The primary roles attributed to the human immunodeficiency virus type 1 (HIV-1) Vpu protein are the degradation of the viral receptor CD4 and the enhancement of virion release. With regard to CD4 downregulation, Vpu has been shown to act as an adapter linking CD4 with the ubiquitin-proteasome machinery via interaction with the F-box protein ␤TrCP. To identify additional cellular ␤TrCP-dependent Vpu targets, we performed quantitative proteomics analyses using the plasma membrane fraction of HeLa cells expressing either wild-type Vpu or a Vpu mutant (S52N/S56N) that does not bind ␤TrCP. One cellular protein, BST-2 (CD317), was consistently underrepresented in the membrane proteome of cells expressing wild-type Vpu compared to the proteome of cells expressing the Vpu mutant. To verify the biological relevance of this phenotype for HIV pathogenesis, we showed that in T cells infected with HIV-1, BST-2 downregulation occurred in a Vpu-dependent manner. Recently, BST-2 has been identified as the interferon-inducible cellular factor Tetherin, which restricts HIV virion release in the absence of Vpu. We address here the unresolved mechanism of Vpu-mediated BST-2 downregulation. Our data show that the presence of wild-type Vpu reduced cell surface and total steady-state BST-2 levels, whereas that of the mutant Vpu had no effect. In addition, treatment of cells with the lysosome acidification inhibitor concanamycin A, but not treatment with the proteasome inhibitor MG132, reduced BST-2 downregulation by wild-type Vpu, thereby suggesting that the presence of Vpu leads to the degradation of BST-2 via an endosome-lysosome degradation pathway. The importance of ␤TrCP in this process was confirmed by demonstrating that in the absence of ␤TrCP, BST-2 levels were restored despite the presence of Vpu. Taken together, these data support the hypothesis that, in similarity to its role in CD4 degradation, Vpu acts as an adapter molecule linking BST-2 to the cellular ubiquitination machinery via ␤TrCP. However, in contrast to the proteasome-dependent degradation of CD4, which occurs in the endoplasmic reticulum, Vpu appears to interact with BST-2 in the trans-Golgi network or in early endosomes, leading to lysosomal degradation of BST-2. Via this action, Vpu could counter the tethering function of BST-2, resulting in enhanced HIV-1 virion release. Interestingly, although HIV-2 does not express Vpu, an isolate known to exhibit enhanced viral egress can downregulate surface BST-2 by an as-yet-unknown mechanism that does not appear to involve degradation. Understanding the molecular mechanisms of both Vpu-dependent and -independent mediated antagonism of BST-2 will be critical for therapeutic strategies that exploit this novel viral function.

K3/MIR1 and K5/MIR2 of Kaposi's sarcoma-associated herpesvirus (KSHV) are viral members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family and contribute to viral immune evasion by directing the conjugation of ubiquitin to immunostimulatory transmembrane proteins. In a quantitative proteomic screen for novel host cell proteins downregulated by viral immunomodulators, we previously observed that K5, as well as the human immunodeficiency virus type 1 (HIV-1) immunomodulator VPU, reduced steady-state levels of bone marrow stromal cell antigen 2 (BST2; also called CD317 or tetherin), suggesting that BST2 might be a novel substrate of K5 and VPU. Recent work revealed that in the absence of VPU, HIV-1 virions are tethered to the plasma membrane in BST2-expressing HeLa cells. By targeting BST2, K5 might thus similarly overcome an innate antiviral host defense mechanism. Here we establish that despite its type II transmembrane topology and carboxy-terminal glycosylphosphatidylinositol (GPI) anchor, BST2 represents a bona fide target of K5 that is downregulated during primary infection by and reactivation of KSHV. Upon exit of the protein from the endoplasmic reticulum, lysines in the short amino-terminal domain of BST2 are ubiquitinated by K5, resulting in rapid degradation of BST2. Ubiquitination of BST2 is required for degradation, since BST2 lacking cytosolic lysines was K5 resistant and ubiquitin depletion by proteasome inhibitors restored BST2 surface expression. Thus, BST2 represents the first type II transmembrane protein targeted by K5 and the first example of a protein that is both ubiquitinated and GPI linked. We further demonstrate that KSHV release is decreased in the absence of K5 in a BST2-dependent manner, suggesting that K5 contributes to the evasion of intracellular antiviral defense programs.Bone marrow stromal cell antigen 2 (BST2) was recently identified as a host cell restriction factor that prevents the release of retroviral and filoviral particles from infected host cells (23). Human immunodeficiency virus type 1 (HIV-1) counteracts this antiviral function of BST2 by expressing the viral auxiliary protein VPU (41, 53). In the absence of VPU, virus particles are prevented from budding off the cellular membrane in cells that express BST2, resulting in virions being tethered to the plasma membrane. BST2 was therefore renamed tetherin (41), although questions still remain as to whether BST2 acts as the actual tether and whether BST2-dependent tethering occurs in all BST2-expressing cell types (36). Independently, BST2 was shown to be induced by type I and type II interferons (IFNs) (7), suggesting that BST2 is part of the innate antiviral response triggered in infected cells.Using a quantitative membrane proteomic approach, we observed that BST2 is underrepresented in plasma membranes from cells expressing not only VPU (14) but also the K5 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) (4). K5 is a viral homologue of a family of cellular transmembrane ubiquitin ligases, term...

The MARCH family of proteins are membrane-associated E3 ubiquitin ligases that down-regulate surface membrane proteins. Expression of MARCH8 in cells causes the ubiquitination and down-regulation of surface CD98 and CD44—cargo proteins that enter cells by clathrin-independent endocytosis and are usually routed to recycling, not degradation.

The interferon-induced BST-2 protein has the unique ability to restrict the egress of HIV-1, Kaposi's sarcoma–associated herpesvirus (KSHV), Ebola virus, and other enveloped viruses. The observation that virions remain attached to the surface of BST-2-expressing cells led to the renaming of BST-2 as “tetherin”. However, viral proteins such as HIV-1 Vpu, simian immunodeficiency virus Nef, and KSHV K5 counteract BST-2, thereby allowing mature virions to readily escape from infected cells. Since the anti-viral function of BST-2 was discovered, there has been an explosion of research into several aspects of this intriguing interplay between host and virus. This review focuses on recent work addressing the molecular mechanisms involved in BST-2 restriction of viral egress and the species-specific countermeasures employed by various viruses.

Cytomegaloviruses are highly host restricted, resulting in cospeciation with their hosts. As a natural pathogen of rhesus macaques (RM), rhesus cytomegalovirus (RhCMV) has therefore emerged as a highly relevant experimental model for pathogenesis and vaccine development due to its close evolutionary relationship to human CMV (HCMV). Most in vivo experiments performed with RhCMV employed strain 68-1 cloned as a bacterial artificial chromosome (BAC). However, the complete genome sequence of the 68-1 BAC has not been determined. Furthermore, the gene content of the RhCMV genome is unknown, and previous open reading frame (ORF) predictions relied solely on uninterrupted ORFs with an arbitrary cutoff of 300 bp. To obtain a more precise picture of the actual proteins encoded by the most commonly used molecular clone of RhCMV, we reevaluated the RhCMV 68-1 BAC genome by whole-genome shotgun sequencing and determined the protein content of the resulting RhCMV virions by proteomics. By comparing the RhCMV genome to those of several related Old World monkey (OWM) CMVs, we were able to filter out many unlikely ORFs and obtain a simplified map of the RhCMV genome. This comparative genomics analysis suggests a high degree of ORF conservation among OWM CMVs, thus decreasing the likelihood that ORFs found only in RhCMV comprise true genes. Moreover, virion proteomics independently validated the revised ORF predictions, since only proteins that were conserved across OWM CMVs could be detected. Taken together, these data suggest a much higher conservation of genome and virion structure between CMVs of humans, apes, and OWMs than previously assumed.

Serp-1 is a secreted myxoma viral serine protease inhibitor (serpin) with proven, highly effective, anti-inflammatory defensive activity during host cell infection, as well as potent immunomodulatory activity in a wide range of animal disease models. Serp-1 binds urokinase-type plasminogen activator (uPA) and the tissue-type PA, plasmin, and factor Xa, requiring uPA receptor (uPAR) for anti-inflammatory activity. To define Serp-1-mediated effects on inflammatory cell activation, we examined the association of Serp-1 with monocytes and T cells, effects on cellular migration, and the role of uPAR-linked integrins and actin-binding proteins in Serp-1 cellular responses. Our results show that Serp-1 associates directly with activated monocytes and T lymphocytes, in part through interaction with uPAR (P<0.001). Serp-1, but not mammalian serpin PA inhibitor-1 (PAI-1), attenuated cellular adhesion to the extracellular matrix. Serp-1 and PAI-1 reduced human monocyte and T cell adhesion (P<0.001) and migration across endothelial monolayers in vitro (P<0.001) and into mouse ascites in vivo (P<0.001). Serp-1 and an inactive Serp-1 mutant Serp-1(SAA) bound equally to human monocytes and T cells, but a highly proinflammatory mutant, Serp-1(Ala(6)), bound less well to monocytes. Serp-1 treatment of monocytes increased expression of filamin B actin-binding protein and reduced CD18 (beta-integrin) expression (P<0.001) in a uPAR-dependent response. Filamin colocalized and co-immunoprecipitated with uPAR, and short interference RNA knock-down of filamin blocked Serp-1 inhibition of monocyte adhesion. We report here that the highly potent, anti-inflammatory activity of Serp-1 is mediated through modification of uPAR-linked beta-integrin and filamin in monocytes, identifying this interaction as a central regulatory axis for inflammation.

Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins.

The thrombolytic serine protease cascade is intricately involved in activation of innate immune responses. The urokinase-type plasminogen activator and receptor form complexes that aid inflammatory cell invasion at sites of arterial injury. Plasminogen activator inhibitor-1 is a mammalian serpin that binds and regulates the urokinase receptor complex. Serp-1, a myxomaviral serpin, also targets the urokinase receptor, displaying profound anti-inflammatory and anti-atherogenic activity in a wide range of animal models. Serp-1 reactive center site mutations, mimicking known mammalian and viral serpins, were constructed in order to define sequences responsible for regulation of inflammation. Thrombosis, inflammation, and plaque growth were assessed after treatment with Serp-1, Serp-1 chimeras, plasminogen activator inhibitor-1, or unrelated viral serpins in plasminogen activator inhibitor or urokinase receptor-deficient mouse aortic transplants. Altering the P1-P1 Arg-Asn sequence compromised Serp-1 protease-inhibitory activity and anti-inflammatory activity in animal models; P1-P1 Ala-Ala mutants were inactive, P1 Met increased remodeling, and P1 Thr increased thrombosis. Substitution of Serp-1 P2-P7 with Ala 6 allowed for inhibition of urokinase but lost plasmin inhibition, unexpectedly inducing a diametrically opposed, proinflammatory response with mononuclear cell activation, thrombosis, and aneurysm formation (p < 0.03). Other serpins did not reproduce Serp-1 activity; plasminogen activator inhibitor-1 increased thrombosis (p < 0.0001), and unrelated viral serpin, CrmA, increased inflammation. Deficiency of urokinase receptor in mouse transplants blocked Serp-1 and chimera activity, in some cases increasing inflammation. In summary, 1) Serp-1 anti-inflammatory activity is highly dependent upon the reactive center loop sequence, and 2) plasmin inhibition is central to anti-inflammatory activity.