Ebola Virus Glycoprotein Counteracts BST-2/Tetherin Restriction in a Sequence-Independent Manner That Does Not Require Tetherin Surface Removal

BST-2/tetherin is an interferon-inducible protein that restricts the release of enveloped viruses from the surface of infected cells by physically linking viral and cellular membranes. It is present at both the cell surface and in a perinuclear region, and viral anti-tetherin factors including HIV-1 Vpu and HIV-2 Env have been shown to decrease the cell surface population. To map the domains of human tetherin necessary for both virus restriction and sensitivity to viral anti-tetherin factors, we constructed a series of tetherin derivatives and assayed their activity. We found that the cytoplasmic tail (CT) and transmembrane (TM) domains of tetherin alone produced its characteristic cellular distribution, while the ectodomain of the protein, which includes a glycosylphosphatidylinositol (GPI) anchor, was sufficient to restrict virus release when presented by the CT/TM regions of a different type II membrane protein. To counteract tetherin restriction and remove it from the cell surface, HIV-1 Vpu required the specific sequence present in the TM domain of human tetherin. In contrast, the HIV-2 Env required only the ectodomain of the protein and was sensitive to a point mutation in this region. Strikingly, the anti-tetherin factor, Ebola virus GP, was able to overcome restriction conferred by both tetherin and a series of functional tetherin derivatives, including a wholly artificial tetherin molecule. Moreover, GP overcame restriction without significantly removing tetherin from the cell surface. These findings suggest that Ebola virus GP uses a novel mechanism to circumvent tetherin restriction.Pathogenic viruses often have evolved mechanisms to neutralize host defenses that act at the cellular level to interfere with the virus life cycle. Such cellular restriction factors have been most extensively characterized for HIV-1 (38) and include the interferon-inducible membrane protein BST-2/HM1.24/CD317/tetherin (28, 40). If unchecked, tetherin blocks the release of newly formed HIV-1 particles from cells by physically tethering them at the cell surface (7, 28, 32, 40). In addition, tetherin has been shown to act against a broad range of enveloped viral particles, including retroviruses, filoviruses, arenaviruses, and herpesviruses (17, 18, 23, 35). In turn, certain viruses that are targeted by tetherin appear to have evolved counteracting activities, and anti-tetherin factors so far identified include HIV-1 Vpu; HIV-2 Env; simian immunodeficiency virus (SIV) Nef, Vpu, and Env proteins; Ebola virus GP; and Kaposi''s sarcoma-associated herpesvirus (KSHV) K5 (11, 16, 18, 20, 23, 28, 36, 40, 44, 45).Tetherin is a homodimeric type II integral membrane protein containing an N-terminal cytoplasmic tail (CT), a single-pass transmembrane domain (TM), an ectodomain-containing predicted coiled-coil regions, two glycoslyation sites, three conserved cysteines, and a C-terminal glycosylphosphatidylinositol (GPI) anchor (2, 19, 31). This unusual topology, with two independent membrane anchors, has led to the suggestion that the retention of virions at the cell surface arises from tetherin''s ability to be inserted simultaneously in both host and viral membranes (28, 32, 41) or, alternatively, that dimers or higher-order complexes of tetherin conferred by the ectodomain mediate this effect (39). Interestingly, an artificial tetherin containing the same structural features as the native protein but constructed from unrelated sequences was able to restrict both HIV-1 and Ebola virus particles (32). This suggests that the viral lipid envelope is the target of tetherin and provides an explanation for tetherin''s broad activity against diverse enveloped viruses.A fraction of tetherin is present at the plasma membrane of cells (9, 14), and it has been proposed that viral anti-tetherin factors function by removing this cell surface fraction (40). This now has been shown to occur in the presence of HIV-1 Vpu (5, 7, 15, 26, 34, 40, 44), HIV-2 Env (5, 20), SIV Env (11), SIV Nef (15), and KSHV K5 (3, 23). In addition, certain anti-tetherin factors also may promote the degradation of tetherin, as has been observed for both HIV-1 Vpu (3, 5, 7, 10, 22, 26, 27) and KSHV K5 (3, 23), although Vpu also appears able to block tetherin restriction in the absence of degradation (8), and no effects on tetherin steady-state levels have been observed in the presence of either the HIV-2 or SIVtan Env (11, 20). Simply keeping tetherin away from the cell surface, or targeting it for degradation, may not be the only mechanism used by anti-tetherin factors, since it also has been reported that Vpu does not affect the levels of surface tetherin or its total cellular levels in certain T-cell lines (27).The interactions between tetherin and viral anti-tetherin factors show evidence of species specificity, suggesting ongoing evolution between viruses and their hosts. HIV-1 Vpu is active against human and chimpanzee tetherin but not other primate tetherins (10, 25, 34, 36, 44, 45), while SIV Nef proteins are active against primate but not human tetherins (16, 36, 44, 45). This suggests that, unlike tetherin restriction, the action of the anti-tetherin factors may involve specific sequence interactions. Indeed, the TM domain has been recognized as a target for HIV-1 Vpu (10, 15, 16, 25, 34), while a single point mutation introduced into the extracellular domain of human tetherin can block its antagonism by the SIVtan Env (11).In the present study, we investigated the roles of the different domains of tetherin in both promoting virus restriction and conferring susceptibility to the anti-tetherin factors encoded by HIV-1, HIV-2, and Ebola virus. We confirmed that tetherin restriction can be conferred by proteins that retain the two distinct membrane anchors, while signals for the cellular localization of the protein reside in the CT/TM domains of the protein. We found that the Vpu protein targets the TM domain of tetherin, while the HIV-2 Env targets the ectodomain of the protein. In contrast, the Ebola virus GP appears to use a non-sequence-specific mechanism to counteract tetherin restriction, since even an artificial tetherin could be successfully overcome by GP expression. Interestingly, Ebola virus GP counteracted tetherin restriction without removing the protein from the cell surface, suggesting that it is possible to overcome this restriction by mechanisms other than blocking tetherin''s cell surface expression.     

Bocavirus Infection Induces Mitochondrion-Mediated Apoptosis and Cell Cycle Arrest at G2/M Phase

Bocavirus is a newly classified genus of the family Parvovirinae. Infection with Bocavirus minute virus of canines (MVC) produces a strong cytopathic effect in permissive Walter Reed/3873D (WRD) canine cells. We have systematically characterized the MVC infection-produced cytopathic effect in WRD cells, namely, the cell death and cell cycle arrest, and carefully examined how MVC infection induces the cytopathic effect. We found that MVC infection induces an apoptotic cell death characterized by Bax translocalization to the mitochondrial outer membrane, disruption of the mitochondrial outer membrane potential, and caspase activation. Moreover, we observed that the activation of caspases occurred only when the MVC genome was replicating, suggesting that replication of the MVC genome induces apoptosis. MVC infection also induced a gradual cell cycle arrest from the S phase in early infection to the G₂/M phase at a later stage, which was confirmed by the upregulation of cyclin B1 and phosphorylation of cdc2. Cell cycle arrest at the G₂/M phase was reproduced by transfection of a nonreplicative NS1 knockout mutant of the MVC infectious clone, as well as by inoculation of UV-irradiated MVC. In contrast with other parvoviruses, only expression of the MVC proteins by transfection did not induce apoptosis or cell cycle arrest. Taken together, our results demonstrate that MVC infection induces a mitochondrion-mediated apoptosis that is dependent on the replication of the viral genome, and the MVC genome per se is able to arrest the cell cycle at the G₂/M phase. Our results may shed light on the molecular pathogenesis of Bocavirus infection in general.The Bocavirus genus is newly classified within the subfamily Parvovirinae of the family Parvoviridae (21). The currently known members of the Bocavirus genus include bovine parvovirus type 1 (BPV1) (17), minute virus of canines (MVC) (57), and the recently identified human bocaviruses (HBoV, HBoV2, and HBoV3) (4, 7, 36).MVC was first recovered from canine fecal samples in 1970 (10). The virus causes respiratory disease with breathing difficulty (14, 32, 49) and enteritis with severe diarrhea (11, 39), which often occurs with coinfection with other viruses (39), spontaneous abortion of fetuses, and death of newborn puppies (14, 29). Pathological lesions in fetuses in experimental infections were found in the lymphoid tissue of the lung and small intestine (14). MVC was isolated and grown in the Walter Reed/3873D (WRD) canine cell line (10), which is derived from a subdermoid cyst of an irradiated male dog (10). The full-length 5.4-kb genome of MVC was recently mapped with palindromic termini (60). Under the control of a single P6 promoter, through the mechanism of alternative splicing and alternative polyadenylation, MVC expresses two nonstructural proteins (NS1 and NP1) and two capsid proteins (VP1 and VP2). Like the NS1 proteins of other parvoviruses, the NS1 of MVC is indispensable for genome replication. The NP1 protein, which is unique to the Bocavirus genus, appears to be critical for optimal viral replication, as the NP1 knockout mutant of MVC suffers from severe impairment of replication (60). A severe cytopathic effect during MVC infection of WRD cells has been documented (10, 60).The HBoV genome has been frequently detected worldwide in respiratory specimens from children under 2 years old with acute respiratory illnesses (2, 34, 55). HBoV is associated with acute expiratory wheezing and pneumonia (3, 34, 55) and is commonly detected in association with other respiratory viruses (34, 55). Further studies are necessary, however, to identify potential associations of HBoV infection with clinical symptoms or disease of acute gastroenteritis (7, 36). The full-length sequence of infectious MVC DNA (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ214110","term_id":"219665308","term_text":"FJ214110"}}FJ214110) that we have reported shows 52.6% identity to HBoV, while the NS1, NP1, and VP1 proteins are 38.5%, 39.9%, and 43.7% identical to those of HBoV, respectively (60).The cytopathic effect induced during parvovirus infection has been widely documented, e.g., in infections with minute virus of mice (MVM) (13), human parvovirus B19 (B19V) (58), parvovirus H-1 (25, 52), and BPV1 (1). In Bocavirus, cell death during BPV1 infection of embryonic bovine tracheal cells has been shown to be achieved through necrosis, independent of apoptosis (1). B19V-induced cell death of primary erythroid progenitor cells has been shown to be mainly mediated by an apoptotic pathway (58) in which the nonstructural protein 11kDa plays a key role (16). In contrast, the MVM-induced cytopathic effect has been revealed to be mediated by NS1 interference with intracellular casein kinase II (CKII) signaling (22, 44, 45), a nonapoptotic cell death. Oncolytic parvovirus H-1 infections can induce either apoptosis or nonapoptotic cell death, depending on the cell type (25, 40). Therefore, the mechanisms underlying parvovirus infection-induced cell death vary, although NS1 has been widely shown to be involved in both apoptotic and nonapoptotic cell death. The nature of the cytopathic effect during Bocavirus MVC infection has not been studied.Parvovirus replication requires infected cells at the S phase. Infection with parvovirus has been revealed to accompany a cell cycle perturbation that mostly leads to an arrest in the S/G₂ phase or the G₂/M phase during infection (30, 33, 42, 47, 65). MVM NS1 expression induces an accumulation of sensitive cells in the S/G₂ phase (6, 46, 47). Whether MVC infection-induced cell death is accompanied by an alternation of cell cycle progression and whether the viral nonstructural protein is involved in these processes have not been addressed.In this study, we found, in contrast with other members of the family Parvoviridae, expression of both the nonstructural and structural proteins of MVC by transfection did not induce cell death or cell cycle arrest. However, the cytopathic effect induced during MVC infection is a replication-coupled, mitochondrion-mediated and caspase-dependent apoptosis, accompanied with a gradual cell cycle arrest from the S phase to the G₂/M phase, which is facilitated by the MVC genome.     

mTORC1-Activated S6K1 Phosphorylates Rictor on Threonine 1135 and Regulates mTORC2 Signaling

The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3α/β (GSK3α/β) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.The mammalian target of rapamycin (mTOR) is an evolutionarily conserved phosphatidylinositol 3-kinase (PI3K)-related Ser/Thr kinase that integrates signals from nutrients, energy sufficiency, and growth factors to regulate cell growth as well as organ and body size in a variety of organisms (reviewed in references 4, 38, 49, and 77). mTOR was discovered as the molecular target of rapamycin, an antifungal agent used clinically as an immunosuppressant and more recently as an anticancer drug (5, 20). Recent evidence indicates that deregulation of the mTOR pathway occurs in a majority of human cancers (12, 18, 25, 46), suggesting that rapamycin analogs may be potent antineoplastic therapeutic agents.mTOR forms two distinct multiprotein complexes, the rapamycin-sensitive and -insensitive mTOR complexes 1 and 2 (mTORC1 and mTORC2), respectively (6, 47). In cells, rapamycin interacts with FKBP12 and targets the FKBP12-rapamycin binding (FRB) domain of mTORC1, thereby inhibiting some of its function (13, 40, 66). mTORC1 is comprised of the mTOR catalytic subunit and four associated proteins, Raptor (regulatory associated protein of mTOR), mLST8 (mammalian lethal with sec13 protein 8), PRAS40 (proline-rich Akt substrate of 40 kDa), and Deptor (28, 43, 44, 47, 59, 73, 74). The small GTPase Rheb (Ras homolog enriched in brain) is a key upstream activator of mTORC1 that is negatively regulated by the tuberous sclerosis complex 1 (TSC1)/TSC2 GTPase-activating protein complex (reviewed in reference 35). mTORC1 is activated by PI3K and Ras signaling through direct phosphorylation and inactivation of TSC2 by Akt, extracellular signal-regulated kinase (ERK), and p90 ribosomal protein S6 kinase (RSK) (11, 37, 48, 53, 63). mTORC1 activity is also regulated at the level of Raptor. Whereas low cellular energy levels negatively regulate mTORC1 activity through phosphorylation of Raptor by AMP-activated protein kinase (AMPK) (27), growth signaling pathways activating the Ras/ERK pathway positively regulate mTORC1 activity through direct phosphorylation of Raptor by RSK (10). More recent evidence has also shown that mTOR itself positively regulates mTORC1 activity by directly phosphorylating Raptor at proline-directed sites (20a, 75). Countertransport of amino acids (55) and amino acid signaling through the Rag GTPases were also shown to regulate mTORC1 activity (45, 65). When activated, mTORC1 phosphorylates two main regulators of mRNA translation and ribosome biogenesis, the AGC (protein kinase A, G, and C) family kinase p70 ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and thus stimulates protein synthesis and cellular growth (50, 60).The second mTOR complex, mTORC2, is comprised of mTOR, Rictor (rapamycin-insensitive companion of mTOR), mSin1 (mammalian stress-activated mitogen-activated protein kinase-interacting protein 1), mLST8, PRR5 (proline-rich region 5), and Deptor (21, 39, 58, 59, 66, 76, 79). Rapamycin does not directly target and inhibit mTORC2, but long-term treatment with this drug was shown to correlate with mTORC2 disassembly and cytoplasmic accumulation of Rictor (21, 39, 62, 79). Whereas mTORC1 regulates hydrophobic motif phosphorylation of S6K1, mTORC2 has been shown to phosphorylate other members of the AGC family of kinases. Biochemical and genetic evidence has demonstrated that mTORC2 phosphorylates Akt at Ser473 (26, 39, 68, 70), thereby contributing to growth factor-mediated Akt activation (6, 7, 52). Deletion or knockdown of the mTORC2 components mTOR, Rictor, mSin1, and mLST8 has a dramatic effect on mTORC2 assembly and Akt phosphorylation at Ser473 (26, 39, 79). mTORC2 was also shown to regulate protein kinase Cα (PKCα) (26, 66) and, more recently, serum- and glucocorticoid-induced protein kinase 1 (SGK1) (4, 22). Recent evidence implicates mTORC2 in the regulation of Akt and PKCα phosphorylation at their turn motifs (19, 36), but whether mTOR directly phosphorylates these sites remains a subject of debate (4).Activation of mTORC1 has been shown to negatively regulate Akt phosphorylation in response to insulin or insulin-like growth factor 1 (IGF1) (reviewed in references 30 and 51). This negative regulation is particularly evident in cell culture models with defects in the TSC1/TSC2 complex, where mTORC1 and S6K1 are constitutively activated. Phosphorylation of insulin receptor substrate-1 (IRS-1) by mTORC1 (72) and its downstream target S6K1 has been shown to decrease its stability and lead to an inability of insulin or IGF1 to activate PI3K and Akt (29, 69). Although the mechanism is unknown, platelet-derived growth factor receptor β (PDGF-Rβ) has been found to be downregulated in TSC1- and TSC2-deficient murine embryonic fibroblasts (MEFs), contributing to a reduction of PI3K signaling (80). Interestingly, inhibition of Akt phosphorylation by mTORC1 has also been observed in the presence of growth factors other than IGF-1, insulin, or PDGF, suggesting that there are other mechanisms by which mTORC1 activation restricts Akt activity in cells (reviewed in references 6 and 31). Recent evidence demonstrates that rapamycin treatment causes a significant increase in Rictor electrophoretic mobility (2, 62), suggesting that phosphorylation of the mTORC2 subunit Rictor may be regulated by mTORC1 or downstream protein kinases.Herein, we demonstrate that Rictor is phosphorylated by S6K1 in response to mTORC1 activation. We demonstrate that Thr1135 is directly phosphorylated by S6K1 and found that a Rictor mutant lacking this phosphorylation site increases Akt phosphorylation induced by growth factor stimulation. Cells expressing the Rictor T1135A mutant were found to have increased Akt signaling to its substrates compared to Rictor wild-type- and T1135D mutant-expressing cells. Together, our results suggest that Rictor integrates mTORC1 signaling via its phosphorylation by S6K1, resulting in the inhibition of mTORC2 and Akt signaling.     

The Adenovirus E4orf4 Protein Induces G2/M Arrest and Cell Death by Blocking Protein Phosphatase 2A Activity Regulated by the B55 Subunit

Human adenovirus E4orf4 protein is toxic in human tumor cells. Its interaction with the Bα subunit of protein phosphatase 2A (PP2A) is critical for cell killing; however, the effect of E4orf4 binding is not known. Bα is one of several mammalian B-type regulatory subunits that form PP2A holoenzymes with A and C subunits. Here we show that E4orf4 protein interacts uniquely with B55 family subunits and that cell killing increases with the level of E4orf4 expression. Evidence suggesting that Bα-specific PP2A activity, measured in vitro against phosphoprotein substrates, is reduced by E4orf4 binding was obtained, and two potential B55-specific PP2A substrates, 4E-BP1 and p70^S6K, were seen to be hypophosphorylated in vivo following expression of E4orf4. Furthermore, treatment of cells with low levels of the phosphatase inhibitor okadaic acid or coexpression of the PP2A inhibitor I₁^PP2A enhanced E4orf4-induced cell killing and G₂/M arrest significantly. These results suggested that E4orf4 toxicity results from the inhibition of B55-specific PP2A holoenzymes, an idea that was strengthened by an observed growth arrest resulting from treatment of H1299 cells with Bα-specific RNA interference. We believe that E4orf4 induces growth arrest resulting in cell death by reducing the global level of B55-specific PP2A activity, thus preventing the dephosphorylation of B55-specific PP2A substrates, including those involved in cell cycle progression.Our research group and others have shown that the 114-residue product of early region E4 of human adenoviruses, termed E4orf4, induces p53-independent cell death in human tumor cells (24, 25, 34-36, 55) and in Saccharomyces cerevisiae (23, 53). E4orf4 protein, which shares no obvious homology with other viral or cellular products, kills a wide range of human cancer cells but is believed to have reduced activity against normal human primary cells (6, 55, 56). Although in some cases E4orf4-expressing cells exhibit characteristics typical of apoptosis, including the presence of irregularly shaped and shrunken nuclei, cytoplasmic vacuolization, and membrane blebbing (24, 25, 50, 55), cell death may more typically be independent of caspase activation (24, 25, 30, 32, 50). With H1299 human non-small-cell lung carcinoma cells, death is characterized by rapid cell rounding, enlargement, release from the surface of culture plates, cell cycle arrest in G₂/M and possibly G₁, and eventually, after an extended period, loss of membrane integrity (30). Both cytoplasmic and nuclear pathways have been observed, the former involving interactions with c-Src family kinases, activation of calpain, and remodeling of the actin cytoskeleton (7, 24, 50, 51, 58). Little is known about the nuclear pathway, which may represent the predominant death-inducing process. Our current evidence suggests that H1299 cells die following prolonged irreversible cell cycle arrest leading to mitotic catastrophe and death by a necrosis-like process (30).E4orf4 is known to associate with the Bα regulatory subunit of protein phosphatase 2A (PP2A) (22, 34), and this interaction appears to be necessary for the majority of E4orf4 toxicity in both yeast (23, 53) and human tumor cells (34, 56). PP2A is an abundant serine-threonine phosphatase involved in regulation of metabolism, splicing, translation, morphogenesis, development, and cell cycle progression (15, 19, 27, 43, 59). PP2A holoenzymes exist as multiple heterotrimeric complexes composed of a catalytic C subunit, an A subunit that functions as a scaffold, and a B-type regulatory subunit. Two forms each of the A and C subunits exist in mammalian cells; however, more than 20 B-type subunits have been identified in three unique classes (B/B55, B′/B56, B″/PR72), plus striatin/SG2NA (sometimes called B‴) (10, 19, 26). Although one group has suggested that E4orf4 protein interacts with one or more members of the B′/B56 class (57), it is generally accepted that interaction with the Bα/B55 subunit (Cdc55 in yeast) is important for induction of cell death in both human tumor cells and yeast (53, 57). Interestingly, a recent report has also suggested that in yeast, growth suppression induced by E4orf4 is mediated only in part by the catalytic C subunit of PP2A (31).In the present report, we show that E4orf4 protein interacts uniquely with members of the B55 class of PP2A B-type subunits, and at sufficient concentrations, it appears to become toxic by reducing dephosphorylation of substrates of B55-containing PP2A holoenzymes. As cell death is preceded by cell cycle arrest, we believe that key substrates may include proteins required for cell cycle progression.     

Isolation of Human Monoclonal Antibodies That Potently Neutralize Human Cytomegalovirus Infection by Targeting Different Epitopes on the gH/gL/UL128-131A Complex

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC₉₀] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.Human cytomegalovirus (HCMV) is a member of the herpesvirus family which is widely distributed in the human population and can cause severe disease in immunocompromised patients and upon infection of the fetus. HCMV infection causes clinical disease in 75% of patients in the first year after transplantation (58), while primary maternal infection is a major cause of congenital birth defects including hearing loss and mental retardation (5, 33, 45). Because of the danger posed by this virus, development of an effective vaccine is considered of highest priority (51).HCMV infection requires initial interaction with the cell surface through binding to heparan sulfate proteoglycans (8) and possibly other surface receptors (12, 23, 64, 65). The virus displays a broad host cell range (24, 53), being able to infect several cell types such as endothelial cells, epithelial cells (including retinal cells), smooth muscle cells, fibroblasts, leukocytes, and dendritic cells (21, 37, 44, 54). Endothelial cell tropism has been regarded as a potential virulence factor that might influence the clinical course of infection (16, 55), whereas infection of leukocytes has been considered a mechanism of viral spread (17, 43, 44). Extensive propagation of HCMV laboratory strains in fibroblasts results in deletions or mutations of genes in the UL131A-128 locus (1, 18, 21, 36, 62, 63), which are associated with the loss of the ability to infect endothelial cells, epithelial cells, and leukocytes (15, 43, 55, 61). Consistent with this notion, mouse monoclonal antibodies (MAbs) to UL128 or UL130 block infection of epithelial and endothelial cells but not of fibroblasts (63). Recently, it has been shown that UL128, UL130, and UL131A assemble with gH and gL to form a five-protein complex (thereafter designated gH/gL/UL128-131A) that is an alternative to the previously described gCIII complex made of gH, gL, and gO (22, 28, 48, 63).In immunocompetent individuals T-cell and antibody responses efficiently control HCMV infection and reduce pathological consequences of maternal-fetal transmission (13, 67), although this is usually not sufficient to eradicate the virus. Albeit with controversial results, HCMV immunoglobulins (Igs) have been administered to transplant patients in association with immunosuppressive treatments for prophylaxis of HCMV disease (56, 57), and a recent report suggests that they may be effective in controlling congenital infection and preventing disease in newborns (32). These products are plasma derivatives with relatively low potency in vitro (46) and have to be administered by intravenous infusion at very high doses in order to deliver sufficient amounts of neutralizing antibodies (4, 9, 32, 56, 57, 66).The whole spectrum of antigens targeted by HCMV-neutralizing antibodies remains poorly characterized. Using specific immunoabsorption to recombinant antigens and neutralization assays using fibroblasts as model target cells, it was estimated that 40 to 70% of the serum neutralizing activity is directed against gB (6). Other studies described human neutralizing antibodies specific for gB, gH, or gM/gN viral glycoproteins (6, 14, 26, 29, 34, 41, 52, 60). Remarkably, we have recently shown that human sera exhibit a more-than-100-fold-higher potency in neutralizing infection of endothelial cells than infection of fibroblasts (20). Similarly, CMV hyperimmunoglobulins have on average 48-fold-higher neutralizing activities against epithelial cell entry than against fibroblast entry (10). However, epitopes that are targeted by the antibodies that comprise epithelial or endothelial cell-specific neutralizing activity of human immune sera remain unknown.In this study we report the isolation of a large panel of human monoclonal antibodies with extraordinarily high potency in neutralizing HCMV infection of endothelial and epithelial cells and myeloid cells. With the exception of a single antibody that recognized a conserved epitope of UL128, all other antibodies recognized conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex.     

Cell-Cell Spread of Human Immunodeficiency Virus Type 1 Overcomes Tetherin/BST-2-Mediated Restriction in T cells

Direct cell-to-cell spread of human immunodeficiency virus type 1 (HIV-1) between T cells at the virological synapse (VS) is an efficient mechanism of viral dissemination. Tetherin (BST-2/CD317) is an interferon-induced, antiretroviral restriction factor that inhibits nascent cell-free particle release. The HIV-1 Vpu protein antagonizes tetherin activity; however, whether tetherin also restricts cell-cell spread is unclear. We performed quantitative cell-to-cell transfer analysis of wild-type (WT) or Vpu-defective HIV-1 in Jurkat and primary CD4⁺ T cells, both of which express endogenous levels of tetherin. We found that Vpu-defective HIV-1 appeared to disseminate more efficiently by cell-to-cell contact between Jurkat cells under conditions where tetherin restricted cell-free virion release. In T cells infected with Vpu-defective HIV-1, tetherin was enriched at the VS, and VS formation was increased compared to the WT, correlating with an accumulation of virus envelope proteins on the cell surface. Increasing tetherin expression with type I interferon had only minor effects on cell-to-cell transmission. Furthermore, small interfering RNA (siRNA)-mediated depletion of tetherin decreased VS formation and cell-to-cell transmission of both Vpu-defective and WT HIV-1. Taken together, these data demonstrate that tetherin does not restrict VS-mediated T cell-to-T cell transfer of Vpu-defective HIV-1 and suggest that under some circumstances tetherin might promote cell-to-cell transfer, either by mediating the accumulation of virions on the cell surface or by regulating integrity of the VS. If so, inhibition of tetherin activity by Vpu may balance requirements for efficient cell-free virion production and cell-to-cell transfer of HIV-1 in the face of antiviral immune responses.Human immunodeficiency virus type 1 can disseminate between and within hosts by cell-free infection or by direct cell-cell spread. Cell-cell spread of HIV-1 between CD4⁺ T cells is an efficient means of viral dissemination (65) and has been estimated to be several orders of magnitude more rapid than cell-free virus infection (6, 8, 41, 64, 74). Cell-cell transmission of HIV-1 takes place at the virological synapse (VS), a multimolecular structure that forms at the interface between an HIV-1-infected T cell and an uninfected target T cell during intercellular contact (27). Related structures that facilitate cell-cell spread of HIV-1 between dendritic cells and T cells (42) and between macrophages and T cells (16, 17) and for cell-cell spread of the related retrovirus human T-cell leukemia virus type 1 (HTLV-1) (24) have also been described. Moreover, more long-range cell-cell transfer can occur via cellular projections, including filopodia (71) and membrane nanotubes (75). The VS is initiated by binding of the HIV-1 envelope glycoprotein (Env), which is expressed on the surfaces of infected T cells, to HIV-1 entry receptors (CD4 and either CXCR4 or CCR5) present on the target cell membrane (6, 22, 27, 41, 61, 73). Interactions between LFA-1 and ICAM-1 and ICAM-3 further stabilize the conjugate interface and, together with Env receptor binding, help trigger the recruitment of viral proteins, CD4/coreceptor, and integrins to the contact site (27, 28, 61). The enrichment of viral and cellular proteins at the VS is an active process, dependent on cytoskeletal remodeling, and in the infected T cell both the actin and tubulin network regulate polarization of HIV-1 proteins at the cell-cell interface, thus directing HIV-1 assembly and egress toward the engaged target cell (27, 29). Virus is transferred by budding into the synaptic cleft, and virions subsequently attach to the target cell membrane to mediate entry, either by fusion at the plasma membrane or possibly following endocytic uptake (2, 22). In this way, the VS promotes more rapid infection kinetics and may enhance HIV-1 pathogenesis in vivo.Cells have evolved a number of barriers to resist invading microorganisms. One mechanism that appears to be particularly important in counteracting HIV-1 infection is a group of interferon-inducible, innate restriction factors that includes TRIM5α, APOBEC3G, and tetherin (38, 49, 69, 79). Tetherin (BST-2/CD317) is a host protein expressed by many cell types, including CD4⁺ T cells, that acts at a late stage of the HIV-1 life cycle to trap (or “tether”) mature virions at the plasma membranes of virus-producing cells, thereby inhibiting cell-free virus release (49, 56, 81). This antiviral activity of tetherin is not restricted to HIV-1, and tetherin can also inhibit the release of other enveloped viruses from infected cells (31, 40, 54, 62). What the cellular function of tetherin is besides its antiviral activity is unclear, but because expression is upregulated following alpha/beta interferon (IFN-α/β) treatment (1) and tetherin can restrict a range of enveloped viruses, tetherin has been postulated to be a broad-acting mediator of the innate immune defense against enveloped viruses.To circumvent restriction of particle release, HIV-1 encodes the 16-kDa accessory protein Vpu, which antagonizes tetherin and restores normal virus budding (47, 78). The molecular mechanisms by which Vpu does this are not entirely clear, but evidence suggests that Vpu may exert its antagonistic function by downregulating tetherin from the cell surface, trapping it in the trans-Golgi network (10) and targeting it for degradation by the proteasome (12, 39, 81) or lysosome (9, 25, 44); however, degradation of tetherin may be dispensable for Vpu activity (13), and in HIV-1-infected T cells, surface downregulation of tetherin has been reported to be minor (45), suggesting that global removal of tetherin from the plasma membrane may not be necessary to antagonize its function.Tetherin-mediated restriction of HIV-1 and antagonism by Vpu have been the focus of much research, and inhibition of cell-free virus infection has been well documented (33, 47-49, 77, 81, 82). In contrast, less studied is the impact of tetherin on direct cell-cell dissemination. For example, it is not clear if tetherin-mediated restriction inhibits T cell-T cell spread as efficiently as cell-free release or whether tetherin affects VS formation. To address these questions, we analyzed Vpu⁺ and Vpu⁻ viruses for their ability to spread directly between Jurkat T cells and primary CD4⁺ T cells in the presence or absence of endogenous tetherin. Our data suggest that tetherin does not restrict HIV-1 in the context of cell-to-cell transmission of virus between T cells expressing endogenous tetherin. Interestingly, we also that observed that Vpu-defective virus may disseminate more efficiently by cell-cell spread at the VS. We postulate that cell-cell spread may favor viral pathogenesis by allowing HIV-1 to disseminate in the presence of tetherin during an interferon-producing innate response.