Structural and functional characterization of human immunodeficiency virus tat protein.

Site-directed mutagenesis was used to identify functional domains present within the human immunodeficiency virus (HIV) tat protein. Transient cotransfection experiments showed that derivatives of tat protein with amino acid substitutions either at the amino-terminal end or at cysteine residue 22, 37, 27, or 25 were no longer able to transactivate HIV long terminal repeat-directed gene expression. Incubation of Tat expressed in Escherichia coli with zinc demonstrated that both authentic Tat and cysteine mutation derivatives could form metal-protein complexes. The tat proteins that contained alterations within the cluster of positively charged amino acid residues retained their ability to transactivate gene expression, albeit at markedly reduced levels. Indirect immunofluorescence showed that the authentic tat protein and the amino-terminal and cysteine substitution mutants all localized in the nucleus, with accumulation being most evident in the nucleolus. In contrast, nuclear accumulation was greatly reduced with the basic-substitution mutations. Consistent with this result, a fusion protein that contained amino acids GRKKR, derived from the basic region, fused to the amino-terminal end of beta-galactosidase also accumulated within the nucleus. These results demonstrate that the 14-kilodalton tat protein contains at least three distinct functional domains affecting localization and transactivation.     

The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels.

Vpu is a small phosphorylated integral membrane protein encoded by the human immunodeficiency virus type 1 genome and found in the endoplasmic reticulum and Golgi membranes of infected cells. It has been linked to roles in virus particle budding and degradation of CD4 in the endoplasmic reticulum. However, the molecular mechanisms employed by Vpu in performance of these functions are unknown. Structural similarities between Vpu and the M2 protein of influenza A virus have raised the question of whether the two proteins are functionally analogous: M2 has been demonstrated to form cation-selective ion channels in phospholipid membranes. In this paper we provide evidence that Vpu, purified after expression in Escherichia coli, also forms ion channels in planar lipid bilayers. The channels are approximately five- to sixfold more permeable to sodium and potassium cations than to chloride or phosphate anions. A bacterial cross-feeding assay was used to demonstrate that Vpu can also form sodium-permeable channels in vivo in the E. coli plasma membrane.     

Differential effects of human immunodeficiency virus type 1 Vpu on the stability of BST-2/tetherin

BST-2/CD317/tetherin is a host factor that inhibits the release of HIV-1 and other unrelated viruses. A current model proposes that BST-2 physically tethers virions to the surface of virus-producing cells. The HIV-1-encoded Vpu protein effectively antagonizes the activity of BST-2. How Vpu accomplishes this task remains unclear; however, it is known that Vpu has the ability to down-modulate BST-2 from the cell surface. Here we analyzed the effects of Vpu on BST-2 by performing a series of kinetic studies with HeLa, 293T, and CEMx174 cells. Our results indicate that the surface downregulation of BST-2 is not due to an accelerated internalization or reduced recycling of internalized BST-2 but instead is caused by interference with the resupply of newly synthesized BST-2 from within the cell. While our data confirm previous reports that the high-level expression of Vpu can cause the endoplasmic reticulum (ER)-associated degradation of BST-2, we found no evidence that Vpu targets endogenous BST-2 in the ER in the course of a viral infection. Instead, we found that Vpu acts in a post-ER compartment and increases the turnover of newly synthesized mature BST-2. Our observation that Vpu does not affect the recycling of BST-2 suggests that Vpu does not act directly at the cell surface but may interfere with the trafficking of newly synthesized BST-2 to the cell surface, resulting in the accelerated targeting of BST-2 to the lysosomal compartment for degradation.     

Determinants of tetherin antagonism in the transmembrane domain of the human immunodeficiency virus type 1 Vpu protein

Tetherin (BST2/CD317) potently restricts the particle release of human immunodeficiency virus type 1 (HIV-1) mutants defective in the accessory gene vpu. Vpu antagonizes tetherin activity and induces its cell surface downregulation and degradation in a manner dependent on the transmembrane (TM) domains of both proteins. We have carried out extensive mutagenesis of the HIV-1 NL4.3 Vpu TM domain to identify three amino acid positions, A14, W22, and, to a lesser extent, A18, that are required for tetherin antagonism. Despite the mutants localizing indistinguishably from the wild-type (wt) protein and maintaining the ability to multimerize, mutation of these positions rendered Vpu incapable of coimmunoprecipitating tetherin or mediating its cell surface downregulation. Interestingly, these amino acid positions are predicted to form one face of the Vpu transmembrane alpha helix and therefore potentially contribute to an interacting surface with the transmembrane domain of tetherin either directly or by modulating the conformation of Vpu oligomers. While the equivalent of W22 is invariant in HIV-1/SIVcpz Vpu proteins, the positions of A14 and A18 are highly conserved among Vpu alleles from HIV-1 groups M and N, but not those from group O or SIVcpz that lack human tetherin (huTetherin)-antagonizing activity, suggesting that they may have contributed to the adaption of HIV-1 to human tetherin.     

Structural and functional analysis of the human immunodeficiency virus type 2 Rev protein.

The Rev proteins of the human immunodeficiency virus (HIV) are necessary for expression of viral structural gene products. Site-directed mutations were made within the HIV-2 rev gene to identify functional domains. We observed that similar to HIV-1 Rev, the HIV-2 Rev protein was phosphorylated, albeit to a much lesser extent than was HIV-1 Rev. We also found that like HIV-1 Rev, HIV-2 Rev localized to the nucleus, with a marked accumulation in the nucleolus. Mutations within a stretch of basic residues prevented both nuclear and nucleolar localization. Furthermore, mutant Rev proteins able to localize in the nucleus but unable to localize in the nucleolus were nonfunctional.     

The human immunodeficiency virus type 1 Nef and Vpu proteins downregulate the natural killer cell-activating ligand PVR

The human immunodeficiency virus type 1 (HIV-1) evades the immune responses of natural killer (NK) cells through mechanisms that have been partially deciphered. Here we show that in HIV-1-infected T lymphocytes, the early viral Nef protein downmodulates PVR (CD155, Necl-5), a ligand for the activating receptor DNAM-1 (CD226) expressed by all NK cells, CD8(+) T cells, and other cell types. This novel Nef activity is conserved by Nef proteins of laboratory HIV-1 strains (NL4-3, SF2) and of a patient-derived virus, but it is not maintained by HIV-2. Nef uses the same motifs to downregulate PVR and HLA-I molecules, likely by the same mechanisms. Indeed, as previously demonstrated for HLA-I, Nef reduces the total amounts of cell-associated PVR. Optimal downregulation of cell surface PVR by Nef also requires the presence of the late viral factor Vpu. In line with PVR reduction, the NK cell-mediated lysis of T cells infected by a wild-type but not Nef-deficient virus is virtually abrogated upon blocking of both DNAM-1 and another activating receptor, NKG2D, previously shown to mediate killing of HIV-infected cells. Together, these data demonstrate that the PVR downmodulation by Nef and Vpu is a strategy evolved by HIV-1 to prevent NK cell-mediated lysis of infected cells. The PVR downregulation reported here has the potential to affect the immune responses of other DNAM-1-positive cells besides NK cells and to alter multiple PVR-mediated cellular processes, such as adhesion and migration, and may thus greatly influence HIV-1 pathogenesis.     

Reciprocal regulatory interaction between human herpesvirus 8 and human immunodeficiency virus type 1

Human herpesvirus 8 (HHV8) is the primary viral etiologic agent in Kaposi's sarcoma (KS). However, individuals dually infected with both HHV8 and human immunodeficiency virus type 1 (HIV-1) show an enhanced prevalence of KS when compared with those singularly infected with HHV8. Host immune suppression conferred by HIV infection cannot wholly explain this increased presentation of KS. To better understand how HHV8 and HIV-1 might interact directly in the pathogenesis of KS, we queried for potential regulatory interactions between the two viruses. Here, we report that HHV8 and HIV-1 reciprocally up-regulate the gene expression of each other. We found that the KIE2 immediate-early gene product of HHV8 interacted synergistically with Tat in activating expression from the HIV-1 long terminal repeat. On the other hand, HIV-1 encoded Tat and Vpr proteins increased intracellular HHV8-specific expression. These results provide molecular insights correlating coinfection with HHV8 and HIV-1 with an unusually high incidence of KS.     

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.     

Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome

Generation of RNA dimeric form of the human immunodeficiency virus type 1 (HIV-1) genome is crucial for viral replication. The dimerization initiation site (DIS) has been identified as a primary sequence that can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem. It has been reported that HIV-1 RNA fragments containing the DIS form two types of dimers, loose dimers and tight dimers. The loose dimers are spontaneously generated at the physiological temperature and converted into tight dimers by the addition of nucleocapsid protein NCp7. To know the biochemical process in this two-step dimerization reaction, we chemically synthesized a 39-mer RNA covering the entire DIS sequence and also a 23-mer RNA covering the self-complementary loop and its flanking stem within the DIS. Electrophoretic dimerization assays demonstrated that the 39-mer RNA reproduced the two-step dimerization process, whereas the 23-mer RNA immediately formed the tight dimer. Furthermore, deletion of the bulge from the 39-mer RNA prevented the NCp7-assisted tight-dimer formation. Therefore, the whole DIS sequence is necessary and sufficient for the two-step dimerization. Our data suggested that the bulge region regulates the stability of the stem and guides the DIS to the two-step dimerization process.     

Detection and characterization of a functional complex of human immunodeficiency virus type 1 integrase and its DNA substrate by UV cross-linking.

To analyze the association of the human immunodeficiency virus type 1 integrase (IN) protein with DNA substrates, we applied a shortwave UV cross-linking method to the in vitro integration system. Three photoadduct bands were detected on sodium dodecyl sulfate-polyacrylamide gels. The appearances of these photoadducts were examined with various substrates and under several incubation conditions. Our data suggest that one of these photoadducts is derived from the sequence- and strand-specific bound state of the early phase of the integration reaction before the 3' processing reaction. We also show that most of the photoadduct complexes are competent for integration even after UV irradiation.     

Vpu and Tsg101 regulate intracellular targeting of the human immunodeficiency virus type 1 core protein precursor Pr55gag

Assembly of human immunodeficiency virus type 1 (HIV-1) is directed by the viral core protein Pr55gag. Depending on the cell type, Pr55gag accumulates either at the plasma membrane or on late endosomes/multivesicular bodies. Intracellular localization of Pr55gag determines the site of virus assembly, but molecular mechanisms that define cell surface or endosomal targeting of Pr55gag are poorly characterized. We have analyzed targeting of newly synthesized Pr55gag in HeLa H1 cells by pulse-chase studies and subcellular fractionations. Our results indicated that Pr55gag was inserted into the plasma membrane and, when coexpressed with the viral accessory protein Vpu, Pr55gag remained at the plasma membrane and virions assembled at this site. In contrast, Pr55gag expressed in the absence of Vpu was initially inserted into the plasma membrane, but subsequently endocytosed, and virus assembly was partially shifted to internal membranes. This endocytosis of Pr55gag required the host protein Tsg101. These results identified a previously unknown role for Vpu and Tsg101 as regulators for the endocytic uptake of Pr55gag and suggested that the site of HIV-1 assembly is determined by factors that regulate the endocytosis of Pr55gag.     

DC-SIGN interactions with human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus

Dendritic cells (DCs) efficiently bind and transmit human immunodeficiency virus (HIV) to cocultured T cells and so may play an important role in HIV transmission. DC-SIGN, a novel C-type lectin that is expressed in DCs, has recently been shown to bind R5 HIV type 1 (HIV-1) strains and a laboratory-adapted X4 strain. To characterize the interaction of DC-SIGN with primate lentiviruses, we investigated the structural determinants of DC-SIGN required for virus binding and transmission to permissive cells. We constructed a panel of DC-SIGN mutants and established conditions which allowed comparable cell surface expression of all mutants. We found that R5, X4, and R5X4 HIV-1 isolates as well as simian immunodeficiency and HIV-2 strains bound to DC-SIGN and could be transmitted to CD4/coreceptor-positive cell types. DC-SIGN contains a single N-linked carbohydrate chain that is important for efficient cell surface expression but is not required for DC-SIGN-mediated virus binding and transmission. In contrast, C-terminal deletions removing either the lectin binding domain or the repeat region abrogated DC-SIGN function. Trypsin-EDTA treatment inhibited DC-SIGN mediated infection, indicating that virus was maintained at the surface of the DC-SIGN-expressing cells used in this study. Finally, quantitative fluorescence-activated cell sorting analysis of AU1-tagged DC-SIGN revealed that the efficiency of virus transmission was strongly affected by variations in DC-SIGN expression levels. Thus, variations in DC-SIGN expression levels on DCs could greatly affect the susceptibility of human individuals to HIV infection.     

The two biological activities of human immunodeficiency virus type 1 Vpu protein involve two separable structural domains.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is an integral membrane phosphoprotein that induces CD4 degradation in the endoplasmic reticulum and enhances virus release from the cell surface. CD4 degradation is specific, requires phosphorylation of Vpu, and involves the interaction between Vpu and the CD4 cytoplasmic domain. In contrast, regulation of virus release is less specific and not restricted to HIV-1 and may be mechanistically-distinct from CD4 degradation. We show here that a mutant of Vpu, Vpu35, lacking most of its cytoplasmic domain has residual biological activity for virus release but is unable to induce CD4 degradation. This finding suggests that the N terminus of Vpu encoding the transmembrane (TM) anchor represents an active domain important for the regulation of virus release but not CD4 degradation. To better define the functions of Vpu's TM anchor and cytoplasmic domain, we designed a mutant, VpuRD, containing a scrambled TM sequence with a conserved amino acid composition and alpha-helical structure. The resulting protein was integrated normally into membranes, was able to form homo-oligomers, and exhibited expression levels, protein stability, and subcellular localization similar to those of wild-type Vpu. Moreover, VpuRD was capable of binding to CD4 and to induce CD4 degradation with wild-type efficiency, confirming proper membrane topology and indicating that the alteration of the Vpu TM domain did not interfere with this function of Vpu. However, VpuRD was unable to enhance the release of virus particles from infected or transfected cells, and virus encoding VpuRD had replication characteristics in T cells indistinguishable from those of a Vpu-deficient HIV-1 isolate. Mutation of the phosphorylation sites in VpuRD resulted in a protein which was unable to perform either function of Vpu. The results of our experiments suggest that the two biological activities of Vpu operate via two distinct molecular mechanisms and involve two different structural domains of the Vpu protein.