Edward B. Stephens

The Interferon-Induced Protein BST-2 Restricts HIV-1 Release and Is Downregulated from the Cell Surface by the Viral Vpu Protein

The HIV-1 accessory protein Vpu counteracts a host factor that restricts virion release from infected cells. Here we show that the interferon-induced cellular protein BST-2/HM1.24/CD317 is such a factor. BST-2 is downregulated from the cell surface by Vpu, and BST-2 is specifically expressed in cells that support the vpu phenotype. Exogenous expression of BST-2 inhibits HIV-1 virion release, while suppression of BST-2 relieves the requirement for Vpu. Downregulation of BST-2 requires both the transmembrane/ion channel domain and conserved serines in the cytoplasmic domain of Vpu. Endogenous BST-2 colocalizes with the HIV-1 structural protein Gag in endosomes and at the plasma membrane, suggesting that BST-2 traps virions within and on infected cells. The unusual structure of BST-2, which includes a transmembrane domain and a lumenal GPI anchor, may allow it to retain nascent enveloped virions on cellular membranes, providing a mechanism of viral restriction counteracted by a specific viral accessory protein.

Vpu Antagonizes BST-2–Mediated Restriction of HIV-1 Release via β-TrCP and Endo-Lysosomal Trafficking

The interferon-induced transmembrane protein BST-2/CD317 (tetherin) restricts the release of diverse enveloped viruses from infected cells. The HIV-1 accessory protein Vpu antagonizes this restriction by an unknown mechanism that likely involves the down-regulation of BST-2 from the cell surface. Here, we show that the optimal removal of BST-2 from the plasma membrane by Vpu requires the cellular protein β-TrCP, a substrate adaptor for a multi-subunit SCF E3 ubiquitin ligase complex and a known Vpu-interacting protein. β-TrCP is also required for the optimal enhancement of virion-release by Vpu. Mutations in the DSGxxS β-TrCP binding-motif of Vpu impair both the down-regulation of BST-2 and the enhancement of virion-release. Such mutations also confer dominant-negative activity, consistent with a model in which Vpu links BST-2 to β-TrCP. Optimal down-regulation of BST-2 from the cell surface by Vpu also requires the endocytic clathrin adaptor AP-2, although the rate of endocytosis is not increased; these data suggest that Vpu induces post-endocytic membrane trafficking events whose net effect is the removal of BST-2 from the cell surface. In addition to its marked effect on cell-surface levels, Vpu modestly decreases the total cellular levels of BST-2. The decreases in cell-surface and intracellular BST-2 are inhibited by bafilomycin A1, an inhibitor of endosomal acidification; these data suggest that Vpu induces late endosomal targeting and partial degradation of BST-2 in lysosomes. The Vpu-mediated decrease in surface expression is associated with reduced co-localization of BST-2 and the virion protein Gag along the plasma membrane. Together, the data support a model in which Vpu co-opts the β-TrCP/SCF E3 ubiquitin ligase complex to induce endosomal trafficking events that remove BST-2 from its site of action as a virion-tethering factor.

Novel targets for HIV therapy.

There are currently 25 drugs belonging to 6 different inhibitor classes approved for the treatment of human immunodeficiency virus (HIV) infection. However, new anti-HIV agents are still needed to confront the emergence of drug resistance and various adverse effects associated with long-term use of antiretroviral therapy. The 21st International Conference on Antiviral Research, held in April 2008 in Montreal, Canada, therefore featured a special session focused on novel targets for HIV therapy. The session included presentations by world-renowned experts in HIV virology and covered a diverse array of potential targets for the development of new classes of HIV therapies. This review contains concise summaries of discussed topics that included Vif-APOBEC3G, LEDGF/p75, TRIM 5alpha, virus assembly and maturation, and Vpu. The described viral and host factors represent some of the most noted examples of recent scientific breakthroughs that are opening unexplored avenues to novel anti-HIV target discovery and validation, and should feed the antiretroviral drug development pipeline in the near future.

HIV-1 Vpu Protein Antagonizes Innate Restriction Factor BST-2 via Lipid-embedded Helix-Helix Interactions

Background: HIV-1 Vpu counteracts the cellular antiviral factor BST-2 via an interaction that maps to the transmembrane domains of each protein. Results: This interaction is detectable by NMR spectroscopy and involves conserved faces of each helix. Conclusion: HIV-1 avoids an innate host defense via a lipid-embedded helix-helix interface. Significance: Intermolecular interactions within the lipid bilayer can be highly specific and shape the host-pathogen relationship. The Vpu protein of HIV-1 antagonizes BST-2 (tetherin), a broad spectrum effector of the innate immune response to viral infection, by an intermolecular interaction that maps genetically to the α-helical transmembrane domains (TMDs) of each protein. Here we utilize NMR spectroscopy to describe key features of the helix-helix pairing that underlies this interaction. The antagonism of BST-2 involves a sequence of three alanines and a tryptophan spaced at four residue intervals within the Vpu TMD helix. Responsiveness to Vpu involves bulky hydrophobic residues in the C-terminal region of the BST-2 TMD helix that likely fit between the alanines on the interactive face of Vpu. These aspects of Vpu and BST-2 form an anti-parallel, lipid-embedded helix-helix interface. Changes in human BST-2 that mimic sequences found in nonhuman primate orthologs unresponsive to Vpu change the tilt angle of the TMD in the lipid bilayer without abrogating its intrinsic ability to interact with Vpu. These data explain the mechanism by which HIV-1 evades a key aspect of innate immunity and the species specificity of Vpu using an anti-parallel helix-helix packing model.

Modulation by Morphine of Viral Set Point in Rhesus Macaques Infected with Simian Immunodeficiency Virus and Simian-Human Immunodeficiency Virus

ABSTRACT Six rhesus macaques were adapted to morphine dependence by injecting three doses of morphine (5 mg/kg of body weight) for a total of 20 weeks. These animals along with six control macaques were infected intravenously with mixture of simian-human immunodeficiency virus KU-1B (SHIVKU-1B), SHIV89.6P, and simian immunodeficiency virus 17E-Fr. Levels of circulating CD4+ T cells and viral loads in the plasma and the cerebrospinal fluid were monitored in these macaques for a period of 12 weeks. Both morphine and control groups showed precipitous loss of CD4+ T cells. However this loss was more prominent in the morphine group at week 2 (P = 0.04). Again both morphine and control groups showed comparable peak plasma viral load at week 2, but the viral set points were higher in the morphine group than that in the control group. Likewise, the extent of virus replication in the cerebral compartment was more pronounced in the morphine group. These results provide a definitive evidence for a positive correlation between morphine and levels of viral replication.

Compartmentalization of Simian Immunodeficiency Virus Replication within Secondary Lymphoid Tissues of Rhesus Macaques Is Linked to Disease Stage and Inversely Related to Localization of Virus-Specific CTL

We previously demonstrated that HIV replication is concentrated in lymph node B cell follicles during chronic infection and that HIV-specific CTL fail to accumulate in large numbers at those sites. It is unknown whether these observations can be generalized to other secondary lymphoid tissues or whether virus compartmentalization occurs in the absence of CTL. We evaluated these questions in SIVmac239-infected rhesus macaques by quantifying SIV RNA+ cells and SIV-specific CTL in situ in spleen, lymph nodes, and intestinal tissues obtained at several stages of infection. During chronic asymptomatic infection prior to simian AIDS, SIV-producing cells were more concentrated in follicular (F) compared with extrafollicular (EF) regions of secondary lymphoid tissues. At day 14 of infection, when CTL have minimal impact on virus replication, there was no compartmentalization of SIV-producing cells. Virus compartmentalization was diminished in animals with simian AIDS, which often have low-frequency CTL responses. SIV-specific CTL were consistently more concentrated within EF regions of lymph node and spleen in chronically infected animals regardless of epitope specificity. Frequencies of SIV-specific CTL within F and EF compartments predicted SIV RNA+ cells within these compartments in a mixed model. Few SIV-specific CTL expressed the F homing molecule CXCR5 in the absence of the EF retention molecule CCR7, possibly accounting for the paucity of F CTL. These findings bolster the hypothesis that B cell follicles are immune privileged sites and suggest that strategies to augment CTL in B cell follicles could lead to improved viral control and possibly a functional cure for HIV infection.

Sequence Note: Comparison of Vpu Sequences from Diverse Geographical Isolates of HIV Type 1 Identifies the Presence of Highly Variable Domains, Additional Invariant Amino Acids, and a Signature Sequence Motif Common to Subtype C Isolates

We compared the Vpu sequences from 101 strains of HIV-1 isolated from diverse geographical regions and various subtypes in order to identify regions of high variability, and those amino acid residues that were highly conserved or invariant. In addition to the highly conserved casein kinase II (CKII) phosphorylation sites, our analysis identified additional invariant residues in the transmembrane domain and in the first and second alpha-helical domains. Our analysis revealed that all subtype C sequences had a conserved LRLL motif at the C terminus that was also found in A/C intersubtype recombinants. While our analysis demonstrated the conservation of CKII domains in HIV-1 group M and O isolates, the number of potential CKII phosphorylation sites was variable in SIVcpz sequences. The results of this study will provide a basis for future mutagenesis studies to examine the role of certain amino acid residues in the structure and function of Vpu.

The proteins of lymphocyte- and macrophage-tropic strains of simian immunodeficiency virus are processed differently in macrophages

Since the pathogenesis of SIVmac disease complex is thought to be explained by the tropism of the infecting virus for either CD4+ T-lymphocytes or macrophages or both types of cells, we compared the infection in primary macaque macrophages with molecularly cloned, lymphocyte-tropic SIVmac239 and a cloned, macrophage-tropic chimeric virus (SIVmac239/17E) whose env gene was derived from brain of a macaque (17E) dying from SIV-induced encephalopathy. SIVmac239/17E caused a productive, syncytial cytopathic infection accompanied by accumulation of virus particles within cytoplasmic vesicles of the macrophages. Pulse-chase and immune precipitation studies showed that both the viral glycoprotein precursor (gp160) and the gag precursor (p57) were cleaved into gp120 and p27, respectively, and both were released into the culture medium of infected cells, although most of the p27 remained cell associated. SIVmac239 also infected macrophages, but in comparison to SIVmac239/17E, minimal virus replication occurred. Immunocytostaining revealed that while occasional syncytia were observed in cultures, the majority of the infected cells were not associated with syncytium formation. Ultrastructural studies did not reveal the accumulation of virions within infected macrophages. Pulse-chase studies showed that both gp160 and p57 were produced but were cleaved inefficiently and only minimal amounts of gp120 and p27 were released into the culture medium, even after prolonged incubation times. The processing of proteins of the two viruses was indistinguishable in lymphocytes. Since these two viruses are identical except for changes within the env gene, these results indicate that efficient assembly and release of SIV from blood-derived macrophages is mediated by changes in the envelope glycoprotein.

The presence of the casein kinase II phosphorylation sites of Vpu enhances the CD4+ T cell loss caused by the simian–human immunodeficiency virus SHIVKU-lbMC33 in pig-tailed macaques

The simian-human immunodeficiency virus (SHIV)/ macaque model for human immunodeficiency virus type 1 has become a useful tool to assess the role of Vpu in lentivirus pathogenesis. In this report, we have mutated the two phosphorylated serine residues of the HIV-1 Vpu to glycine residues and have reconstructed a SHIV expressing this nonphosphorylated Vpu (SHIV(S52,56G)). Expression studies revealed that this protein was localized to the same intracellular compartment as wild-type Vpu. To determine if this virus was pathogenic, four pig-tailed macaques were inoculated with SHIV(S52,56G) and virus burdens and circulating CD4(+) T cells monitored up to 1 year. Our results indicate that SHIV(S52,56G) caused rapid loss in the circulating CD4(+) T cells within 3 weeks of inoculation in one macaque (CC8X), while the other three macaques developed no or gradual numbers of CD4(+) T cells and a wasting syndrome. Histological examination of tissues revealed that macaque CC8X had lesions in lymphoid tissues (spleen, lymph nodes, and thymus) that were typical for macaques inoculated with pathogenic parental SHIV(KU-1bMC33) and had no lesions within the CNS. To rule out that macaque CC8X had selected for a virus in which there was reversion of the glycine residues at positions 52 and 56 to serine residues and/or compensating mutations occurred in other genes associated with CD4 down-regulation, sequence analysis was performed on amplified vpu sequences isolated from PBMC and from several lymphoid tissues at necropsy. Sequence analysis revealed a reversion of the glycine residues back to serine residues in this macaque. The other macaques maintained low virus burdens, with one macaque (P003) developing a wasting syndrome between months 9 and 11. Histological examination of tissues from this macaque revealed a thymus with severe atrophy that was similar to that of a previously reported macaque inoculated with a SHIV lacking vpu (Virology 293, 2002, 252). Sequence analysis revealed no reversion of the glycine residues in the vpu sequences isolated from this macaque. These results contrast with those from four macaques inoculated with the parental pathogenic SHIV(KU-1bMC33), all of which developed severe CD4(+) T cell loss within 1 month after inoculation. Taken together, these results indicate that casein kinase II phosphorylation sites of Vpu contributes to the pathogenicity of the SHIV(KU-1bMC33) and suggest that the SHIV(KU-1bMC33)/pig-tailed macaque model will be useful in analyzing amino acids/domains of Vpu that contribute to the pathogenesis of HIV-1.

Initial characterization of viral sequences from a SHIV-inoculated pig-tailed macaque that developed AIDS.

Abstract: In this study, we report on the derivation of a pathogenic SIV‐HIV chimeric virus (SHIV) and the initial characterization of the viral sequences from the first (macaque PPc) of a series of pig‐tailed macaques that developed CD4+ T cell loss and AIDS. Viral genes were amplified by PCR from the brain, lymphoid, and kidney tissues and their sequences compared to the original SHIV used to initiate passages in macaques. Our results show that the vpu gene, which was nonfunctional in the original SHIV, now coded for functional protein in macaque PPc. The tat and rev genes had no consensus changes but the nef gene had 4–5 consensus changes, depending on the tissue examined. The gp 120 gene had the highest number of nucleotide and amino acid substitution rates that varied from 0.64% to 1.44% and 1.17% to 3.71%, respectively, again depending on the tissue examined. These results suggest that a constellation of changes accumulated at the genomic level during the derivation of a SHIV that was pathogenic for pig‐tailed macaques.