Assembly of infectious HIV-1 in human epithelial and T-lymphoblastic cell lines

The canonical view of the ultimate steps of HIV-1 replication is that virus assembly and budding are taking place at the plasma membrane of infected cells. Surprisingly, recent studies revealed that these steps also occur on endosomal membranes in the interior of infected cells, such as macrophages. This prompted us to revisit the site of HIV-1 assembly in human epithelial-like cells and in infected human T-lymphoblastic cells. To address this question, we investigated the intracellular location of the major viral structural components of HIV-1, namely Gag, Env and the genomic RNA. Using a sub-cellular fractionation method, as well as immuno-confocal and electron microscopy, we show that Gag, the Env glycoproteins and the genomic RNA accumulate in late endosomes that contain infectious HIV-1 particles. In epithelial-like 293T cells, HIV-1 assembles and buds both at the plasma membrane and in endosomes, while in chronically infected human T lymphocytes, viral assembly mostly occurs within the cell where large amounts of infectious virions accumulate in endosomal compartments. In addition, HIV-1 release could be enhanced by ionomycin, a drug stimulating calcium-dependent exocytosis. These results favour the view that newly made Gag molecules associate with the genomic RNA in the cytosol, then viral core complexes can be targeted to late endosomes together with Env, where infectious HIV-1 are made and subsequently released by exocytosis.     

Cell-dependent replication potentials of HIV-1 gag mutants

An infectious molecular clone of human immunodeficiency virus type 1 (HIV-1), designated pNLaiKH, which is tropic for both lymphocytic and monocytic cells, was constructed. To study the early function of HIV-1 Gag proteins in two types of cells, the mutations known to give host cell-dependent early defects were introduced into pNLaiKH, and the replication potentials and defective replication sites in the cells of the resultant mutants were monitored. All mutants grew in some lymphocytic cells, but not at all in monocytic cells. A nucleocapsid mutant was found to be defective at an early replication phase in all the cell lines to various extent, as expected. In contrast, a matrix mutant and a capsid mutant displayed a replication defect in a producer-cell-dependent manner. These results demonstrated that complex interactions of cell factors and Gag proteins are involved in an early process of HIV-1 replication.     

Novel activities of cyclophilin A and cyclosporin A during HIV-1 infection of primary lymphocytes and macrophages

Studies conducted in cell lines indicate that cyclophilin A (CypA) is a component of HIV type 1 (HIV-1) virions, and that when CypA incorporation into virions is inhibited by treatment of infected cells with the immunosuppressive agent cyclosporin A (CsA), HIV-1 infection also is inhibited. Because HIV-1 particles assemble along a different pathway and incorporate different host proteins in macrophages than in other cell types, we investigated CypA and CsA activities in HIV-1-infected primary human macrophages, compared with primary human lymphocytes. We tested virus protein production, virion composition and infectivity, and progress through the virus life cycle under perturbation by drug treatment or mutagenesis in infected cells from multiple donors. Our findings from both primary cell types are different from that previously reported in transformed cells and show that the amount of CypA incorporated into virions is variable and that CsA inhibits HIV-1 infection at both early and late phases of virus replication, the stage affected is determined by the sequence of HIV-1 Gag. Because the cell type infected determines the identity of host proteins active in HIV-1 replication and can influence the activity of some viral inhibitors, infection of transformed cells may not recapitulate infection of the native targets of HIV-1.     

Cells with High Cyclophilin A Content Support Replication of Human Immunodeficiency Virus Type 1 Gag Mutants with Decreased Ability To Incorporate Cyclophilin A

Gag polyprotein-mediated incorporation of cellular cyclophilin A (CyPA) into virions is essential for the formation of infectious human immunodeficiency virus type 1 (HIV-1) virions. Either a point mutation in Gag (P222A) or drugs which bind CyPA decrease virion incorporation of CyPA and interfere with HIV-1 replication. We have found that lymphoid cells varied greatly in their CyPA content and that cells with high CyPA content supported the replication of P222A HIV-1 Gag mutants. These experiments demonstrated that a higher cellular CyPA content of some cells was able to compensate for the decreased binding affinity of P222A mutant Gag for CyPA, allowing virus replication to occur.     

Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

Human immunodeficiency virus type 1 (HIV-1) Gag and the cellular protein cyclophilin A form an essential complex in the virion core: virions produced by proviruses encoding Gag mutants with decreased cyclophilin A affinity exhibit attenuated infectivity, as do virions produced in the presence of the competitive inhibitor cyclosporine. The A224E Gag mutant has no effect on cyclophilin A affinity but renders HIV-1 replication cyclosporine resistant in Jurkat T cells. In contrast, A224E mutant virus is dead in H9 T cells, although replication is rescued by cyclosporine or by expression in cis of a Gag mutant that decreases cyclophilin A-affinity. The observation that disruption of the Gag-cyclophilin A interaction rescues A224E mutant replication in H9 cells prompted experiments which revealed that, relative to Jurkat cells, H9 cells express greater quantities of cyclophilin A. The resulting larger quantity of cyclophilin A shown to be packaged into virions produced by H9 cells is presumably disruptive to the A224E mutant virion core. Further evidence that increased cyclophilin A expression in H9 cells is of functional relevance was provided by the finding that Gag mutants with decreased cyclophilin A affinity are dead in Jurkat cells but capable of replication in H9 cells. Similarly, cyclosporine concentrations which inhibit wild-type HIV-1 replication in Jurkat cells stimulate HIV-1 replication in H9 cells. These results suggest that HIV-1 virion infectivity imposes narrow constraints upon cyclophilin A stoichiometry in virions and that infectivity is finely tuned by host cyclophilin A expression levels.     

CRM1, an RNA transporter, is a major species-specific restriction factor of human T cell leukemia virus type 1 (HTLV-1) in rat cells

Rat ortholog of human CRM1 has been found to be responsible for the poor activity of viral Rex protein, which is essential for RNA export of human T cell leukemia virus type 1 (HTLV-1). Here, we examined the species-specific barrier of HTLV-1 by establishing rat cell lines, including both adherent and CD4(+) T cells, which express human CRM1 at physiological levels. We demonstrated that expression of human CRM1 in rat cells is not harmful to cell growth and is sufficient to restore the synthesis of the viral structural proteins, Gag and Env, at levels similar to those in human cells. Gag precursor proteins were efficiently processed to the mature forms in rat cells and released into the culture medium as sedimentable viral particles. An HTLV-1 pseudovirus infection system suggested that the released virus particles are fully infectious. Our newly developed reporter cell system revealed that Env proteins produced in rat cells are fully fusogenic, which is the basis for cell-cell HTLV-1 infection. Moreover, we show that the early steps in infection, from post-entry uncoating to integration into the host chromosomes, occur efficiently in rat cells. These results, in conjunction with reports describing efficient entry of HTLV-1 into rat cells, may indicate that HTLV-1 is unique in that its major species-specific barrier is determined by CRM1 at a viral RNA export step. These observations will enable us to construct a transgenic rat model expressing human CRM1 that is sensitive to HTLV-1 infection.     

Functional exchange of an oncoretrovirus and a lentivirus matrix protein.

To map functional domains in the retroviral Gag protein we have constructed chimeric viruses where regions of the murine leukemia virus (MuLV) Gag protein have been replaced with analogous sequences from human immunodeficiency virus type 1 (HIV-1). Here we describe the chimeric virus MuLV(MAHIV) which contains the HIV-1 matrix (MA) protein in place of the MuLV MA. MuLV(MAHIV) is infectious but grows at a reduced rate compared with wild-type MuLV. We found that the partial defect in replication of the chimeric virus is at a late stage in the viral life cycle. The MuLV(MAHIV) Gag proteins are distributed aberrantly within cells and are not associated with cellular membranes. Unlike MuLV, HIV-1 is able to integrate into growth-arrested cells. Incorporation of the HIV-1 MA, which is known to play a role in infection of nondividing cells, does not enable MuLV(MAHIV) to be expressed in growth-arrested cells. While it possesses no amino acid homology, we found that the HIV-1 MA can efficiently replace the MuLV matrix protein in infection.     

The Late Domain of Human Immunodeficiency Virus Type 1 p6 Promotes Virus Release in a Cell Type-Dependent Manner

The p6 domain of human immunodeficiency virus type 1 (HIV-1) is located at the C terminus of the Gag precursor protein Pr55(Gag). Previous studies indicated that p6 plays a critical role in HIV-1 particle budding from virus-expressing HeLa cells. In this study, we performed a detailed mutational analysis of the N terminus of p6 to map the sequences required for efficient virus release. We observed that the highly conserved P-T/S-A-P motif located near the N terminus of p6 is remarkably sensitive to change; even conservative mutations in this sequence imposed profound virus release defects in HeLa cells. In contrast, single and double amino acid substitutions outside the P-T/S-A-P motif had no significant effect on particle release. The introduction of stop codons one or two residues beyond the P-T/S-A-P motif markedly impaired virion release, whereas truncation four residues beyond P-T/S-A-P had no effect on particle production in HeLa cells. By examining the effects of p6 mutation in biological and biochemical analyses and by electron microscopy, we defined the role of p6 in particle release and virus replication in a panel of T-cell and adherent cell lines and in primary lymphocytes and monocyte-derived macrophages. We demonstrated that the effects of p6 mutation on virus replication are markedly cell type dependent. Intriguingly, even in T-cell lines and primary lymphocytes in which p6 mutations block virus replication, these changes had little or no effect on particle release. However, p6-mutant particles produced in T-cell lines and primary lymphocytes exhibited a defect in virion-virion detachment, resulting in the production of tethered chains of virions. Virus release in monocyte-derived macrophages was markedly inhibited by p6 mutation. To examine further the cell type-specific virus release defect in HeLa versus T cells, transient heterokaryons were produced between HeLa cells and the Jurkat T-cell line. These heterokaryons display a T-cell-like phenotype with respect to the requirement for p6 in particle release. The results described here define the role of p6 in virus replication in a wide range of cell types and reveal a strong cell type-dependent requirement for p6 in virus particle budding.     

High levels of anti-human immunodeficiency virus type 1 (HIV-1) memory cytotoxic T-lymphocyte activity and low viral load are associated with lack of disease in HIV-1-infected long-term nonprogressors.

Lack of disease in long-term nonprogressors with human immunodeficiency virus type 1 (HIV-1) infection was strongly associated with very low copy numbers of HIV-1 DNA and RNA in peripheral blood mononuclear cells and plasma and the presence of high levels of anti-HIV-1 CD8+ memory cytotoxic T lymphocytes specific for Gag, Pol, and Env, compared with levels present in intermediate and advanced progressors. CD8+ memory cytotoxic T lymphocytes may have an important role in controlling HIV-1 replication and preventing disease in long-term nonprogressors.     

Mutations in the matrix protein of human immunodeficiency virus type 1 inhibit surface expression and virion incorporation of viral envelope glycoproteins in CD4+ T lymphocytes.

Highly conserved amino acids in the second helix structure of the human immunodeficiency virus type 1 (HIV-1) MA protein were identified to be critical for the incorporation of viral Env proteins into HIV-1 virions from transfected COS-7 cells. The effects of these MA mutations on viral replication in the HIV-1 natural target cells, CD4+ T lymphocytes, were evaluated by using a newly developed system. In CD4+ T lymphocytes, mutations in the MA domain of HIV-1 Gag also inhibited the incorporation of viral Env proteins into mature HIV-1 virions. Furthermore, mutations in the MA domain of HIV-1 Gag reduced surface expression of viral Env proteins in CD4+ T lymphocytes. The synthesis of gp160 and cleavage of gp160 to gp120 were not significantly affected by MA mutations. On the other hand, the stability of gp120 in MA mutant-infected cells was significantly reduced compared to that in the parental wild-type virus-infected cells. These results suggest that functional interaction between HIV-1 Gag and Env proteins is not only critical for efficient incorporation of Env proteins into mature virions but also important for proper intracellular transport and stable surface expression of viral Env proteins in infected CD4+ T lymphocytes. A single amino acid substitution in MA abolished virus infectivity in dividing CD4+ T lymphocytes without significantly affecting virus assembly, virus release, or incorporation of Gag-Pol and Env proteins, suggesting that in addition to its functional role in virus assembly, the MA protein of HIV-1 also plays an important role in other steps of virus replication.     

Cellular compartments of human immunodeficiency virus type 1 replication in vivo: determination by presence of virion-associated host proteins and impact of opportunistic infection

Antigens derived from host cells are detectable in the envelope of human immunodeficiency virus type 1 (HIV-1) and result in a distinctive viral phenotype reflecting that of the host cell. An immunomagnetic capture assay targeting discriminatory host proteins was developed to differentiate between HIV-1 derived from macrophages and lymphocytes. HIV-1 propagated in macrophages or lymphocytes in vitro was selectively captured by monoclonal antibodies directed against the virally incorporated cell-type-specific host markers CD36 (macrophages) and CD26 (lymphocytes). Furthermore, by targeting these markers, virus of defined cellular origin was selectively captured from a mixed pool of in vitro-propagated viruses. This technique was further refined in order to determine the impact of opportunistic infection on HIV-1 expression from these cellular compartments in vivo. Analysis of cell-free virus purified from plasma of patients with HIV-1 infection suggested that in those with an opportunistic infection, viral replication occurred in activated lymphocytes. Interestingly, there was also significant replication in activated macrophages in those patients with untreated pulmonary tuberculosis. Thus, in addition to lymphocytes, the macrophage cellular pool may serve as an important source of cell-free HIV-1 in patients with opportunistic infections that lead to marked macrophage activation. This novel viral capture technique may allow researchers to address a wide range of important questions regarding virus-host dynamics.     

Roles of the interactions between Env and Gag proteins in the HIV-1 replication cycle

The Env and Gag proteins of HIV-1 are the two major structural proteins of this retrovirus. The interactions between Env and Gag proteins and their regulation in HIV-1 are required for several steps of the replication cycle, involving not only virus assembly, specifically Env incorporation, but also entry steps after virus maturation. A large number of host factors and certain membrane microdomains appear to engage both in transport/trafficking of Env and/or Gag proteins, and in the interactions of these two proteins. The present review briefly summarizes our current knowledge regarding the roles of the interactions between Env and Gag proteins in the virus replication cycle.     

The cellular protein lyric interacts with HIV-1 Gag

Human immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein driving assembly and release of virions from infected cells. Gag alone is capable of self-assembly in vitro, but host factors have been shown to play a role in efficient viral replication and particle morphogenesis within the living cell. In a series of affinity purification experiments, we identified the cellular protein Lyric to be an HIV-1 Gag-interacting protein. Lyric was previously described to be an HIV-inducible gene and is involved in various signaling pathways. Gag interacts with endogenous Lyric via its matrix (MA) and nucleocapsid (NC) domains. This interaction requires Gag multimerization and Lyric amino acids 101 to 289. Endogenous Lyric is incorporated into HIV-1 virions and is cleaved by the viral protease. Gag-Lyric interaction was also observed for murine leukemia virus and equine infectious anemia virus, suggesting that it represents a conserved feature among retroviruses. Expression of the Gag binding domain of Lyric increased Gag expression levels and viral infectivity, whereas expression of a Lyric mutant lacking the Gag binding site resulted in lower Gag expression and decreased viral infectivity. The results of the current study identify Lyric to be a cellular interaction partner of HIV-1 Gag and hint at a potential role in regulating infectivity. Further experiments are needed to elucidate the precise role of this interaction.     

Human Discs Large is a new negative regulator of human immunodeficiency virus-1 infectivity

Human immunodeficiency virus (HIV)-1 replication is positively or negatively regulated through multiple interactions with host cell proteins. We report here that human Discs Large (Dlg1), a scaffold protein recruited beneath the plasma membrane and involved in the assembly of multiprotein complexes, restricts HIV-1 infectivity. The endogenous Dlg1 and HIV-1 Gag polyprotein spontaneously interact in HIV-1-chronically infected T cells. Depleting endogenous Dlg1 in either adherent cells or T cells does not affect Gag maturation, production, or release, but it enhances the infectivity of progeny viruses five- to sixfold. Conversely, overexpression of Dlg1 reduces virus infectivity by ~80%. Higher virus infectivity upon Dlg1 depletion correlates with increased Env content in cells and virions, whereas the amount of virus-associated Gag or genomic RNA remains identical. Dlg1 knockdown is also associated with the redistribution and colocalization of Gag and Env toward CD63 and CD82 positive vesicle-like structures, including structures that seem to still be connected to the plasma membrane. This study identifies both a new negative regulator that targets the very late steps of the HIV-1 life cycle, and an assembly pathway that optimizes HIV-1 infectivity.     

Mutation of dileucine-like motifs in the human immunodeficiency virus type 1 capsid disrupts virus assembly, gag-gag interactions, gag-membrane binding, and virion maturation

The human immunodeficiency virus type 1 (HIV-1) Gag precursor protein Pr55(Gag) drives the assembly and release of virus-like particles in the infected cell. The capsid (CA) domain of Gag plays an important role in these processes by promoting Gag-Gag interactions during assembly. The C-terminal domain (CTD) of CA contains two dileucine-like motifs (L189/L190 and I201/L202) implicated in regulating the localization of Gag to multivesicular bodies (MVBs). These dileucine-like motifs are located in the vicinity of the CTD dimer interface, a region of CA critical for Gag-Gag interactions during virus assembly and CA-CA interactions during core formation. To study the importance of the CA dileucine-like motifs in various aspects of HIV-1 replication, we introduced a series of mutations into these motifs in the context of a full-length, infectious HIV-1 molecular clone. CA mutants LL189,190AA and IL201,202AA were both severely impaired in virus particle production because of a variety of defects in the binding of Gag to membrane, Gag multimerization, and CA folding. In contrast to the model suggesting that the CA dileucine-like motifs regulate MVB targeting, the IL201,202AA mutation did not alter Gag localization to the MVB in either HeLa cells or macrophages. Revertants of single-amino-acid substitution mutants were obtained that no longer contained dileucine-like motifs but were nevertheless fully replication competent. The varied phenotypes of the mutants reported here provide novel insights into the interplay among Gag multimerization, membrane binding, virus assembly, CA dimerization, particle maturation, and virion infectivity.     

Macrophages archive HIV-1 virions for dissemination in trans

Viruses have evolved various strategies in order to persist within the host. To date, most information on mechanisms of HIV-1 persistence has been derived from studies with lymphocytes, but there is little information regarding mechanisms that govern HIV-1 persistence in macrophages. It has previously been demonstrated that virus assembly in macrophages occurs in cytoplasmic vesicles, which exhibit the characteristics of multivesicular bodies or late endosomes. The infectious stability of virions that assemble intracellularly in macrophages has not been evaluated. We demonstrate that virions assembling intracellularly in primary macrophages retain infectivity for extended intervals. Infectious virus was recovered directly from cytoplasmic lysates of macrophages and could be transmitted from macrophages to peripheral blood lymphocytes in trans 6 weeks after ongoing viral replication was blocked. Cell-associated virus decayed significantly from 1 to 2 weeks post infection, but decreased minimally thereafter. The persistence of intracellular virions did not require the viral accessory proteins Vpu or Nef. The stable sequestration of infectious virions within cytoplasmic compartments of macrophages may represent an additional mechanism for viral persistence in HIV-1-infected individuals.