The cytoplasmic domain of CD4 is sufficient for its down-regulation from the cell surface by human immunodeficiency virus type 1 Nef.

Human immunodeficiency virus type 1 Nef down-regulates surface expression of murine and human CD4 but not human CD8. We recently reported that the cytoplasmic domain of CD4 is required for its down-regulation by Nef. Using a chimeric molecule composed of the extracellular and transmembrane domains of human CD8 fused to the cytoplasmic domain of human CD4, we show here that the cytoplasmic domain of CD4 is sufficient for down-regulation by Nef. Since the cytoplasmic domain of CD4 is also the site of its association with p56lck, we used a series of CD4 mutants to determine whether the regions of the cytoplasmic domain of CD4 required for down-regulation by Nef are the same as those required for p56lck binding. Our results indicate that the portion of the cytoplasmic domain required for the down-regulation of CD4 by Nef overlaps with the binding site of p56lck, but the cysteine residues which are essential for the association of CD4 with p56lck are not required. This observation raised the possibility that Nef competes with p56lck for binding to CD4. However, under conditions which are considerably milder than those permissive for coimmunoprecipitation of CD4 and p56lck, we found no evidence for an association between Nef and CD4. While a decrease in total CD4 was observed in lysates of cells expressing Nef, the levels of p56lck were not significantly affected. Pulse-chase experiments further revealed a decrease in the half-life of CD4 in Nef-expressing cells. These results show that the decrease in surface CD4 expression induced by Nef is mediated at least in part by a decrease in the half-life of CD4 protein. These results also indicate that a large portion of p56lck is free of CD4 in T cells expressing Nef, which could have a significant effect on T-cell function.     

Direct in vitro binding of full-length human immunodeficiency virus type 1 Nef protein to CD4 cytoplasmic domain

The Nef protein of the simian and human immunodeficiency viruses is known to directly bind and downregulate the CD4 receptor. Although the molecular mechanism is well understood, direct binding of Nef and CD4 is difficult to demonstrate and is believed to be of low affinity. Applying nuclear magnetic resonance and fluorescence spectroscopy, we biophysically reevaluated the CD4-Nef complex and found the dissociation constant to be in the submicromolar range. We conclude that additional, so far disregarded residues in the N terminus of Nef are important for interaction with CD4.     

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.     

Reduced cell surface expression of processed human immunodeficiency virus type 1 envelope glycoprotein in the presence of Nef.

nef genes from two laboratory grown human immunodeficiency virus type 1 (HIV-1) strains and from two proviruses that had not been propagated in vitro were introduced into CD4+ lymphoblastoid CEM cells. The stable expression of all four Nef proteins was associated with an almost complete abrogation of CD4 cell surface localization. The consequences of the presence of Nef on gp160 cleavage, gp120 surface localization, and envelope-induced cytopathic effect were examined in CEM cells in which the HIV-1 env gene was expressed from a vaccinia virus vector. The presence of Nef did not modify the processing of gp160 into its subunits but resulted in a significant decrease of cell surface levels of gp120, associated with a dramatic reduction of the fusion-mediated cell death. Surface levels of mutant envelope glycoproteins unable to bind CD4 were not altered in Nef-expressing cells, suggesting that the phenomenon was CD4 dependent. The intracellular accumulation of fully processed envelope glycoproteins could significantly delay the cytopathic effect associated with envelope surface expression in HIV-infected cells and may be relevant to the selective advantage associated with Nef during the in vivo infectious process.     

Functional characterization of the human immunodeficiency virus type 1 Nef acidic domain

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef downregulates major histocompatibility complex class I (MHC-I) from the cell surface. It has been proposed that the direct interaction of the acidic cluster (AC) of Nef, (62)EEEE(65), with the furin binding region (fbr) of PACS-1 is crucial for this Nef function. Contrary to this proposal, evidence is presented here that the four glutamates in Nef do not functionally engage the PACS-1 fbr. (i) The binding of Nef to the PACS-1 fbr in vitro is much weaker than the binding of the canonical furin AC to the PACS-1 fbr. (ii) The mutation of two of the four glutamates in Nef's AC to alanines does not alter Nef's ability to downregulate MHC-I, and triply mutated Nefs exhibit 50% activity. (iii) The introduction of lysine into the AC has little effect on Nef function. (iv) The mutation of all four glutamates to alanine does debilitate Nef MHC-I downregulation, but this quadruple mutation also impairs the ability of Nef to regulate p21-activated protein kinase and enhance viral particle infectivity. (v) The replacement of the Nef AC with the bona fide AC from furin results in the loss of the expected regulatory properties of the furin AC. (vi) The insertion of the conformation-disrupting amino acid proline into the Nef AC does not disrupt MHC-I downregulation. Our results are consistent with an alternative model in which (62)EEEE(65) plays a stabilizing role in the formation of a ternary complex between Nef, the MHC-I cytoplasmic domain, and AP-1.     

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.     

Phosphorylation-dependent down-modulation of CD4 requires a specific structure within the cytoplasmic domain of CD4.

Several structural features of the cytoplasmic domain of CD4 including phosphorylation of Ser-408 have been shown to be important in its endocytosis (Shin, J., Doyle, C., Yang, Z., Kappes, D., and Strominger, J.L. (1990) EMBO J. 9, 425-434). A series of cytoplasmic domain truncations have now indicated that the membrane proximal region of the cytoplasmic domain from Arg-396 to Lys-417 is sufficient for phorbol ester-induced internalization; this segment is predicted to be an alpha-helix. The severe impairment of endocytosis resulting from the mutation Ser-408 to Ala-408 is largely restored by a compensating mutation Ala-404 to Ser-404; phosphorylation of Ser-404 has been directly demonstrated. Furthermore, mutation of Met-407, Ile-410, Leu-413, or Leu-414 to a hydrophilic residue eliminated CD4 endocytosis as did domain truncation at Arg-412. Ser-408 was normally phosphorylated in all of these mutants, suggesting that other residues in this region, including the four hydrophobic amino acids, are also required for CD4 endocytosis. Immunofluorescence microscopy following staining of intact and permeabilized cells showed that all endocytosis defective mutants indeed remained on the cell surface even after phorbol ester treatment, while wild type CD4 was endocytosed and degraded in lysosomes. These data indicate that endocytosis requiring residues 397-417 and binding of lymphocyte tyrosine kinase at residues 417-429 are functions of independent segments of the cytoplasmic region and lead to a hypothesis regarding some features of the endocytic process.     

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.     

Dissociation of the CD4 downregulation and viral infectivity enhancement functions of human immunodeficiency virus type 1 Nef.

Recent evidence indicates that the nef gene of human immunodeficiency virus type 1 augments rather than inhibits viral replication in both cell culture and in vivo models. In addition, nef alters various normal cellular processes, including the display of CD4 on the cell surface. However, it remains unknown whether the enhancement of infectivity and the downregulation of CD4 represent linked or independent biologic properties of this single protein. In the present studies, mutational analyses were performed to define structure-function relationships within the Nef protein that mediate these effects. To assess the functional consequences of these mutations, sensitive and reliable assays were developed to quantitate the viral infectivity enhancement and CD4 downregulation functions of Nef. The results indicate that membrane-targeting sequences at the N terminus of Nef are important for both functions of Nef, while certain other conserved regions are dispensable for both functions. A conserved proline-X-X repeat segment in the central core of the protein, which is reminiscent of an SH3-binding domain, is critical for the enhancement of infectivity function but is dispensable for CD4 downregulation. However, the downregulation of CD4 by Nef appears to involve a two-step process requiring the initial dissociation of p56lck from CD4 to permit engagement of the endocytic apparatus by CD4. Together, these findings demonstrate that the infectivity enhancement and CD4 downregulation activities of human immunodeficiency virus type 1 Nef can be dissociated. Thus, these processes may be independent of one another in the viral replication cycle.     

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.     

Truncation of the human immunodeficiency virus type 1 transmembrane glycoprotein cytoplasmic domain blocks virus infectivity.

Human immunodeficiency virus type 1 contains a transmembrane glycoprotein with an unusually long cytoplasmic domain. To determine the role of this domain in virus replication, a series of single nucleotide changes that result in the insertion of premature termination codons throughout the cytoplasmic domain has been constructed. These mutations delete from 6 to 192 amino acids from the carboxy terminus of gp41 and do not affect the amino acid sequence of the regulatory proteins encoded by rev and tat. The effects of these mutations on glycoprotein biosynthesis and function as well as on virus infectivity have been examined in the context of a glycoprotein expression vector and the viral genome. All of the mutant glycoproteins were synthesized, processed, and transported to the cell surface in a manner similar to that of the wild-type glycoprotein. With the exception of mutants that remove the membrane anchor domain, all of the mutant glycoproteins retained the ability to cause fusion of CD4-bearing cells. However, deletion of more than 19 amino acids from the C terminus of gp41 blocked the ability of mutant virions to infect cells. This defect in virus infectivity appeared to be due at least in part to a failure of the virus to efficiently incorporate the truncated glycoprotein. Similar data were obtained for mutations in two different env genes and two different target cell lines. These results indicate that the cytoplasmic domain of gp41 plays a critical role during virus assembly and entry in the life cycle of human immunodeficiency virus type 1.     

The human immunodeficiency virus type 1 Vpu protein specifically binds to the cytoplasmic domain of CD4: implications for the mechanism of degradation.

We have recently demonstrated that coexpression of Vpu and CD4 in HeLa cells results in the degradation of CD4 in the endoplasmic reticulum. The sensitivity of CD4 to Vpu-mediated degradation is conferred by the presence of specific sequences located between amino acids 402 and 420 in the CD4 cytoplasmic domain. Using an in vitro translation system, we also showed that degradation of CD4 by Vpu requires the two proteins to be present in the same membrane compartment. Although these results suggest that spatial proximity between CD4 and Vpu may be critical in triggering degradation, it remains unknown whether the two molecules have the ability to interact with each other. In order to better define the mechanisms involved in CD4 degradation, we investigated the existence and functional relevance of direct interactions between CD4 and Vpu. Coimmunoprecipitation experiments showed that Vpu specifically binds to the cytoplasmic tail of CD4. This phenomenon is relevant to the mechanism of CD4 degradation since the ability of CD8/CD4 chimeric molecules and various CD4 mutants to form complexes with Vpu correlates with their sensitivity to degradation. Accordingly, we found that amino acid residues in the CD4 cytoplasmic tail previously shown to be important for degradation are necessary for Vpu binding. We further demonstrate that a deletion mutant of Vpu as well as a phosphorylation mutant, both biologically inactive with regard to CD4 degradation, retained the capacity to interact with the CD4 cytoplasmic domain. Taken together, these results indicate that Vpu binding is necessary to trigger CD4 degradation. However, the binding to target molecules is not sufficient per se to cause degradation. Interaction between CD4 and Vpu is thus likely to be an early event critical in triggering a multistep process leading to CD4 degradation.     

Downregulation of CD4 by human immunodeficiency virus type 1 Nef is dependent on clathrin and involves direct interaction of Nef with the AP2 clathrin adaptor

Nef, an accessory protein of human and simian immunodeficiency viruses, is a critical determinant of pathogenesis that promotes the progression from infection to AIDS. The pathogenic effects of Nef are in large part dependent on its ability to downregulate the macrophage and T-cell coreceptor, CD4. It has been proposed that Nef induces downregulation by linking the cytosolic tail of CD4 to components of the host-cell protein trafficking machinery. To identify these components, we developed a novel Nef-CD4 downregulation system in Drosophila melanogaster S2 cells. We found that human immunodeficiency virus type 1 (HIV-1) Nef downregulates human CD4 in S2 cells and that this process is subject to the same sequence requirements as in human cells. An RNA interference screen targeting protein trafficking genes in S2 cells revealed a requirement for clathrin and the clathrin-associated, plasma membrane-localized AP2 complex in the downregulation of CD4. The requirement for AP2 was confirmed in the human cell line HeLa. We also used a yeast three-hybrid system and glutathione S-transferase pull-down analyses to demonstrate a robust, direct interaction between HIV-1 Nef and AP2. This interaction requires a dileucine motif in Nef that is also essential for downregulation of CD4. Together, these results support a model in which HIV-1 Nef downregulates CD4 by promoting its accelerated endocytosis by a clathrin/AP2 pathway.     

Restoration of cell surface CD4 expression in human immunodeficiency virus type 1-infected cells by treatment with a Tat antagonist.

Productive infection of T lymphocytes with human immunodeficiency virus type 1 (HIV-1) is accompanied by a diminution of surface CD4 receptors. Treatment of chronically HIV-1-infected CD4-negative T cells in vitro with the Tat antagonist Ro 5-3335 resulted in a drug dose-dependent decrease in virus protein production and a reciprocal increase in surface CD4 display. The drug-treated cells remained viable, showed significantly reduced levels of the full-length and spliced HIV-1 mRNAs as detected by Northern (RNA) blot hybridization, and maintained integrated HIV-1 DNA. In immunoprecipitation studies with drug-treated cells, the levels of free 55-kDa CD4 protein increased and gp160 complexed with CD4 decreased in amount. These results show for the first time that certain cytopathogenic effects of chronic HIV-1 infection can be reversed by suppressing virus expression.     

Producer-cell modification of human immunodeficiency virus type 1: Nef is a virion protein.

Type 1 human immunodeficiency viruses encoding mutated nef reading frames are 10- to 30-fold less infectious than are isogenic viruses in which the nef gene is intact. This defect in infectivity causes nef-negative viruses to grow at an attenuated rate in vitro. To investigate the mechanism of Nef-mediated enhancement of viral growth rate and infectivity, a complementation analysis of nef mutant viruses was performed. To provide Nef in trans upon viral infection, a CEM derivative cell line (designated CLN) that expresses Nef under the control of the viral long terminal repeat was constructed. When nef-negative virus was grown in CLN cells, its growth rate was restored to wild-type levels. However, the output of nef-negative virus during the first 72 h after infection of CLN cells was not restored, suggesting that provision of Nef within the newly infected cell does not enhance the productivity of a nef-negative provirus. The genetically nef-negative virions produced by the CLN cells, however, were restored to wild-type levels of infectivity as measured in a syncytium formation assay in which CD4-expressing HeLa cells were targets. These trans-complemented, genetically nef-negative virions yielded wild-type levels of viral output following a single cycle of replication in primary CD4 T cells as well as in parental CEM cells. To define the determinants for producer cell modification of virions by Nef, the role of myristoylation was investigated. Virus that encodes a myristoylation-negative nef was as impaired in infectivity as was virus encoding a deleted nef gene. Because myristoylation is required for both membrane association of Nef and optimal viral infectivity, the possibility that Nef protein is included in the virion was investigated. Wild-type virions were purified by filtration and exclusion chromatography. A Western blot (immunoblot) of the eluate fractions revealed a correlation between peak Nef signal and peak levels of p24 antigen. Although virion-associated Nef was detected in part as the 27-kDa full-length protein, the majority of immunoreactive protein was detected as a 20-kDa isoform. nef-negative virus lacked both 27- and 20-kDa immunoreactive species. Production of wild-type virions in the presence of a specific inhibitor of the human immunodeficiency virus type 1 protease resulted in virions which contained only 27-kDa full-length Nef protein. These data indicate that Nef is a virion protein which is processed by the viral protease into a 20-kDa isoform within the virion particle.     

Integration is not necessary for expression of human immunodeficiency virus type 1 protein products.

A common feature in the life cycle of cytocidal retroviruses, including human immunodeficiency virus type 1 (HIV-1), is the accumulation of large amounts of unintegrated viral DNA. As yet, the role of unintegrated viral DNA in the cytopathogenesis of cytocidal retrovirus infections remains unresolved. HIV-1 mutants which were deleted in the integrase/endonuclease gene and which were unable to establish an integrated form of the virus were constructed. Despite an inability to integrate, these mutants were fully competent templates for HIV-1 core and envelope antigen production. HIV-1 antigen could be detected in the supernatants of lymphocyte cultures infected with HIV-1 integrase mutants. However, an inability to rescue infectious virus from these cultures indicated that HIV-1 integration was required for the production of infectious HIV-1. On the basis of the ability of unintegrated HIV-1 DNA to serve as a template for HIV-1 antigen production, it is plausible that unintegrated viral DNA can contribute to the HIV-1 antigen pool during HIV-1 replication.