cell and its accumulation at the plasma membrane. Since this method is microscopy based, it allows one to quantitate Gag across the cytoplasm, from the nuclear periphery to plasma membrane, at the single-cell level.

Analysis of ABCA1 and Cholesterol Efflux in HIV-Infected Cells.

Cholesterol is an essential component of the cellular membranes and, by extension, of the HIV envelope membrane, which is derived from the host cell plasma membrane. Depletion of the cellular cholesterol has an inhibitory effect on HIV assembly, reduces infectivity of the produced virions, and makes the cell less susceptible to HIV infection. It is not surprising that the virus has evolved to gain access to cellular proteins regulating cholesterol metabolism. One of the key mechanisms used by HIV to maintain high levels of cholesterol in infected cells is Nef-mediated inhibition of cholesterol efflux and the cholesterol transporter responsible for this process, ABCA1. In this chapter, we describe methods to investigate these effects of HIV-1 infection.