Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex.

Replication of HIV-1 in non-dividing and slowly proliferating cell populations depends on active import of the viral pre-integration complex (PIC) into the cell nucleus. While it is commonly accepted that this process is mediated by an interaction between the HIV-1 PIC and the cellular nuclear import machinery, controversial results have been reported concerning the mechanisms of this interaction. Here, we demonstrate that a recently identified nuclear localization signal within the HIV-1 matrix protein (MA), MA NLS-2, together with previously described MA NLS-1, mediates nuclear import of the HIV-1 PIC. Inactivation of both MA NLSs precluded nuclear translocation of MA and rendered the virus defective in nuclear import and replication in non-dividing macrophage cultures, even when functional Vpr and integrase (IN), two more viral proteins implicated in HIV-1 nuclear import, were present. Taken together, these results indicate that Vpr does not function as an independent nuclear import factor and demonstrate that HIV-1 MA, by virtue of its two nuclear localization signals, regulates HIV-1 nuclear import.

HIV-1 nuclear import: in search of a leader.

The ability of HIV-1 to use host cell nuclear import machinery to translocate the viral pre-integration complex into the cell nucleus is the critical determinant in the replication of the virus in non-dividing cells, such as macrophages. In this review, we describe the viral and cellular factors involved in this process. The available data suggest that the process of HIV-1 nuclear import is driven by interaction between nuclear localization signals (NLSs) present on viral proteins matrix and integrase and the cellular NLS receptor, karyopherin alpha. However, this interaction by itself is weak and insufficient to insure effective import of the pre-integration complex. Viral protein R (Vpr) functions to increase the affinity of interaction between viral NLSs and karyopherin alpha, thus substantially enhancing the karyophilic potential of the pre-integration complex. Interestingly, some cells, in particular HeLa, seem to contain a factor that can substitute for the Vpr's activity, making HIV-1 replication in such cells Vpr-independent. We also describe a class of novel anti-HIV compounds that target the NLSs of HIV-1 and effectively block viral replication in T cells and macrophages.

CNI-H0294, a nuclear importation inhibitor of the human immunodeficiency virus type 1 genome, abrogates virus replication in infected activated peripheral blood mononuclear cells.

Active nuclear importation of the human immunodeficiency virus (HIV) type 1 (HIV-1) preintegration complex (PIC) is required for the productive infection of nondividing cells, but it is believed to be dispensable for the infection of proliferating cells, such as activated T lymphocytes. To investigate this question, we exploited the properties of the small arylene bis (methyl ketone) compound CNI-H0294. We have previously shown that this compound associated with the HIV-1 matrix protein nuclear localization sequence and blocked binding of the HIV-1 PIC to yeast karyopherin alpha. CNI-H0294 abrogated nuclear importation of the HIV-1 genome in macrophages and effectively inhibited infection of nondividing cells. In this study we demonstrate that CNI-H0294 inhibits binding of the HIV-1 PIC to human karyopherin alpha and reduces nuclear importation of the viral genome in primary peripheral blood mononuclear cells (PBMCs). We also demonstrate that CNI-H0294 inhibits acute infection of PBMC cultures in vitro with a primary isolate of HIV-1 and reduces virus replication and virus load in cultures of endogenously infected PBMCs from seropositive individuals. Thus, as for infection of nondividing, terminally differentiated macrophages, HIV-1 uses active nuclear importation of the virus genome to infect activated CD4+ T cells. These results support nuclear importation as a novel target and CNI-H0294 and its derivatives as novel compounds for therapeutic intervention in HIV infection and AIDS.

HIV-1 nuclear import: in search of a leader.

The ability of HIV-1 to use host cell nuclear import machinery to translocate the viral preintegration complex into the cell nucleus is the critical determinant in the replication of the virus in non-dividing cells, such as macrophages. In this review, we describe the viral and cellular factors involved in this process. The available data suggest that the process of HIV-1 nuclear import is driven by interaction between nuclear localization signals (NLSs) present on viral proteins matrix and integrase and the cellular NLS receptor, karyopherin alpha. However, this interaction by itself is weak and insufficient to insure effective import of the preintegration complex. Viral protein R (Vpr) functions to increase the affinity of interaction between viral NLSs and karyopherin alpha, thus substantially enhancing the karyophilic potential of the preintegration complex. Interestingly, some cells, in particular HeLa, seem to contain a factor which can substitute for the Vpr's activity, making HIV-1 replication in such cells Vpr-independent. We also describe a class of novel anti-HIV compounds which target the NLSs of HIV-1 and effectively block viral replication in T cells and macrophages.

TCR-independent CD28-mediated gene expression in peripheral blood lymphocytes from donors chronically infected with HIV-1.

Complete activation of peripheral blood T cells requires both T-cell receptor (TCR) stimulation and CD28 costimulation. Signalling pathways associated specifically with CD28 are not well understood, however, because ligation of CD28 in the absence of TCR stimulation does not give rise to cellular responses in normal cells. In peripheral blood lymphocytes (PBL) from donors chronically infected with human immunodeficiency virus-1 (HIV-1), CD28 can induce viral replication through an alternative pathway that does not require TCR ligation. We have exploited this observation to study CD28-mediated signal transduction using reverse transcriptase-mediated polymerase chain reaction (RT-PCR) to amplify viral RNA. Independent ligation of CD28 on donor PBL induced expression of the HIV-1 tat gene but not the interleukin-2 (IL-2) gene. Viral induction did not occur following pretreatment of cells with actinomycin D, suggesting it was mediated through transcriptional activation of the viral long terminal repeat (LTR). tat was induced in the presence of the protein kinase C inhibitor H-7, but was inhibited by cyclosporin A. Our results demonstrate that CD28 is linked directly to specific signalling pathways leading to de novo induction of genes in PBL.

Differential expression and sequence-specific interaction of karyopherin alpha with nuclear localization sequences.

The process of nuclear protein transport requires the interaction of several different proteins, either directly or indirectly with nuclear localization or targeting sequences (NLS). Recently, a number of karyopherins alpha, or NLS-binding proteins, have been identified. We have found that the karyopherins hSRP1 and hSRP1alpha are differentially expressed in various leukocyte cell lines and could be induced in normal human peripheral blood lymphocytes. We show that the two karyopherins bind with varied specificities in a sequence specific manner to different NLSs and that the sequence specificity is modulated by other cytosolic proteins. There was a correlation between binding of karyopherins alpha to different NLSs and their ability to be imported into the nucleus. Taken together, these data provide evidence for multiple levels of control of the nuclear import process.

IL-7 up-regulates HIV-1 replication in naturally infected peripheral blood mononuclear cells.

We have investigated the effect of exogenous IL-7 on replication of HIV-1 in PBMCs isolated from asymptomatic, chronically infected donors. We show that IL-7 induced virus replication and increased proviral DNA levels in CD8- PBMC cultures. IL-7 also increased the levels of doubly spliced HIV-1 tat RNA in these cultures. In comparison, IL-2 induced lower levels of virus production than IL-7, but had a more pronounced effect on cell proliferation. The IL-7-mediated increase in virus replication was not inhibited by neutralizing mAbs to IL-1 beta, IL-2, IL-6, or TNF-alpha, and was only partially dependent on ligation of the T cell accessory molecule CD28. CD8+ cells inhibited the increase in viral replication following IL-7 stimulation, but did not prevent virus replication following ligation of CD3 in the presence of IL-7. The data shows that IL-7 regulates HIV-1 replication in naturally infected PBMCs.

Anti-idiotypic antibody to the V3 domain of gp120 binds to vimentin: a possible role of intermediate filaments in the early steps of HIV-1 infection cycle.

Although the CD4 molecule is the major cellular receptor for human immunodeficiency virus (HIV), several lines of evidence suggest participation of additional molecules that are engaged after the binding of HIV to the CD4 receptor and that may facilitate viral entry into the target cell. Some of the post-CD4 binding, perfusion events involve the third hypervariable region (V3 loop) of the viral envelope protein gp120. To identify cellular proteins that interact with the V3 loop, we chose as a probe an antiidiotypic monoclonal antibody (MAb), anti-id2, which was prepared against the neutralizing MAb 110.4 that binds the V3 domain in the envelope glycoprotein gp120 of the LAI isolate of HIV-1. Anti-id2 reacted specifically with a 55- to 60-kDa protein in human T cell and monocytoid cell lines, and in a mouse melanoma cell line. This protein was identified immunologically and by protein sequence analysis as vimentin, an intermediate filament protein of lymphoid and other cells of mesodermal origin. Antiserum raised against vimentin inhibited nuclear translocation of HIV-1 DNA following infection of monocytes and CD4+ T cells with live virus, and reduced the amount of HIV-1 gag-specific RNA in the nuclei of monocytes following inoculation with HIV-1 pseudovirions. These data suggest that vimentin may participate in the early steps of HIV-1 replication, perhaps during the uptake of HIV-1 preintegration complexes into the nuclear compartment.

Human immunodeficiency virus type 1 infection of CD4+ T cells down-regulates the expression of CD28: effect on T cell activation and cytokine production.

Infection with human immunodeficiency virus type 1 (HIV-1) results in dysregulation of normal T cell function. To study the effects of HIV-1 at the cellular level, primary T cell lines were generated by alloantigen stimulation of CD4+ T cells collected from peripheral blood of HIV-1-infected donors. Using Epstein-Barr virus-infected B lymphocytes (EBV-LCL) as a source of alloantigen, the T cell lines were expanded in vitro for 7 weeks. Uninfected T cell lines were cultured in parallel. Virus was inducible from the infected lines with stimulation, and complete infection was achieved after 4-7 weeks depending on the line. The down-modulation of CD28 expression correlated with virus replication and spread. Furthermore, CD28 mRNA was not inducible in the infected lines after stimulation with alloantigen. Loss of CD28 correlated with reduced responsiveness to costimulation with a monoclonal antibody to CD28 following similar engagement of the CD3 protein. In contrast, activation with alloantigen was not affected. HIV-1 infection and down-modulation of CD28 did not alter the relative levels of IL-2, IFN-gamma, and IL-4 mRNA. Production of the various cytokine mRNAs following alloantigen stimulation was inhibited by CTLA4Ig and thus remained under the regulation of CD80 and CD86 expressed on the EBV-LCL. Taken together, our data suggest that dysregulation of normal T cell function associated with HIV-1 infection may result in part form the loss of CD28 expression.

Costimulation of CD4+ T cells via CD28 modulates human immunodeficiency virus type 1 infection and replication in vitro.

Stimulation of human immunodeficiency virus type 1 (HIV-1)-infected donor peripheral blood mononuclear cells (PBMCs) via the TCR-CD3 complex induces HIV-1 production in vitro (Zarling JM, et al.: Nature [London] 1990;347:92; Haffar OK, et al.: J Virol 1992;66:4279; Moran PM, et al.: AIDS Res Hum Retroviruses 1993;9:455). However, in addition to the primary stimulatory signal delivered through the TCR-CD3 complex, optimal T cell activation requires secondary or costimulatory signals delivered via various T cell accessory proteins (Alton A, et al.: Adv Immunol 1990;48:227). In this article we explore the role of costimulation of T cells via CD28 in HIV-1 replication. Ligation of CD28 with either a CD28-specific MAb or by coculture of PBMCs with Chinese hamster ovary (CHO) cell lines stably expressing either of the CD28 counterreceptors, B7-1 (CD80) or B7-2 (CD86), concomitant with stimulation via CD3, results in increased virus replication compared to stimulation via CD3 alone. CD28 ligation also augments de novo infection of CD3-stimulated seronegative donor PBMCs with cell-free virus. Increased virus replication following CD28 ligation is not solely attributed to increased levels of endogenous IL-2, because addition of an anti-IL-2-neutralizing antibody only partially inhibits the response. In contrast, interfering with the interaction between CD28 and its counterreceptors on antigen-presenting cells (APCs) using CTLA4Ig effectively inhibits virus replication. At high concentrations CTLA4Ig also reduces cell proliferation. These in vitro results suggest that CD28 plays a central role in HIV-1 replication and that interfering with the CD28 costimulatory pathway may modify the course of HIV-1 infection.

HIV-1 down-regulates CD4 costimulation of TCR/CD3-directed tyrosine phosphorylation through CD4/p56lck dissociation.

One consequence of HIV type 1 (HIV-1) infection is the gradual loss of responsiveness of T lymphocytes to Ags both in vitro and in vivo. It has been suggested that the underlying mechanism that contributes to this T cell dysfunction before CD4+ cell decline involves down-regulation of surface receptors, alterations in intracellular redox status, interference by viral Ags, and later in infection, the absence or alteration of specific cytokine production. In this report, we demonstrate that infection of the T-lymphocytic cell line H9 with the LAI isolate of HIV-1 results in profoundly altered regulation of CD4-induced costimulation of TCR/CD3-directed signaling. TCR/CD3-induced tyrosine phosphorylation of the intracellular enzyme phospholipase-C gamma 1 and the surface receptor/substrates CD5 and CD6 was unaffected by virus infection, whereas augmented responses normally observed after the co-ligation of CD4 with TCR/CD3 on T lymphocytes were absent in HIV-1-infected H9 cells. Costimulation of TCR/CD3-induced signaling via MHC class II molecules was also down-regulated in virally infected cells. TCR/CD3 and HLA-DR receptor expression remained intact in infected cultures for at least 3 wk, whereas CD4 surface expression was gradually lost but maintained for up to 1 wk, suggesting that the absence of costimulation early in infection was not surface receptor density-dependent. In HIV-1-infected cells, CD4 was not physically linked with its associated tyrosine kinase p56lck, whereas normal levels of p56lck were readily recovered from the cellular cytoplasm. Similar observations were noted in cultures of H9 cells infected with a field isolate of HIV-1 obtained from cultured PBMC from an infected donor. HIV-1 infection of T lymphocytes thus down-regulates potentially critical early signal transduction events by a mechanism that appears to involve interference of CD4 receptor association with p56lck. A potential outcome of these biochemical effects may include the limited responsiveness of infected T cells to antigenic stimulation observed during HIV-1 infection.

Dual specificity of a monoclonal anti-idiotypic antibody for HIV-1 neutralizing monoclonals 110.3 and 110.4 as well as the V3 loop of gp120.

Monoclonal antibodies (MAbs) 110.3 and 110.4 bind an epitope at the tip of the third hypervariable region (V3) of the envelope protein gp120 of human immunodeficiency virus type 1 (HIV-1). These MAbs inhibit HIV-induced syncytium formation and neutralize cell-free virus infection. Anti-idiotypic MAb alpha-id8, generated against 110.3, was found to mimic the V3 loop of gp120, as demonstrated by competition ELISA and by the generation of anti-anti-idiotypic sera which bound gp120 and a peptide representing the tip of the V3 loop. Interestingly, alpha-id8 itself also reacted specifically with both gp120 and the V3 loop peptide. Thus, alpha-id8 both mimics and binds directly to the V3 loop, suggesting that the V3 loop of gp120 may associate with itself.

Characterization of human immunodeficiency virus type 1 Pr55gag membrane association in a cell-free system: requirement for a C-terminal domain.

Association of the human immunodeficiency virus type 1 (HIV-1) gag polyprotein precursor with cellular membranes is necessary for assembly of virions. We used in vitro synthesized HIV-1 gag to study its association with isolated cellular membranes. Rabbit reticulocyte lysates programmed with HIV-1 gag mRNA incorporated [35S]methionine and [3H]myristate into two predominant species of 55 kDa and 40 kDa. Radioimmunoprecipitation with HIV-1-specific antibodies suggested that the 55-kDa protein represented the polyprotein precursor (Pr55gag), while the 40-kDa protein was a mixture of N- or C-terminal truncations of the gag precursor. The Pr55gag protein bound to cellular membranes, while the 40-kDa mixed protein species did not. Membrane binding studies with C terminus-truncated and point mutants revealed that the seven-amino acid sequence located between the two Cys-His arrays in the nucleocapsid region was necessary for stable association to occur. Therefore, we propose that signals in addition to myristate are required for the membrane association of HIV-1 gag proteins and that these signals include a domain in the nucleocapsid protein.

Costimulation of T-cell activation and virus production by B7 antigen on activated CD4+ T cells from human immunodeficiency virus type 1-infected donors.

Infection with the human immunodeficiency virus type 1 (HIV-1) requires T-cell activation. Recent studies have shown that interactions of the T-lymphocyte receptors CD28 and CTLA-4 with their counter receptor, B7, on antigen-presenting cells are required for optimal T-cell activation. Here we show that HIV-1 infection is associated with decreased expression of CD28 and increased expression of B7 on CD4+ T-cell lines generated from seropositive donors by alloantigen stimulation. Loss of CD28 expression was not seen on CD4+ T-cell lines from seronegative donors, but up-regulation of B7 expression was observed upon more prolonged culture. Both T-cell proliferation and interleukin 2 mRNA accumulation in HIV-1-infected cultures required costimulation with exogenous B7 because these events were blocked by CTLA4Ig, a soluble form of CTLA-4 that binds B7 with high avidity. In contrast, levels of HIV-1 RNA were not affected by CTLA4Ig, indicating that regulation of virus transcription in these cultures did not depend upon CD28-B7 engagement. Infected T cells could present alloantigen to fresh, uninfected CD4+ T cells, leading to increased proliferation and virus spread to the activated cells. Both of these events were blocked by CTLA4Ig. Thus, chronic activation of HIV-1-infected CD4+ T cells reduces expression of CD28 and increases expression of B7, thereby enabling these T cells to become antigen-presenting cells for uninfected CD4+ T cells; this might be another mechanism for HIV-1 transmission via T-cell-T-cell contact.

Inhibition of virus production in peripheral blood mononuclear cells from human immunodeficiency virus (HIV) type 1-seropositive donors by treatment with recombinant HIV-like particles.

We have previously reported on the assembly of recombinant human immunodeficiency virus (HIV)-like particles that contain gag structural proteins and present env glycoproteins gp120 and gp41 on their surfaces (O. Haffar,. J. Garriques, B. Travis, P. Moran, J. Zarling, and S.-L. Hu, J. Virol. 64:2653-2659, 1990). On the basis of their structures, we hypothesized that the recombinant particles would interfere with virus infection and tested our hypothesis in vitro by using peripheral blood mononuclear cells (PBMC) from HIV type 1-seropositive donors. Addition of the recombinant particles to PBMC concomitant with stimulation by anti-CD3 inhibited virus production, as determined by reduced levels of p24 in the culture supernatants. This inhibition of p24 production correlated with lower levels of cell-associated viral DNA. Several lines of evidence suggested that the recombinant particles exerted their antiviral effects primarily by inhibiting virus production from latently infected cells and not by inhibiting subsequent virus spread. Importantly, CD4+ T-cell stimulation by specific antigen or by anti-CD3 was not inhibited by treatment with the recombinant particles. This apparent selective inhibition of virus replication in infected PBMC represents a novel property of the recombinant HIV-like particles.

HIV-specific humoral and cellular immunity in rabbits vaccinated with recombinant human immunodeficiency virus-like gag-env particles.

Recombinant human immunodeficiency virus type-1 (HIV-1)-like gag-env particles produced in mammalian cells were inoculated into two New Zealand white rabbits. In parallel, two control rabbits were inoculated with the homologous HIV-1 virions inactivated by ultra violet light (uv) and psoralen treatments. The humoral and cellular immune responses to HIV-1 were evaluated for both groups of animals. Recombinant particles elicited humoral immunity that was specific for all the viral structural proteins. The antibodies recognized both denatured and nondenatured proteins. Moreover, the sera neutralized the in vitro infectivity of the homologous virus in CEM cells. Importantly, the recombinant particles also generated a T helper response by priming with the HIV proteins. Similar results were observed with inactivated virus immunization. Therefore, our results suggest that the recombinant HIV-like particles elicit functional humoral immunity as well as cellular immunity and represent a novel vaccine candidate for AIDS.

The cytoplasmic tail of HIV-1 gp160 contains regions that associate with cellular membranes.

The HIV-1 envelope glycoprotein gp160 associates with cellular membranes via a discrete transmembrane domain. Unlike other retroviral envelope proteins, however, gp160 also forms a secondary association with the lipid bilayer mediated by one or more regions located in the cytoplasmic tail. We have expressed the full cytoplasmic tail sequence of gp160, as a fusion protein with the HSV-1 glycoprotein D signal sequence, transiently in a human embryonic kidney cell line. Our results show that in the absence of any defined transmembrane domain or stop transfer sequence, the protein corresponding to the cytoplasmic tail of HIV-1 gp160 formed stable interactions with cellular membranes that mediated its export to the cell surface.

The carboxy terminus of human immunodeficiency virus type 1 gp160 limits its proteolytic processing and transport in transfected cell lines.

Mutagenesis of the transmembrane domain and cytoplasmic tail of human immunodeficiency virus type 1 envelope glycoprotein gp160 revealed that its intracellular transport and processing in transfected cell lines were modulated by a functional domain included in the carboxy-terminal sequence consisting of residues 751 to 856.

Expression and immunogenicity of the extracellular domain of the human immunodeficiency virus type 1 envelope glycoprotein, gp160.

The envelope glycoprotein of human immunodeficiency virus type 1 is synthesized as a precursor, gp160, that subsequently is cleaved to yield mature gp120 and gp41. In these studies, the gene encoding gp160 was mutagenized so as direct the synthesis of a truncated protein consisting of the extracellular domains of both gp120 and gp41. The variant protein, termed sgp160, consisted of 458 amino acids of gp120 and 172 amino acids of gp41. To facilitate protein purification, the normal polyglycoprotein processing site between gp120 and gp41 was deleted through the use of site-directed mutagenesis. This allowed for the synthesis of a molecule that could be purified by affinity chromatography, using acid elution, without dissociation of the gp120 polypeptide from the gp41 polypeptide. The conformation of the sgp160 variant appeared to be functionally relevant, as reflected by its ability to bind to CD4 with an affinity comparable to that of the variant rgp120. The structure of the sgp160-containing polypeptide differed from that of rgp120 in that it tended to form high-molecular-weight aggregates that could be dissociated to monomers and dimers in the presence of reducing agents. Antibodies against the sgp160 protein reacted with authentic virus-derived gp160, gp120, and gp41; neutralized viral infectivity; and inhibited the binding of rgp120 to CD4. Rabbit antibodies to the sgp160 protein differed from those raised against rgp120 in that they were enriched for populations that blocked CD4 binding but did not prevent human immunodeficiency virus type 1-induced syncytium formation.