Human immunodeficiency virus type 1 Nef-induced CD4 cell surface downregulation is inhibited by ikarugamycin

One well-characterized in vitro function of Nef is its ability to remove CD4, the human immunodeficiency virus (HIV) receptor, from the cell surface. Nef accomplishes this by accelerating the internalization and degradation of CD4. Current models propose that Nef promotes CD4 internalization via an increased association of CD4 with clathrin-coated pits (CCP). Here, we investigated the effect of a naturally occurring antiprotozoan antibiotic, ikarugamycin (IKA), on CD4 cell surface expression in human monocytic cells stably expressing HIV type 1 SF2 Nef. IKA was able to efficiently restore CD4 cell surface expression in Nef-expressing cells without affecting either CD4 synthesis or Nef expression. In addition, we demonstrate that IKA is also capable of efficiently blocking CD4 down-modulation in response to phorbol myristate acetate. Our data suggest that IKA may be an efficient and useful inhibitor of CCP-dependent endocytosis.     

Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4.

CD4 is an integral membrane glycoprotein which is known as the human immunodeficiency virus (HIV) receptor for infection of human cells. The protein is synthesized in the endoplasmic reticulum (ER) and subsequently transported to the cell surface via the Golgi complex. HIV infection of CD4+ cells leads to downmodulation of cell surface CD4, due at least in part to the formation of stable intracellular complexes between CD4 and the HIV type 1 (HIV-1) Env precursor polyprotein gp160. This process "traps" both proteins in the ER, leading to reduced surface expression of CD4 and reduced processing of gp160 to gp120 and gp41. We have recently demonstrated that the presence of the HIV-1-encoded integral membrane protein Vpu can reduce the formation of Env-CD4 complexes, resulting in increased gp160 processing and decreased CD4 stability. We have studied the effect of Vpu on CD4 stability and found that Vpu induces rapid degradation of CD4, reducing the half-life of CD4 from 6 h to 12 min. By using a CD4-binding mutant of gp160, we were able to show that this Vpu-induced degradation of CD4 requires retention of CD4 in the ER, which is normally accomplished through its binding to gp160. The involvement of gp160 in the induction of CD4 degradation is restricted to its function as a CD4 trap, since, in the absence of Env, an ER retention mutant of CD4, as well as wild-type CD4 in cultures treated with brefeldin A, a drug that blocks transport of proteins from the ER, is degraded in the presence of Vpu.     

ARF1 regulates Nef-induced CD4 degradation

BACKGROUND: The HIV Nef protein downregulates CD4 through sequential connection with clathrin-coated pits and the COP1 coatomer, resulting in accelerated endocytosis and lysosomal targeting. RESULTS: Here we report that the small GTPase ARF1 controls the Nef-induced, COP-mediated late-endosomal targeting of CD4. We find that Nef binds ARF1 directly and can recruit the GTPase onto endosomal membranes. Furthermore, a complex comprising Nef, ARF1, and betaCOP can be immunoprecipitated from cells expressing the viral protein. Residues in a C-terminal loop of the viral protein facilitate both these interactions and the targeting of Nef and CD4 to acidic late endosomes, whereas other residues primarily involved in mediating CD4 endocytosis are dispensable for this process. Finally, a dominant-negative ARF1 mutant blocks the migration of the Nef-CD4 complex to lysosomes. CONCLUSIONS: Our results support a model in which ARF1 is the immediate downstream partner of Nef for CD4 lysosomal targeting.     

Silencing of both beta-TrCP1 and HOS (beta-TrCP2) is required to suppress human immunodeficiency virus type 1 Vpu-mediated CD4 down-modulation

The human immunodeficiency virus type 1 (HIV-1) Vpu protein interacts with CD4 within the endoplasmic reticula of infected cells and targets CD4 for degradation through interaction with β-TrCP1. Mammals possess a homologue of β-TrCP1, HOS, which is also named β-TrCP2. We show by coimmunoprecipitation experiments that β-TrCP2 binds Vpu and is able to induce CD4 down-modulation as efficiently as β-TrCP1. In two different cell lines, HeLa CD4⁺ and Jurkat, Vpu-mediated CD4 down-modulation could not be reversed through the individual silencing of endogenous β-TrCP1 or β-TrCP2 but instead required the two genes to be silenced simultaneously.     

Human immunodeficiency virus Nef induces rapid internalization of the T-cell coreceptor CD8alphabeta

Human immunodeficiency virus (HIV) Nef is a membrane-associated protein decreasing surface expression of CD4, CD28, and major histocompatibility complex class I on infected cells. We report that Nef strongly down-modulates surface expression of the beta-chain of the CD8alphabeta receptor by accelerated endocytosis, while CD8 alpha-chain expression is less affected. By mutational analysis of the cytoplasmic tail of the CD8 beta-chain, an FMK amino acid motif was shown to be critical for Nef-induced endocytosis. Although independent of CD4, endocytosis of the CD8 beta-chain was abrogated by the same mutations in Nef that affect CD4 down-regulation, suggesting common molecular interactions. The ability to down-regulate the human CD8 beta-chain was conserved in HIV-1, HIV-2, and simian immunodeficiency virus SIVmac239 Nef and required an intact AP-2 complex. The Nef-mediated internalization of receptors, such as CD4, major histocompatibility complex class I, CD28, and CD8alphabeta, may contribute to the subversion of the host immune system and progression towards AIDS.     

Human immunodeficiency virus type 1 Vpu protein induces degradation of CD4 in vitro: the cytoplasmic domain of CD4 contributes to Vpu sensitivity.

CD4 is an integral membrane glycoprotein which functions as the human immunodeficiency virus (HIV) receptor for infection of human host cells. We have recently demonstrated that Vpu, an HIV type 1 (HIV-1) encoded integral membrane phosphoprotein, induces rapid degradation of CD4 in the endoplasmic reticulum. In this report, we describe an in vitro model system that allowed us to define important parameters for Vpu-dependent CD4 degradation. The rate of CD4 decay in rabbit reticulocyte lysate was approximately one-third of that observed previously in tissue culture experiments in the presence of Vpu (40 versus 12 min) and required no other HIV-1 encoded proteins. Degradation was contingent on the presence of microsomal membranes in the assay and the coexpression of Vpu and CD4 in the same membrane compartment. By using the in vitro degradation assay, the effects of specific mutations in CD4, including C-terminal truncations and glycosylation mutants, were analyzed. The results of these experiments indicate that Vpu has the capacity to induce degradation of glycosylated as well as nonglycosylated membrane-associated CD4. Truncation of 13 C-terminal amino acids of CD4 did not affect the ability of Vpu to induce its degradation. However, the removal of 32 amino acids from the C-terminus of CD4 completely abolished sensitivity to Vpu. This suggests that Vpu targets specific sequences in the cytoplasmic domain of CD4 to induce its degradation. We also analyzed the effects of mutations in Vpu on its biological activity in the in vitro CD4 degradation assay. The results of these experiments suggest that sequences critical for this function of Vpu are located in its hydrophilic C-terminal domain.     

Human immunodeficiency virus type 1 envelope gp120 is cleaved after incubation with recombinant soluble CD4.

Human immunodeficiency virus type 1 (HIV-1) infects human CD4+ cells by a high-affinity interaction between its envelope glycoprotein gp120 and the CD4 molecule on the cell surface. Subsequent virus entry into the cells involves other steps, one of which could be cleavage of the gp120 followed by virus-cell fusion. The envelope gp120 is highly variable among different HIV-1 isolates, but conserved amino acid sequence motifs that contain potential proteolytic cleavage sites can be found. Following incubation with a soluble form of CD4, we demonstrate that gp120 of highly purified HIV-1 preparations is, without addition of exogenous proteinase, cleaved most likely in the V3 loop, yielding two proteins of 50 and 70 kDa. The extent of gp120 proteolysis is HIV-1 strain dependent and correlates with the recombinant soluble CD4 sensitivity to neutralization of the particular strain. The origin of the proteolytic activity in the virus preparations remains unclear. The results support the hypothesis that cleavage of gp120 is required for HIV infection of cells.     

Human immunodeficiency virus type 1 Vpr protein does not modulate surface expression of the CD4 receptor

The CD4 receptor is required for the entry of human immunodeficiency virus (HIV) into target cells. It has long been known that Nef, Env, and Vpu participate in the removal of the viral receptor from the cell surface. Recently, it has been proposed that the HIV type 1 (HIV-1) Vpr protein may also play a role in the downmodulation of CD4 from the surfaces of infected cells (L. Conti, B. Varano, M. C. Gauzzi, P. Matarrese, M. Federico, W. Malorani, F. Belardelli, and S. Gessani, J. Virol. 74:10207-10211, 2000). To investigate the possible role of Vpr in the downregulation of the viral receptor Vpr alleles from HIV-1 and simian immunodeficiency virus were transiently expressed in transformed T cells and in 293T fibroblasts, and their ability to modulate surface CD4 was evaluated. All Vpr alleles efficiently arrested cells in the G(2) stage of the cell cycle. However, none of the tested Vpr proteins altered the expression of CD4 on the cell surface. In comparison, HIV-1 Nef efficiently downmodulated surface CD4 in all the experimental settings. Transformed T cells and primary lymphocytes were challenged with wild-type, Nef-defective, and Vpr-defective viruses. A significant reduction in the HIV-induced downmodulation of surface CD4 was observed in viruses lacking Nef. However, Vpr-deletion-containing viruses showed no defect in their ability to remove CD4 from the surfaces of infected cells. Our results indicate that Vpr does not play a role in the HIV-induced downmodulation of the CD4 receptor.     

Human immunodeficiency virus type 1 Nef induces accumulation of CD4 in early endosomes.

We have studied the fate of CD4 in CEM T cells expressing a human immunodeficiency virus type 1 HIV-1 Nef protein. Nef triggered a rapid endocytosis and a degradation of CD4, while most of the p56lck was upheld at the cell membrane. In the presence of Nef, CD4 accumulated in acidic intracellular vesicles that were not stained by antibodies against rab6, a marker of the Golgi apparatus complex. Detection of transferrin in CD4-containing vesicles showed that CD4 was trapped in early endosomes, without significant accumulation of CD4 in late endocytic compartments. Internalization pathways taken by CD4 in Nef+ cells may therefore be different from those observed after treatment with phorbol esters.     

Lentiviral vectors interfering with virus-induced CD4 down-modulation potently block human immunodeficiency virus type 1 replication in primary lymphocytes

CD4 down-modulation is essential for the production of human immunodeficiency virus (HIV) infectious particles. Disease progression correlates with enhanced viral induced CD4 down-modulation, and a subset of long-term nonprogressors carry viruses defective in this function. Despite multiple pieces of evidence highlighting the importance of this function in viral pathogenesis in vivo, to date, HIV-induced CD4 down-modulation has not been used as a target for intervention. We describe here HIV-based vectors that deliver truncated CD4 molecules resistant to down-modulation by the viral products Nef and Vpu. Infection of cells previously transduced with these vectors proceeded normally, and viral particles were released in normal amounts. However, the infectivity of the released virions was reduced 1,000-fold. Lentiviral vectors expressing truncated CD4 molecules were efficient at blocking HIV-1 infectivity and replication in several cell lines and in CD4-positive primary lymphocytes. The findings presented here provide proof-of-principle that approaches targeting the virus-induced CD4 down-modulation may constitute the basis for novel anti-HIV therapies.     

Contribution of Vpu, Env, and Nef to CD4 down-modulation and resistance of human immunodeficiency virus type 1-infected T cells to superinfection

Human immunodeficiency virus type 1 (HIV-1) utilizes Vpu, Env, and Nef to down-modulate its primary CD4 receptor from the cell surface, and this function seems to be critical for the pathogenesis of AIDS. The physiological relevance of CD4 down-modulation, however, is currently not well understood. In the present study, we analyzed the kinetics of CD4 down-modulation and the susceptibility of HIV-1-infected T cells to superinfection using proviral HIV-1 constructs containing individual and combined defects in vpu, env, and nef and expressing red or green fluorescent proteins. T cells infected with HIV-1 mutants containing functional nef genes expressed low surface levels of CD4 from the first moment that viral gene expression became detectable. In comparison, Vpu and Env had only minor to moderate effects on CD4 during later stages of infection. Consistent with these quantitative differences, Nef inhibited superinfection more efficiently than Vpu and Env. Notably, nef alleles from AIDS patients were more effective in preventing superinfection than those derived from a nonprogressor of HIV-1 infection. Our data suggest that protection against X4-tropic HIV-1 superinfection involves both CD4-independent and CD4-dependent mechanisms of HIV-1 Nef. X4 was effectively down-regulated by simian immunodeficiency virus and HIV-2 but not by HIV-1 Nef proteins. Thus, maximal protection seems to involve an as-yet-unknown mechanism that is independent of CD4 or coreceptor down-modulation. Finally, we demonstrate that superinfected primary T cells show enhanced levels of apoptosis. Accordingly, one reason that HIV-1 inhibits CD4 surface expression and superinfection is to prevent premature cell death in order to expand the period of effective virus production.     

Human immunodeficiency virus type 1 Vpu has a CD4- and an envelope glycoprotein-independent function.

Vpu is a 16-kDa membrane-associated phosphoprotein that is expressed from the same, singly spliced message as the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor, gp160. Previous studies suggest that Vpu functions in the late stages of viral replication, possibly in virus egression from the cell. Recently, it has been demonstrated that Vpu functions to allow gp160 to be more efficiently processed by disrupting CD4-gp160 complexes generated by transfection of HeLa cells. We show here that the lack of expression of intact Vpu results in a 90% reduction in infectious virus produced over a single round of replication from HeLa cells in the absence of CD4 expression. This reduction persists when HIV-1 particles are pseudotyped with the HIV-2 or amphotropic murine leukemia virus envelope glycoprotein. Pulse-chase analysis of HIV-1 capsid protein (p24) in the absence of CD4 and envelope glycoprotein demonstrates that the rate of virus release is reduced when Vpu is not expressed. Our findings indicate that Vpu has a function involving particle release not dependent on CD4 or envelope glycoprotein expression.     

Unintegrated human immunodeficiency virus type 1 DNA in chronically infected cell lines is not correlated with surface CD4 expression.

Using a polymerase chain reaction-based assay on total cell lysates, we have detected unintegrated human immunodeficiency virus type 1 (HIV-1) DNA in chronically infected T-lymphocytic (ACH-2, J1) and promyelocytic (OM-10.1) cell lines. Treatment with 3'-azido-3'-deoxythymidine (AZT) or soluble CD4 inhibited accumulation of unintegrated viral DNA about 10-fold within 72 h; removal of AZT permitted recovery to pretreatment levels within 72 h. Our results indicate that unintegrated HIV-1 DNA is unstable in these cell lines and originates from a continuous process of reinfection. OM-10.1 cells had relatively high levels of surface CD4 by flow cytometry and high levels of unintegrated viral DNA by polymerase chain reaction. ACH-2 cells had very low levels of both surface CD4 and unintegrated viral DNA. However, J1 cells, with surface CD4 below the level of detection of flow cytometry had a high level of unintegrated viral DNA similar to that of OM-10.1 cells. This implies that the number of CD4 receptors is not rate limiting for reinfection.     

Mapping genetic determinants for human immunodeficiency virus type 1 resistance to soluble CD4.

Neutralization of human immunodeficiency virus type 1 (HIV-1) infection with soluble CD4 (sCD4) can be achieved over a broad range of concentrations for different virus strains. Laboratory virus strains passaged in transformed T-cell lines are typically sensitive to sCD4 neutralization, whereas primary virus isolates require over 100-fold-higher sCD4 concentrations. Using recombinant viruses generated from a laboratory strain, HIV-1NL4-3, and a primary macrophagetropic strain, HIV-1JR-FL, we mapped a region of gp120 important for determining sensitivity to sCD4 neutralization. This same region has previously been defined as important for macrophage and transformed T-cell line tropism and includes the V3 neutralization domain but does not include regions of gp120 that have been shown to be most important for CD4 binding.     

The negative effect of human immunodeficiency virus type 1 Nef on cell surface CD4 expression is not species specific and requires the cytoplasmic domain of CD4.

The nef gene product of human immunodeficiency virus type 1 has been shown to induce CD4 downregulation from the surface of human cells. To determine if this effect is species specific, we used a retroviral vector to transduce the human immunodeficiency virus type 1 nef gene into murine cells expressing human, chimpanzee, or murine CD4. Our results indicate that Nef induces cell surface downregulation of all three molecules. We also determined that Nef is functional in murine T cells and induces downregulation of both murine CD4 and CD8 (Ly-2) from the cell surface. In contrast, Nef does not downregulate cell surface expression of human CD8 in either murine or human cells. By using a mutant of human CD4 lacking its cytoplasmic domain and a human CD4/CD8 chimera, we determined that the cytoplasmic domain of CD4 is required for its downregulation by Nef. Transduction with a control vector had no effect on CD4 cell surface levels, indicating that retroviral transduction by itself has no significant effect on the cell surface levels of CD4. These results show that the observed downregulation of CD4 by Nef is independent of human-specific factors, is not species specific, and requires the cytoplasmic domain of CD4.     

Human transformed trophoblast-derived cells lacking CD4 receptor exhibit restricted permissiveness for human immunodeficiency virus type 1.

We investigated the nature of interaction of the malignantly transformed cell lines of trophoblast origin BeWo, JAR, and JEG-3 with three different human immunodeficiency virus type 1 (HIV-1) isolates (RF, 3B, and NDK). After inoculation with cell-free virus, the persistence of infection was determined for 1 month by monitoring the presence of viral DNA in the cells by the polymerase chain reaction (PCR). Furthermore, the infectious virus in the culture supernatant was assayed with CEM-SS cells, and attempts to rescue the virus by cocultivation with CEM-SS cells were made. Appraised on the basis of the relative amount of viral DNA and the frequency of positive cocultivation. JEG-3 was the most permissive and BeWo was the least permissive cell line. However, when the cells were transfected with two biologically active molecular clones of HIV-1, the BRU and NDK isolates, all three cell lines turned out to support the production of mature virus progeny to the same extent. The abundance of viral DNA sequences in the infected cells varied with the isolate, showing an overall decline from RF to NDK. The amount of viral DNA in the cells and its expression decreased during the period of observation; this decrease was mirrored in an erosion of the virus recovery rate at cocultivation from 71% recovery on day 8 to failure of isolation on day 32. None of the cell lines expressed detectable amounts of cell surface CD4 molecules when assayed by flow microfluorometry and direct radioimmunoassay. Northern (RNA) blot hybridization analysis of both the total RNA and the mRNA did not reveal any CD4-specific message: nonetheless, by using the PCR, sequences specifically related to the CD4 gene were uncovered. The data demonstrate that the trophoblast-derived cell lines are susceptible to infection with HIV and that they support transient viral replication in the initial phases of infection. However, the latent form of infection may persist over a period of several weeks.     

Human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus Nef use distinct but overlapping target sites for downregulation of cell surface CD4.

Although the Nef proteins encoded by human immunodeficiency virus type 1 (HIV-1) and simian immuno-deficiency virus (SIV) are known to induce the efficient internalization and degradation of cell surface CD4, it remains unclear whether this process involves a direct interaction between Nef and CD4. Here, we report that CD4 downregulation by HIV-1 and SIV Nef requires distinct but overlapping target sites within the CD4 intracytoplasmic domain. In particular, mutation of a glutamic acid residue located at CD4 residue 405 or of arginine and methionine residues located, respectively, at residue 406 and 407 results in a mutant CD4 protein that is efficiently downregulated by HIV-1 Nef but refractory to downregulation by SIV Nef. However, both HIV-1 and SIV Nef require an isoleucine located at residue 410 and the dileucine motif found at CD4 residues 413 and 414. CD4 downregulation induced by the Nef protein encoded by HIV-2 is shown to require a CD4 target sequence that is similar to, but distinct from, that observed with SIV Nef. These data explain the previous finding that the murine CD4 protein, which has an alanine at residue 405, is refractory to downregulation by SIV, but not HIV-1, Nef (J. L. Foster, S.J. Anderson, A. L. B. Frazier, and J. V. Garcia, Virology 201:373-379, 1994). In addition, these observations provide strong genetic support for the hypothesis that the Nef-mediated downregulation of cell surface CD4 requires a direct Nef-CD4 interaction.     

CD4 down-modulation during infection of human T cells with human immunodeficiency virus type 1 involves independent activities of vpu, env, and nef.

The human immunodeficiency virus type 1 (HIV-1) genes vpu, env, and nef have all been implicated in modulating the levels of cell surface CD4 on infected cells. To quantitatively assess the relative contribution of each gene product to the regulation of CD4 during HIV infection of Jurkat T cells and peripheral blood mononuclear cells, we have developed an infectious HIV reporter system which expresses different combinations of these genes. To distinguish infected cells in the early or late stages of infection from uninfected cells, these viruses were designed to express human placental alkaline phosphatase with the kinetics of either early or late viral genes. Flow cytometry to detect placental alkaline phosphatase and CD4 in infected cells showed that vpu, env, and nef are independently capable of down-modulation of CD4. As predicted by their respective expression patterns, nef down-modulated CD4 rapidly during the early phase of virus infection whereas vpu and env functioned late in the infection. In both Jurkat cells and peripheral blood mononuclear cells, a combination of the three genes was more efficient than any one or two genes, demonstrating that all three genes are required to achieve maximal CD4 down-modulation. In primary cells, down-modulation of CD4 was less efficient than in Jurkat cells and there was a stronger dependence on nef function for reducing cell surface CD4. HIV therefore has three genes that are able to independently down-modulate CD4; together, they can eliminate the bulk of cell surface CD4.