Amy J. Andrew

Vpu enhances HIV-1 virus release in the absence of Bst-2 cell surface down-modulation and intracellular depletion

HIV-1 Vpu enhances the release of virions from infected cells. Recent work identified Bst-2/CD317/tetherin as a host factor whose inhibitory activity on viral release is counteracted by Vpu. A current working model proposes that Bst-2 inhibits virus release by tethering viral particles to the cell surface. Here, we analyzed endogenous Bst-2 with respect to its effect on virus release from HeLa cells, T cells, and macrophages. We noted significant cell type-dependent variation in Bst-2 expression. Vpu caused a reduction in Bst-2 expression in transfected HeLa cells and long-term infected macrophages. However, Vpu expression did not result in cell surface down-modulation of Bst-2 or a reduction in intracellular Bst-2 expression in CEMx174 or H9 cells, yet virus replication in these cells was Vpu-responsive. Surprisingly, Bst-2 was undetectable in cell-free virions that were recovered from the surface of HeLa cells by physical shearing, suggesting that a tethering model may not explain all of the functional properties of Bst-2. Taken together we conclude that enhancement of virus release by Vpu does not, at least in CEMx174 and H9 cells, require cell surface down-modulation or intracellular depletion of Bst-2, nor does it entail exclusion of Bst-2 from viral particles.

J protein cochaperone of the mitochondrial inner membrane required for protein import into the mitochondrial matrix

The major Hsp70 of the mitochondrial matrix (Ssc1 in yeast) is critically important for the translocation of proteins from the cytosol, across the mitochondrial inner membrane, and into the matrix. Tim44, a peripheral inner membrane protein with limited sequence similarity to the J domain of J-type cochaperones, tethers Ssc1 to the import channel. Here we report that, unlike a J protein, Tim44 does not stimulate the ATPase activity of Ssc1, nor does it affect the stimulation by either a known mitochondrial J protein or a peptide substrate. Thus, we conclude that Tim44 does not function as a J protein cochaperone of Ssc1; rather, it tethers Ssc1 to the import channel through interactions independent of those critical for J protein function. However, a previously unstudied essential gene, PAM18, encodes an 18-kDa protein that contains a J domain and is localized to the mitochondrial inner membrane. Pam18 stimulates the ATPase activity of Ssc1; depletion of Pam18 in vivo disrupts import of proteins into the mitochondrial matrix. We propose that Pam18 is the J protein partner for Ssc1 at the import channel and is critical for Ssc1s function in protein import.

Iron-induced oligomerization of yeast frataxin homologue Yfh1 is dispensable in vivo

The neurodegenerative disease Friedreichs ataxia is caused by reduced levels of frataxin, a mitochondrial matrix protein. The in vivo role of frataxin is under debate. Frataxin, as well as its yeast homologue Yfh1, binds multiple iron atoms as an oligomer and has been proposed to function as a crucial iron‐storage protein. We identified a mutant Yfh1 defective in iron‐induced oligomerization. This mutant protein was able to replace functionally wild‐type Yfh1, even when expressed at low levels, when mitochondrial iron levels were high and in mutant strains having deletions of genes that had synthetic growth defects with a YFH1 deletion. The ability of an oligomerization‐deficient Yfh1 to function in vivo suggests that oligomerization, and thus oligomerization‐induced iron storage, is not a critical function of Yfh1. Rather, the capacity of this oligomerization‐deficient mutant to interact with the Isu protein suggests a more direct role of Yfh1 in iron–sulphur cluster biogenesis.

Characterization of the Interaction between the J-protein Jac1p and the Scaffold for Fe-S Cluster Biogenesis, Isu1p

Jac1p is a conserved, specialized J-protein that functions with Hsp70 in Fe-S cluster biogenesis in mitochondria of the yeast Saccharomyces cerevisiae. Although Jac1p as well as its specialized Hsp70 partner, Ssq1p, binds directly to the Fe-S cluster scaffold protein Isu, the Jac1p-Isu1p interaction is not well understood. Here we report that a C-terminal fragment of Jac1p lacking its J-domain is sufficient for interaction with Isu1p, and amino acid alterations in this domain affect interaction with Isu1p but not Ssq1p. In vivo, such JAC1 mutations had no obvious phenotypic effect. However, when present in combination with a mutation in SSQ1 that causes an alteration in the substrate binding cleft, growth was significantly compromised. Wild type Jac1p and Isu1p cooperatively stimulate the ATPase activity of Ssq1p. Jac1p mutant protein is only slightly compromised in this regard. Our in vivo and in vitro results indicate that independent interaction of Jac1p and the Isu client protein with Hsp70 is sufficient for robust growth under standard laboratory conditions. However, our results also support the idea that Isu protein can be “targeted” to Ssq1p after forming a complex with Jac1p. We propose that Isu protein targeting may be particularly important when environmental conditions place high demands on Fe-S cluster biogenesis or in organisms lacking specialized Hsp70s for Fe-S cluster biogenesis.

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

ABSTRACT 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.

C-terminal Hydrophobic Region in Human Bone Marrow Stromal Cell Antigen 2 (BST-2)/Tetherin Protein Functions as Second Transmembrane Motif

Background: BST-2/tetherin inhibits virus release by tethering virions to the cell surface. Results: The putative GPI anchor signal of BST can function as a TM region and can be replaced by heterologous TM segments. Conclusion: BST-2 contains a second TM region instead of a GPI anchor. Significance: Understanding the molecular structure of BST-2 is crucial to understanding how the protein inhibits detachment of many enveloped viruses. BST-2/CD317/HM1.24/tetherin is a host factor that inhibits the release of HIV-1 and other enveloped viruses. Structurally, tetherin consists of an N-terminal transmembrane (TM) region, a central coiled coil motif, and a putative C-terminal glycosylphosphatidylinositol (GPI) anchor motif. A current working model proposes that BST-2 inhibits virus release by physically tethering viral particles to the cell surface via its TM motif and GPI anchor. Here we analyzed the functional importance of the C-terminal GPI anchor motif in BST-2. We replaced the GPI anchor motif in BST-2 with the TM regions of several surface markers and found that the TM motifs of CD40 and transferrin receptor, but not that of CD45, could functionally substitute for a GPI anchor in BST-2. Conversely, replacing the TM region of CD4 by the putative GPI anchor signal of human BST-2 resulted in proper membrane targeting and surface expression of the chimeric protein, indicating that the BST-2 GPI anchor signal can function as a bona fide TM region. In fact, attempts to demonstrate GPI anchor modification of human BST-2 by biochemical methods failed. Our results demonstrate that the putative C-terminal GPI anchor motif in human BST-2 fulfills the requirements of a bona fide TM motif, leading us to propose that human BST-2 may in fact contain a second TM segment rather than a GPI anchor.

HIV-1 Vpu targets cell surface markers CD4 and BST-2 through distinct mechanisms.

Vpu is a small integral membrane protein encoded by HIV-1 and some SIV isolates. The protein is known to induce degradation of the viral receptor molecule CD4 and to enhance the release of newly formed virions from the cell surface. Vpu accomplishes these two functions through two distinct mechanisms. In the case of CD4, Vpu acts as a molecular adaptor to connect CD4 to an E3 ubiquitin ligase complex resulting in CD4 degradation by cellular proteasomes. This requires signals located in Vpus cytoplasmic domain. Enhancement of virus release on the other hand involves the neutralization of a cellular host factor, BST-2 (also known as CD317, HM1.24, or tetherin) and requires Vpus TM domain. The current review discusses recent advances on the role of Vpu in controlling degradation of CD4 and in regulating virus release.

Posttranslational regulation of the scaffold for Fe-S cluster biogenesis, Isu.

Isu, the scaffold protein on which Fe-S clusters are built in the mitochondrial matrix, plays a central role in the biogenesis of Fe-S cluster proteins. We report that the reduction in the activity of several components of the cluster biogenesis system, including the specialized Hsp70 Ssq1, causes a 15-20-fold up-regulation of Isu. This up-regulation results from changes at both the transcriptional and posttranslational level: an increase in ISU mRNA levels and in stability of ISU protein. Its biological importance is demonstrated by the fact that cells lacking Ssq1 grow poorly when Isu levels are prevented from rising above those found in wild-type cells. Of the biogenesis factors tested, Nfs1, the sulfur donor, was unique. Little increase in Isu levels occurred when Nfs1 was depleted. However, its presence was required for the up-regulation caused by reduction in activity of other components. Our results are consistent with the existence of a mechanism to increase the stability of Isu, and thus its level, that is dependent on the presence of the cysteine desulfurase Nfs1.

The Interferon-Inducible Host Factor Bone Marrow Stromal Antigen 2/Tetherin Restricts Virion Release, but Is It Actually a Viral Restriction Factor?

Viruses face a variety of obstacles when infecting a new host. The past few years have brought exciting new insights into the function of restriction factors, which form part of the hosts innate immune system. One of the most recently identified restriction factors is bone marrow stromal antigen 2 (BST-2)/tetherin. BST-2 is an interferon-inducible gene whose expression dramatically reduces the release of viruses from infected cells. This effect of BST-2 is not specific to human immunodeficiency virus but affects a broad range of enveloped viruses. Since the identification of BST-2 as a restriction factor in 2008, much progress has been made in understanding the molecular properties and functional characteristics of this host factor. The goal of this review was to provide an update on our current understanding of the role of BST-2 in regulating virus release and to discuss its role in controlling virus spread during productive infection with special emphasis on human immunodeficiency virus-1.

Antibody-Mediated Enhancement of HIV-1 and HIV-2 Production from BST-2/Tetherin-Positive Cells

ABSTRACT BST-2/CD317/HM1.24/tetherin is a B-cell antigen overexpressed on the surface of myeloma cell lines and on neoplastic plasma cells of patients with multiple myeloma. Antibodies to BST-2 are in clinical trial for the treatment of multiple myeloma and are considered for the treatment of solid tumors with high BST-2 antigen levels. Functionally, BST-2 restricts the secretion of retroviruses, including human immunodeficiency virus type 1, as well as members of the herpesvirus, filovirus, and arenavirus families, presumably by tethering nascent virions to the cell surface. Here we report that BST-2 antibody treatment facilitates virus release from BST-2+ cells by interfering with the tethering activity of BST-2. BST-2 antibodies were unable to release already tethered virions and were most effective when added early during virus production. BST-2 antibody treatment did not affect BST-2 dimerization and did not reduce the cell surface expression of BST-2. Interestingly, BST-2 antibody treatment reduced the nonspecific shedding of BST-2 and limited the encapsidation of BST-2 into virions. Finally, flotation analyses indicate that BST-2 antibodies affect the distribution of BST-2 within membrane rafts. Our data suggest that BST-2 antibody treatment may enhance virus release by inducing a redistribution of BST-2 at the cell surface, thus preventing it from accumulating at the sites of virus budding.