Bending of the BST‐2 coiled‐coil during viral budding

BST‐2/tetherin is a human extracellular transmembrane protein that serves as a host defense factor against HIV‐1 and other viruses by inhibiting viral spreading. Structurally, BST‐2 is a homo‐dimeric coiled‐coil that is connected to the host cell membrane by N and C terminal transmembrane anchors. The C‐terminal membrane anchor of BST‐2 is inserted into the budding virus while the N‐terminal membrane anchor remains in the host cell membrane creating a viral tether. The structural mechanism of viral budding and tethering as mediated by BST‐2 is not clear. To more fully describe the mechanism of viral tethering, we created a model of BST‐2 embedded in a membrane and used steered molecular dynamics to simulate the transition from the host cell membrane associated form to the cell‐virus membrane bridging form. We observed that BST‐2 did not transition as a rigid structure, but instead bent at positions with a reduced interface between the helices of the coiled‐coil. The simulations for the human BST‐2 were then compared with simulations on the mouse homolog, which has no apparent weak spots. We observed that the mouse homolog spread the bending across the ectodomain, rather than breaking at discrete points as observed with the human homolog. These simulations support previous biochemical and cellular work suggesting some flexibility in the coiled‐coil is necessary for viral tethering, while also highlighting how subtle changes in protein sequence can influence the dynamics and stability of proteins with overall similar structure.     

Human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements

Human cytomegalovirus (HCMV) replicates in the nuclei of infected cells. Successful replication therefore depends on particle movements between the cell cortex and nucleus during entry and egress. To visualize HCMV particles in living cells, we have generated a recombinant HCMV expressing enhanced green fluorescent protein (EGFP) fused to the C terminus of the capsid-associated tegument protein pUL32 (pp150). The resulting UL32-EGFP-HCMV was analyzed by immunofluorescence, electron microscopy, immunoblotting, confocal microscopy, and time-lapse microscopy to evaluate the growth properties of this virus and the dynamics of particle movements. UL32-EGFP-HCMV replicated similarly to wild-type virus in fibroblast cultures. Green fluorescent virus particles were released from infected cells. The fluorescence stayed associated with particles during viral entry, and fluorescent progeny particles appeared in the nucleus at 44 h after infection. Surprisingly, strict colocalization of pUL32 and the major capsid protein pUL86 within nuclear inclusions indicated that incorporation of pUL32 into nascent HCMV particles occurred simultaneously with or immediately after assembly of the capsid. A slow transport of nuclear particles towards the nuclear margin was demonstrated. Within the cytoplasm, most particles performed irregular short-distance movements, while a smaller fraction of particles performed centripetal and centrifugal long-distance movements. Although numerous particles accumulated in the cytoplasm, release of particles from infected cells was a rare event, consistent with a release rate of about 1 infectious unit per h per cell in HCMV-infected fibroblasts as calculated from single-step growth curves. UL32-EGFP-HCMV will be useful for further investigations into the entry, maturation, and release of this virus.     

PUL21a-Cyclin A2 Interaction is Required to Protect Human Cytomegalovirus-Infected Cells from the Deleterious Consequences of Mitotic Entry

Entry into mitosis is accompanied by dramatic changes in cellular architecture, metabolism and gene expression. Many viruses have evolved cell cycle arrest strategies to prevent mitotic entry, presumably to ensure sustained, uninterrupted viral replication. Here we show for human cytomegalovirus (HCMV) what happens if the viral cell cycle arrest mechanism is disabled and cells engaged in viral replication enter into unscheduled mitosis. We made use of an HCMV mutant that, due to a defective Cyclin A2 binding motif in its UL21a gene product (pUL21a), has lost its ability to down-regulate Cyclin A2 and, therefore, to arrest cells at the G1/S transition. Cyclin A2 up-regulation in infected cells not only triggered the onset of cellular DNA synthesis, but also promoted the accumulation and nuclear translocation of Cyclin B1-CDK1, premature chromatin condensation and mitotic entry. The infected cells were able to enter metaphase as shown by nuclear lamina disassembly and, often irregular, metaphase spindle formation. However, anaphase onset was blocked by the still intact anaphase promoting complex/cyclosome (APC/C) inhibitory function of pUL21a. Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions. Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments. The prolonged metaphase arrest in infected cells coincided with precocious sister chromatid separation and progressive fragmentation of the chromosomal material. We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle. Unscheduled mitotic entry during the course of the HCMV replication has fatal consequences, leading to abortive infection and cell death.     

A new class of receptor for herpes simplex virus has heptad repeat motifs that are common to membrane fusion proteins

We isolated a human cDNA by expression cloning and characterized its gene product as a new human protein that enables entry and infection of herpes simplex virus (HSV). The gene, designated hfl-B5, encodes a type II cell surface membrane protein, B5, that is broadly expressed in human primary tissue and cell lines. It contains a high-scoring heptad repeat at the extracellular C terminus that is predicted to form an alpha-helix for coiled coils like those in cellular SNAREs or in some viral fusion proteins. A synthetic 30-mer peptide that has the same sequence as the heptad repeat alpha-helix blocks HSV infection of B5-expressing porcine cells and human HEp-2 cells. Transient expression of human B5 in HEp-2 cells results in increased polykarocyte formation even in the absence of viral proteins. The B5 protein fulfills all criteria as a receptor or coreceptor for HSV entry. Use by HSV of a human cellular receptor, such as B5, that contains putative membrane fusion domains provides an example where a pathogenic virus with broad tropism has usurped a widely expressed cellular protein to function in infection at events that lead to membrane fusion.     

The human cytomegalovirus IE86 protein can block cell cycle progression after inducing transition into the S phase of permissive cells

Human cytomegalovirus (HCMV) infection of permissive cells has been reported to induce a cell cycle halt. One or more viral proteins may be involved in halting progression at different stages of the cell cycle. We investigated how HCMV infection, and specifically IE86 protein expression, affects the cell cycles of permissive and nonpermissive cells. We used a recombinant virus that expresses the green fluorescent protein (GFP) to determine the effects of HCMV on the cell cycle of permissive cells. Fluorescence by GFP allowed us to select for only productively infected cells. Replication-defective adenovirus vectors expressing the IE72 or IE86 protein were also used to efficiently transduce 95% or more of the cells. The adenovirus-expressed IE86 protein was determined to be functional by demonstrating negative autoregulation of the major immediate-early promoter and activation of an early viral promoter in the context of the viral genome. To eliminate adenovirus protein effects, plasmids expressing GFP for fluorescent selection of only transfected cells and wild-type IE86 protein or a mutant IE86 protein were tested in permissive and nonpermissive cells. HCMV infection induced the entry of U373 cells into the S phase. All permissive cells infected with HCMV were blocked in cell cycle progression and could not divide. After either transduction or transfection and IE86 protein expression, the number of all permissive or nonpermissive cell types in the S phase increased significantly, but the cells could no longer divide. The IE72 protein did not have a significant effect on the S phase. Since IE86 protein inhibits cell cycle progression, the IE2 gene in a human fibroblast IE86 protein-expressing cell line was sequenced. The IE86 protein in these retrovirus-transduced cells has mutations in a critical region of the viral protein. The locations of the mutations and the function of the IE86 protein in controlling cell cycle progression are discussed.     

The Cellular Proteins Grb2 and DDX3 Are Increased upon Human Cytomegalovirus Infection and Act in a Proviral Fashion

While it is well established that human cytomegalovirus (HCMV) upregulates many cellular proteins and incorporates several of them into its virion, little is known about the functional relevance of such virus-host interactions. Two cellular proteins, Grb2 and DDX3, gained our interest as they appeared enriched in virion particles and this incorporation depended on the viral tegument protein pp65, suggesting a functional relevance. We therefore tested whether the level of these proteins is altered upon HCMV infection and whether they support viral replication. Immunoblotting analyses of cellular fractions showed increased levels of both proteins in infected cells with a maximum at 2 d p.i. and a reduction of the soluble Grb2 fraction. Knockdown of either gene by transfection of siRNAs reduced viral spread not only of the cell culture adapted HCMV strain TB40/E but also of recent clinical isolates. Apparently, Grb2 and DDX3 are proviral cellular factors that are upregulated in infected cells.     

Molecular Mechanism of BST2/Tetherin Downregulation by K5/MIR2 of Kaposi's Sarcoma-Associated Herpesvirus

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, termed membrane-associated RING-CH (MARCH) proteins (3), that mediate the ubiquitination of the cytoplasmic portion of transmembrane proteins (reviewed in reference 40). Each member of this family targets a subset of cellular membrane proteins with both unique and shared specificities (4, 56). One of the functions of cellular MARCH proteins is to modulate antigen presentation by mediating the ubiquitin-dependent turnover of major histocompatibility complex (MHC) class II molecules in dendritic cells, B cells, and monocytes/macrophages (43, 52). In contrast, viral homologues of MARCH proteins encoded by KSHV, murine herpesvirus 68, and the leporipoxvirus myxomavirus all share the ability to mediate the destruction of MHC-I (reviewed in reference 16) but not MHC-II molecules. Thus, one of the functions of the viral proteins is to promote viral escape from immune clearance by CD8⁺ T lymphocytes (50). Furthermore, each viral MARCH homologue specifically eliminates additional host cell proteins, so each plays multiple roles in viral pathogenesis. KSHV carries two viral MARCH proteins, K3 and K5, also known as MIR1 and MIR2, which both support viral escape from T-cell, NK-cell, and NKT-cell recognition by eliminating the corresponding ligands from the surfaces of infected cells (reviewed in reference 10). In endothelial cells (ECs), K5 additionally downregulates EC-specific adhesion molecules that play an essential role in the formation of adhesive platforms and adherens junctions (31, 32). Since Kaposi''s sarcoma is a tumor of EC origin, K5 might thus also contribute to tumorigenesis by disrupting normal EC barrier function and by modulating the interaction of ECs with inflammatory leukocytes.The downregulation of BST2 by K5 further suggests that K5 also counteracts innate antiviral responses, which might benefit KSHV. However, most transmembrane proteins targeted by viral or cellular MARCH proteins are type I transmembrane proteins that belong to the immunoglobulin superfamily. In contrast, BST2 is a type II transmembrane protein that is also glycosylphosphatidylinositol (GPI) anchored (25). Thus, BST2 has a short cytoplasmic amino terminus followed by an outside-in transmembrane domain, a large glycosylated extracellular portion, and a GPI anchor. The additional propensity of BST2 to form homodimers (44) was speculated to be crucial for the tethering function of BST2 in that self-association of BST2 molecules in the viral envelope with plasma membrane BST2 could prevent viral exit (19). The unusual topology of BST2 and its multimerization raised the question of whether BST2 is a bona fide target of K5 or whether its downregulation is a downstream effect of K5 eliminating other transmembrane proteins. Additionally, it is not clear whether BST2 would be downregulated in the context of a normal viral infection and, particularly, whether virally expressed K5 would be able to overcome the high expression levels of BST2 observed upon IFN induction. We now demonstrate that KSHV efficiently downregulates IFN-induced BST2 both during primary infection and upon reactivation from latency in ECs. IFN-induced BST2 is ubiquitinated by K5 upon exiting the endoplasmic reticulum (ER) and is rapidly degraded by a pathway that is sensitive to proteasome inhibitors but resistant to inhibitors of lysosomal acidification. These data suggest that despite its unusual topology, BST2 is directly targeted by K5. We further demonstrate that BST2 reduces KSHV release upon inhibition of K5 expression by small interfering RNA (siRNA), suggesting that BST2 is part of the IFN-induced innate immune response to KSHV. Thus, in addition to contributing to viral evasion of cellular immune responses and remodeling EC function, K5 also counteracts the innate immune defense of the host cell.     

Tegument Proteins of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a common, medically relevant human herpesvirus. The tegument layer of herpesvirus virions lies between the genome-containing capsids and the viral envelope. Proteins within the tegument layer of herpesviruses are released into the cell upon entry when the viral envelope fuses with the cell membrane. These proteins are fully formed and active and control viral entry, gene expression, and immune evasion. Most tegument proteins accumulate to high levels during later stages of infection, when they direct the assembly and egress of progeny virions. Thus, viral tegument proteins play critical roles at the very earliest and very last steps of the HCMV lytic replication cycle. This review summarizes HCMV tegument composition and structure as well as the known and speculated functions of viral tegument proteins. Important directions for future investigation and the challenges that lie ahead are identified and discussed.     

Up-regulation of Fas ligand expression by human cytomegalovirus immediate-early gene product 2: a novel mechanism in cytomegalovirus-induced apoptosis in human retina

Human CMV (HCMV) is an important pathogen that causes widespread diseases in immunocompromised individuals. Among the opportunistic HCMV infections, HCMV retinitis is most common in transplant recipients and AIDS patients. It often leads to blindness if left untreated. The question as to how HCMV infection causes retinal pathogenesis remains unresolved. Here, we report that viral immediate-early gene product 2 (IE2), but not IE1, up-regulates the Fas ligand (FasL) expression in HCMV-infected human retinal pigment epithelium cells. Increased secretion of FasL from virally infected cells into cultured medium was observed upon HCMV infection. The capability of such cell-free medium to induce apoptosis of Fas (CD95)-expressing Jurkat cells further implies that Fas-FasL interaction might mediate cell death in the lesion of HCMV retinitis. To support this idea, we observed augmented soluble FasL levels in vitreous from AIDS patients with HCMV retinitis as compared with that from AIDS patients without HCMV infection. In addition, by in situ hybridization and immunohistochemistry, we detected enhanced signals of FasL, the existence of viral IE Ags and apoptotic cells at the same sites in the lesion of HCMV-infected retina. These results strongly suggest that IE2 induction of FasL expression in human retina might be an important event that takes place in the early stage of infection and finally leads to visual loss in individuals affiliated with HCMV retinitis.     

Cutting edge: paradoxical roles of BST2/tetherin in promoting type I IFN response and viral infection

Bone marrow stromal Ag 2 (BST2) is a transmembrane protein that prevents virus release from infected cells. It was also reported that BST2 inhibits type I IFN production by plasmacytoid dendritic cells. To determine BST2 impact on antiviral responses in vivo, we generated BST2(-/-) mice. Following infection with a murine retrovirus, BST2(-/-) mice had slightly elevated viral loads; however, infection with other enveloped viruses revealed unexpected roles of BST2. BST2(-/-) mice showed reduced type I IFN production by plasmacytoid dendritic cells. Moreover, BST2(-/-) mice had lower viral titers in lungs following intranasal infection with vesicular stomatitis virus expressing OVA and influenza B and increased numbers of virus-specific CD8 T cells in the lungs, suggesting that BST2 may facilitate entry and/or replication of enveloped viruses and modulate priming of CD8 T cells. These findings suggest complex roles of BST2 beyond retroviral control in vivo, possibly reflecting the involvement of BST2 in endocytosis and intracellular trafficking of viruses, viral nucleic acids, and Ags.     

Herpes simplex virus type 1 enters human epidermal keratinocytes, but not neurons, via a pH-dependent endocytic pathway

Herpes simplex virus (HSV) enters some laboratory cell lines via a pH-dependent, endocytic mechanism. We investigated whether this entry pathway is used in human cell types relevant to pathogenesis. Three different classes of lysosomotropic agents, which raise endosomal pH, blocked HSV entry into primary and transformed human keratinocytes, but not into human neurons or neuroblastoma lines. In keratinocytes, incoming HSV particles colocalized with markers of endocytic uptake. Treatment with the isoflavone genistein, an inhibitor of protein tyrosine kinases, reduced the delivery of incoming viral particles to the nuclear periphery and virus-induced gene expression in keratinocytes but not neurons. Moreover, in keratinocyte monolayer islets, HSV infected both the inner and outer cells in a genistein-sensitive manner, suggesting viral endocytosis from both basolateral and apical plasma membrane surfaces. Together, the results indicate that HSV enters human epidermal keratinocytes, but not neurons, by a low-pH, endocytic pathway that is dependent on host tyrosine phosphorylation. Thus, HSV utilizes fundamentally different cellular entry pathways to infect important target cell populations.     

Human cytomegalovirus infection of caco-2 cells occurs at the basolateral membrane and is differentiation state dependent

Epithelial cells are known to be a major target for human cytomegalovirus (HCMV) infection; however, the analysis of virus-cell interactions has been difficult to approach due to the lack of in vitro models. In this study, we established a polarized epithelial cell model using a colon epithelial cell-derived cell line (Caco-2) that is susceptible to HCMV infection at early stages of cellular differentiation. Infection of polarized cells was restricted to the basolateral surface whereas virus was released apically, which was consistent with the apical and not basolateral surface localization of two essential viral glycoproteins, gB and gH. HCMV infection resulted in the development of a cytopathology characteristic of HCMV infection of colon epithelium in vivo, and infection did not spread from cell to cell. The inability of HCMV to infect Caco-2 cells at late stages of differentiation was due to a restriction at the level of viral entry and was consistent with the sequestration of a cellular receptor for HCMV. These observations provide the first evidence that restriction of HCMV replication in epithelial cells is due to a receptor-mediated phenomenon.     

BST2/Tetherin Inhibits Dengue Virus Release from Human Hepatoma Cells

Type I interferons (IFN) have been shown to play an important role for inhibiting Dengue virus (DENV) infection. Identifying IFN-induced cellular proteins are essential for understanding its mechanisms against DENV. Here we established stable Huh7-derived cell lines expressing the IFN-induced cell membrane protein BST2 (Huh7-BST2) or its variant bearing a V5 tag at the C-terminal (Huh7-BST5CV5). These cell lines were infected with DENV to determine proteins modulating their anti-DENV response. We found that expression of BST2 did not affect the efficiency of DENV infection and intracellular replication. Rather, it significantly reduced the virion yield of the infected cells, particularly at low MOI infection. In addition, BST2 also decreased the foci formation and the size of infectious foci in cultured Huh7 monolayers with media containing methocellulose. The addition of the V5 tag at C-terminal inhibited the GPI modification of BST2 and blocked its shift from endoplasm to cytoplastic membrane. BST2CV5 did not affect DENV infection and foci formation in Huh7 cells but reduced virion yield by 1 log at low MOI infection. Interestingly, intracellular BST2CV5 expression was reduced by high level of DENV production.

Conclusion

Our results imply that BST2 is a functional mediator of the IFN response against DENV infection. BST2 inhibits the release of DENV virions from Huh7 cells and limits viral cell-to-cell transmission. BST2CV5 variant is unable to inhibit DENV release but impairs viral infection in cells.     

Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2

The distribution of the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor, an angiotensin-converting enzyme 2 (ACE2), does not strictly correlate with SARS-CoV cell tropism in lungs; therefore, other cellular factors have been predicted to be required for activation of virus infection. In the present study, we identified transmembrane protease serine 2 (TMPRSS2), whose expression does correlate with SARS-CoV infection in the upper lobe of the lung. In Vero cells expressing TMPRSS2, large syncytia were induced by SARS-CoV infection. Further, the lysosome-tropic reagents failed to inhibit, whereas the heptad repeat peptide efficiently inhibited viral entry into cells, suggesting that TMPRSS2 affects the S protein at the cell surface and induces virus-plasma membrane fusion. On the other hand, production of virus in TMPRSS2-expressing cells did not result in S-protein cleavage or increased infectivity of the resulting virus. Thus, TMPRSS2 affects the entry of virus but not other phases of virus replication. We hypothesized that the spatial orientation of TMPRSS2 vis-a-vis S protein is a key mechanism underling this phenomenon. To test this, the TMPRSS2 and S proteins were expressed in cells labeled with fluorescent probes of different colors, and the cell-cell fusion between these cells was tested. Results indicate that TMPRSS2 needs to be expressed in the opposing (target) cell membrane to activate S protein rather than in the producer cell, as found for influenza A virus and metapneumoviruses. This is the first report of TMPRSS2 being required in the target cell for activation of a viral fusion protein but not for the S protein synthesized in and transported to the surface of cells. Our findings suggest that the TMPRSS2 expressed in lung tissues may be a determinant of viral tropism and pathogenicity at the initial site of SARS-CoV infection.     

Functional genetic analysis of rhesus cytomegalovirus: Rh01 is an epithelial cell tropism factor

Rhesus cytomegalovirus (RhCMV) is an emerging model for human cytomegalovirus (HCMV) pathogenesis that facilitates experimental CMV infection of a natural primate host closely related to humans. We have generated a library of RhCMV mutants with lesions in genes whose HCMV orthologues have been characterized as nonessential for replication in human fibroblasts, and we characterized their replication in rhesus fibroblasts and epithelial cells. The RhCMV mutants grew well in fibroblasts, as predicted by earlier studies with HCMV. However, mutations in four genes caused replication defects in rhesus retinal pigment epithelial cells: Rh01 (an HCMV TRL1 orthologue), Rh159 (HCMV UL148), Rh160 (HCMV UL132), and Rh203 (HCMV US22). Growth of the Rh01-deficient mutant was examined in detail. After entry into epithelial cells, the mutant expressed representative viral proteins, accumulated viral DNA, and generated infectious virus, but it failed to spread efficiently. We conclude that Rh01 is a cell tropism determinant that has the potential to dramatically affect virus spread and pathogenesis.