Structure, Function, and Interactions of the HIV-1 Capsid Protein.

After budding, the human immunodeficiency virus (HIV) must 'mature' into an infectious viral particle. Viral maturation requires proteolytic processing of the Gag polyprotein at the matrix-capsid junction, which liberates the capsid (CA) domain to condense from the spherical protein coat of the immature virus into the conical core of the mature virus. We propose that upon proteolysis, the amino-terminal end of the capsid refolds into a beta-hairpin/helix structure that is stabilized by formation of a salt bridge between the processed amino-terminus (Pro1) and a highly conserved aspartate residue (Asp51). The refolded amino-terminus then creates a new CA-CA interface that is essential for assembling the condensed conical core. Consistent with this model, we found that recombinant capsid proteins with as few as four matrix residues fused to their amino-termini formed spheres in vitro, but that removing these residues refolded the capsid amino-terminus and redirected protein assembly from spheres to cylinders. Moreover, point mutations throughout the putative CA-CA interface blocked capsid assembly in vitro, core assembly in vivo and viral infectivity. Disruption of the conserved amino-terminal capsid salt bridge also abolished the infectivity of Moloney murine leukemia viral particles, suggesting that lenti- and oncoviruses mature via analogous pathways.

Human Immunodeficiency virus type 1 (HIV-1) is a lentivirus that infects non-dividing cells of the immune system. In non-dividing cells, nuclear import of the viral genome occurs through the nuclear pore complex (NPC), a large macromolecular assembly that forms a channel of ~40 nm across the nuclear envelope. This process requires interactions between NPC components known as nucleoporins and CA (capsid) proteins of HIV-1 post-fusion complexes. In mature HIV-1 virions, multiple copies of CA assemble into a lattice of hexamers and pentamers, forming a cone-shaped capsid core of ~60 nm in width and ~110 nm in length encasing the viral genome. Because of its large size, it was generally believed that the capsid core entirely or partially disassembled prior to its translocation through the NPC. However, nuclear entry and uncoating are rare events and challenging to characterize and therefore subject to a long-standing debate. Here, cryo-electron tomography (cryo-ET) on cryo-focused ion beam (FIB) milled T-cells was combined with subtomogram averaging (SA) to study the ultrastructure of HIV-1 capsids in the process of nuclear entry. Using a HIV-1 variant arrested at the nuclear pore due to a defect in binding of the CA lattice to the host cell protein CPSF6, snapshots of HIV-1 nuclear entry at multiple stages were captured. Reverse transcription-competent HIV-1 complexes were identified in the cytosol, docking to and within the NPC, and in the nucleoplasm of T cells. Surprisingly, in the cytosol and at the NPC, the viral complexes retained cone-shaped capsids highly resembling in size and geometry the mature capsid cores within intact virions. The density at their interior suggested that they were associated with the viral genome. The cone-shape capsids deeply enter into the NPC central channel with their narrow ends. The hexameric lattices of these capsids were intact or nearly intact. These findings argued against the current models of uncoating in the cytosol or at the NPC and rather supported translocation of intact HIV-1 capsids through the NPC. Instead, HIV-1 capsids uncoat in the nucleus of T cells. Inside the nucleus, tube-shape fragments still containing few lattice elements were observed. The lack of density at their inside suggested that the viral genome was released from these complexes. Uncoating may thus occur through the partial opening and remodeling of the hexameric lattices of HIV-1 capsids, rather than a step-wise disassembly. To address how an intact HIV-1 capsid can enter the central channel of NPC, the human NPC from T cells was structurally analyzed in cellulo by SA. The NPC overall structure was not altered upon infection but rather dilated in comparison to the structure previously obtained from purified envelopes. The diameter of the central channel of NPC in T cells corresponded to ~64 nm, suggesting that the translocation of intact HIV-1 capsid through the NPC is geometrically possible. The dilated conformation of NPCs in T cells is independent of HIV-1 infection, but represents a physiological condition of actively transporting NPCs.

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein binds to cyclophilin A and incorporates this cellular peptidyl prolyl-isomerase into virions. Disruption of cyclophilin A incorporation, either by gag mutations or by cyclosporine A, inhibits virion infectivity, indicating that cyclophilin A plays an essential role in the HIV-1 life cycle. Using assays for packaging of cyclophilin A into virions and for viral replication sensitivity to cyclosporine A, as well as information gleaned from the alignment of Gag residues encoded by representative viral isolates, we demonstrate that of the five lineages of primate immunodeficiency viruses, only HIV-1 requires cyclophilin A for replication. Cloned viral isolates from clades A, B, and D of HIV-1 group M, as well as a phylogenetically related isolate from chimpanzee, all require cyclophilin A for replication. In contrast, the replication of two outlier (group O) HIV-1 isolates is unaffected by concentrations of cyclosporine A which disrupt cyclophilin A incorporation into virions, indicating that these viruses are capable of replicating independently of cyclophilin A. These studies identify the first phenotypic difference between HIV-1 group M and group O and are consistent with phylogenetic studies suggesting that the two HIV-1 groups were introduced into human populations via separate zoonotic transmission events.

Human immunodeficiency virus type 1 (HIV-1) proviral mutants that lack viral regulatory genes are unable to replicate unless rescued by complementation in trans. Structurally intact virus can be produced by infecting recombinant cell lines expressing the deficient genes. A HIV-1 mutant functionally defective in tat and rev (vIIIB delta Tat/Rev), which replicates only in a recombinant T-cell line expressing tat and rev (CEMTART), is described in this report. Infection of the CEMTART cell line with vIIIB delta Tat/Rev permits the complete HIV-1 life cycle, including cytopathology, decreased expression of CD4, and production of viral structural proteins, to be biologically contained. Culture supernatants from infected CEMTART contain virus that is able to replicate only in uninfected CEMTART. No reversion of vIIIB delta Tat/Rev to wild-type HIV-1 was observed as measured either by sequencing proviral vIIIB delta Tat/Rev or by detecting the ability of vIIIB delta Tat/Rev to replicate in CEM or activated CD4-bearing T lymphocytes. Defective HIV-1 mutants produced by trans complementation of essential genes permit infection and analysis of defined genotypes on cellular function and phenotype. Authentic HIV-1 structural proteins and infected cells can be prepared in mass, and agents that interfere with the HIV-1 life cycle can be studied on a large scale with minimum risk of exposing workers to virulent HIV-1.

Human immunodeficiency virus type 1 (HIV-1) acquires several host cell membrane proteins when it buds from infected cells. To study the effect of virally incorporated host-derived ICAM-1 glycoproteins on the biology of HIV-1, we have developed a transient expression system that has enabled us to produce virus particles differing only in the absence or the presence of virion-bound ICAM-1. By using a single-round infection assay based on an ICAM-1-negative target T-cell line stably transfected with an HIV-1 long terminal repeat driven luciferase gene construct, we have been able to demonstrate that the acquisition of host-derived ICAM-1 by HIV-1 has functional significance, since it leads to a pronounced increase in viral infectivity (4.6- to 9.8-fold) in an ICAM-1/LFA-1-dependent fashion, as shown by blocking with anti-ICAM-1 and -LFA-1 antibodies. The same potentiating effect on viral infectivity was also observed with monocytoid cells. Studies of the kinetics of infection revealed that the positive effect mediated by virally embedded host cell membrane ICAM-1 is due to an increase in the efficiency of early steps in the viral life cycle. These results provide new insights into how incorporation of host proteins can modulate the biological properties of HIV-1. Our findings have direct clinical relevance, considering that ICAM-1 is expressed on the surface of virus-infected cells and, more importantly, that host-derived ICAM-1 has been shown to be acquired by clinical HIV-1 isolates grown on primary mononuclear cells. These data justify a more complete analysis of the other putative role(s) that virally incorporated ICAM-1 may play in the life cycle of HIV-1, for example, at the level of neutralization sensitivity.

HIV-1 incorporates and proteolytically processes human NDR1 and NDR2 serine-threonine kinases

Previous findings indicated that HLA-DR is probably one of the most abundant cellular constituents incorporated within the human immunodeficiency virus type 1 (HIV-1) envelope. Given that the life-cycle of HIV-1 has been reported to be modulated by virion-bound host HLA-DR, an improved version of a virus capture technique was developed to assess the degree of HLA-DR incorporation in several clinical isolates of HIV-1 derived from primary human peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDM). Analysis of virus stocks purified from PBMCs and MDM indicated that primary isolates of HIV-1 bearing distinct tropism (i.e. T-, macrophage-, and dual-tropic) all incorporate host cell membrane HLA-DR protein. The amount of incorporated HLA-DR varies among the primary HIV-1 isolates tested. Propagation of some clinical HIV-1 isolates in either autologous PBMCs or MDM resulted in differential incorporation of virion-bound cellular HLA-DR depending on the nature of the virus producer cells. Differences in the degree of HLA-DR incorporation were also noticed when macrophage-tropic isolates of HIV-1 were produced in MDM from different donors. Altogether these data show that the efficiency of HLA-DR incorporation into the envelope of primary isolates of HIV-1 is a multifactorial phenomenon since it is affected by the virus isolate itself, the nature of host cells (i.e. PBMCs or MDM) and the donor source.

Many viruses have evolved novel means of exploiting host defense mechanisms for their own survival. This exploitation may be best exemplified by the interrelationships between certain viruses and the host cytokine networks. Many viruses, including the human immunodeficiency virus type-1 (HIV-1), rely on the liberation and cellular action of host immune cytokines to expand their host cell range, to regulate their cellular expression, and to maintain their dormant state until the proper extracellular conditions arise. As again exemplified by HIV-1, viruses may also take an active role regulating cytokine expression and cell surface cytokine receptors. Because the viral life cycle, and in particular the HIV-1 life cycle, is so intertwined with cytokine regulatory networks, these networks represent potential points for therapeutic intervention. As our understanding of cellular cytokine pathways involved in viral infection and replication continues to expand, so too will our ability to design rational anti-viral therapies to alter multiple steps along the viral life cycle.

The latency of human immunodeficiency virus type 1 (HIV-1) is a major barrier to achieving an HIV-1 cure, as antiretroviral therapy does not target the latent virus. Virus-host interactions play an essential role in various stages of the HIV-1 lifecycle. Exploring the interaction between host factors and HIV-1 infection is critical for developing new HIV-1 treatment strategies. Yes-associated protein (YAP) is a key co-transcription factor in the Hippo signaling pathway, which regulates the occurrence and development of various diseases, including cellular metabolism, cancer, immunity, and viral infection. In this study, we first confirmed that YAP gene expression in patients with acquired immune deficiency syndrome (AIDS) was significantly lower than that in the healthy control group, as determined using the GEO2R online tool. Furthermore, YAP was identified as a negative regulator of HIV-1 transcription by mediating K33- and K48-linked ubiquitination and proteasomal degradation of Tat. Here, we further confirmed that the YAP TAD domain recruited ubiquitin-like with PHD and RING finger domain 1 (UHRF1) to mediate Tat's ubiquitination and degradation by the screening of the BioGRID database combined with IP-MS analysis. The conserved lysine residues K28, K29, and K41 on Tat were critical acceptor sites for ubiquitination and proteasomal degradation. Our findings revealed that YAP promotes the suppression of HIV-1 transcription and the maintenance of HIV-1 latency, providing novel insights into virus-host interactions for regulating HIV-1 latency.

Human astrocytes can be infected with human immunodeficiency virus type 1 (HIV-1) in vitro and in vivo, but, in contrast to T lymphocytes and macrophages, virus expression is inefficient. To investigate the HIV-1 life cycle in human fetal astrocytes, we infected cells with HIV-1 pseudotyped with envelope glycoproteins of either amphotropic murine leukemia virus or vesicular stomatitis virus. Infection by both pseudotypes was productive and long lasting and reached a peak of 68% infected cells and 1.7 microg of viral p24 per ml of culture supernatant 7 days after virus inoculation and then continued with gradually declining levels of virus expression through 7 weeks of follow-up. This contrasted with less than 0.1% HIV-1 antigen-positive cells and 400 pg of extracellular p24 per ml at the peak of astrocyte infection with native HIV-1. Cell viability and growth kinetics were similar in infected and control cells. Northern blot analysis revealed the presence of major HIV-1 RNA species of 9, 4, and 2 kb in astrocytes exposed to pseudotyped (but not wild-type) HIV-1 at 2, 14, and 28 days after infection. Consistent with productive infection, the 9- and 4-kb viral transcripts in astrocytes infected by pseudotyped HIV-1 were as abundant as the 2-kb mRNA during 4 weeks of follow-up, and both structural and regulatory viral proteins were detected in infected cells by immunoblotting or cell staining. The progeny virus released by these cells was infectious. These results indicate that the major barrier to HIV-1 infection of primary astrocytes is at virus entry and that astrocytes have no intrinsic intracellular restriction to efficient HIV-1 replication.

Human immunodeficiency virus type 1 (HIV-1) incorporates the cellular peptidyl-prolyl cis-trans isomerase cyclophilin A (CyPA), the cytosolic receptor for the immunosuppressant cyclosporin A (CsA). CsA inhibits the incorporation of CyPA and reduces HIV-1 virion infectivity but is inactive against closely related primate lentiviruses that do not interact with CyPA. The incorporation of CyPA into HIV-1 virions is mediated by a specific interaction with a proline-containing, solvent-exposed loop in the capsid (CA) domain of the Gag polyprotein. CsA, which disrupts the interaction with CA, binds at the active site of CyPA. To test whether active-site residues are also involved in the interaction with HIV-1 CA, we used a panel of previously characterized active-site mutants of human CyPA. Expression vectors for epitope-tagged wild-type and mutant CyPA were transfected into COS-gamma cells along with HIV-1 proviral DNA, and the virions produced were analyzed for the presence of tagged proteins. Cotransfection of the wild-type expression vector led to the incorporation of readily detectable amounts of epitope-tagged CyPA into HIV-1 virions. One CyPA mutant with a substantially decreased sensitivity to CsA was incorporated with wild-type efficiency, demonstrating that the requirements for binding to CsA and to HIV-1 CA are not identical. The remaining six CyPA mutants were incorporated with markedly reduced efficiency, providing in vivo evidence that HIV-1 CA interacts with the active site of CyPA.

The genome of human immunodeficiency virus type 1 (HIV-1) encodes at least six proteins involved in regulation as well as the structural proteins Gag, Pol, and Env. The interplay of the various regulators generates early and late transcriptional phases in the HIV-1 life cycle; the earliest RNA is enriched in subgenomic species, and the genomic transcript appears at the later stage of infection. We investigated the nature of the mRNAs expressed in the early stages of infection when the 2 kilobase subgenomic species predominate. RNA was analyzed in the early phase of a one-step growth cycle of HIV-1 infection in T-lymphoid and monocytic cell lines by using PCR amplification of in vitro-synthesized viral cDNAs. In both cell lines, expression of Tat-, Rev-, and Nef-specific messages appeared simultaneously and could be detected within 8-12 hr of infection but in different amounts with a predominance of Nef-specific message. The Env-specific message could be detected as early as the Rev-specific message, indicating that expression of at least small amounts of the singly spliced message could occur before the accumulation of Rev.

Properties, Functions, and Drug Targeting of the Multifunctional Nucleocapsid Protein of the Human Immunodeficiency Virus

The TRIM5alpha (tripartite motif 5alpha protein) has been linked to the cross-species restriction in human immunodeficiency virus type 1 (HIV-1) infection of non-human cells, but the mechanism by which this occurs remains to be fully elucidated. Here we demonstrate that the capsid (CA) protein of HIV-1 is more rapidly degraded in cells expressing monkey TRIM5alpha than in cells expressing human TRIM5alpha. Other proteins encoded by Gag and Pol are not subject to TRIM5alpha-mediated accelerated degradation. The accelerated CA degradation by TRIM5alpha apparently occurs via a nonproteosomal pathway. TRIM5alpha selectively accelerates degradation of the CA population, which reached the cytosol of restrictive cells, but not the CA population, which ended into the vesicular compartment. Given that cytosolic CA represents "productively" entered cores, whereas vesicular CA represents "nonproductively" entered cores, our findings suggest that TRIM5alpha interrupts the infectious pathway of HIV-1 by acting on the incoming cytosolic CA. The mode of viral entry does not influence the accelerated degradation of cytosolic CA by TRIM5alpha. Thus, this study reveals a correlation between TRIM5alpha-mediated HIV-1 restriction and a selective degradation of cytosolic CA normally associated with productive viral entry.

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses harbor short peptide motifs in Gag that promote the release of infectious virions. These motifs, known as late assembly (L) domains, recruit a cellular budding machinery that is required for the formation of multivesicular bodies (MVBs). The primary L domain of HIV-1 maps to a PTAP motif in the p6 region of Gag and engages the MVB pathway by binding to Tsg101. Additionally, HIV-1 p6 harbors an auxiliary L domain that binds to the V domain of ALIX, another component of the MVB pathway. We now show that ALIX also binds to the nucleocapsid (NC) domain of HIV-1 Gag and that ALIX and its isolated Bro1 domain can be specifically packaged into viral particles via NC. The interaction with ALIX depended on the zinc fingers of NC, which mediate the specific packaging of genomic viral RNA, but was not disrupted by nuclease treatment. We also observed that HIV-1 zinc finger mutants were defective for particle production and exhibited a similar defect in Gag processing as a PTAP deletion mutant. The effects of the zinc finger and PTAP mutations were not additive, suggesting a functional relationship between NC and p6. However, in contrast to the PTAP deletion mutant, the double mutants could not be rescued by overexpressing ALIX, further supporting the notion that NC plays a role in virus release.