Vaccinia Virus Proteins A52 and B14 Share a Bcl-2–Like Fold but Have Evolved to Inhibit NF-κB rather than Apoptosis

Vaccinia virus (VACV), the prototype poxvirus, encodes numerous proteins that modulate the host response to infection. Two such proteins, B14 and A52, act inside infected cells to inhibit activation of NF-κB, thereby blocking the production of pro-inflammatory cytokines. We have solved the crystal structures of A52 and B14 at 1.9 Å and 2.7 Å resolution, respectively. Strikingly, both these proteins adopt a Bcl-2–like fold despite sharing no significant sequence similarity with other viral or cellular Bcl-2–like proteins. Unlike cellular and viral Bcl-2–like proteins described previously, A52 and B14 lack a surface groove for binding BH3 peptides from pro-apoptotic Bcl-2–like proteins and they do not modulate apoptosis. Structure-based phylogenetic analysis of 32 cellular and viral Bcl-2–like protein structures reveals that A52 and B14 are more closely related to each other and to VACV N1 and myxoma virus M11 than they are to other viral or cellular Bcl-2–like proteins. This suggests that a progenitor poxvirus acquired a gene encoding a Bcl-2–like protein and, over the course of evolution, gene duplication events have allowed the virus to exploit this Bcl-2 scaffold for interfering with distinct host signalling pathways.     

Poxviruses package viral redox proteins in lateral bodies and modulate the host oxidative response

All poxviruses contain a set of proteinaceous structures termed lateral bodies (LB) that deliver viral effector proteins into the host cytosol during virus entry. To date, the spatial proteotype of LBs remains unknown. Using the prototypic poxvirus, vaccinia virus (VACV), we employed a quantitative comparative mass spectrometry strategy to determine the poxvirus LB proteome. We identified a large population of candidate cellular proteins, the majority being mitochondrial, and 15 candidate viral LB proteins. Strikingly, one-third of these are VACV redox proteins whose LB residency could be confirmed using super-resolution microscopy. We show that VACV infection exerts an anti-oxidative effect on host cells and that artificial induction of oxidative stress impacts early and late gene expression as well as virion production. Using targeted repression and/or deletion viruses we found that deletion of individual LB-redox proteins was insufficient for host redox modulation suggesting there may be functional redundancy. In addition to defining the spatial proteotype of VACV LBs, these findings implicate poxvirus redox proteins as potential modulators of host oxidative anti-viral responses and provide a solid starting point for future investigations into the role of LB resident proteins in host immunomodulation.     

Structural biology of the Bcl-2 family and its mimicry by viral proteins

Intrinsic apoptosis in mammals is regulated by protein–protein interactions among the B-cell lymphoma-2 (Bcl-2) family. The sequences, structures and binding specificity between pro-survival Bcl-2 proteins and their pro-apoptotic Bcl-2 homology 3 motif only (BH3-only) protein antagonists are now well understood. In contrast, our understanding of the mode of action of Bax and Bak, the two necessary proteins for apoptosis is incomplete. Bax and Bak are isostructural with pro-survival Bcl-2 proteins and also interact with BH3-only proteins, albeit weakly. Two sites have been identified; the in-groove interaction analogous to the pro-survival BH3-only interaction and a site on the opposite molecular face. Interaction of Bax or Bak with activator BH3-only proteins and mitochondrial membranes triggers a series of ill-defined conformational changes initiating their oligomerization and mitochondrial outer membrane permeabilization. Many actions of the mammalian pro-survival Bcl-2 family are mimicked by viruses. By expressing proteins mimicking mammalian pro-survival Bcl-2 family proteins, viruses neutralize death-inducing members of the Bcl-2 family and evade host cell apoptosis during replication. Remarkably, structural elements are preserved in viral Bcl-2 proteins even though there is in many cases little discernible sequence conservation with their mammalian counterparts. Some viral Bcl-2 proteins are dimeric, but they have distinct structures to those observed for mammalian Bcl-2 proteins. Furthermore, viral Bcl-2 proteins modulate innate immune responses regulated by NF-κB through an interface separate from the canonical BH3-binding groove. Our increasing structural understanding of the viral Bcl-2 proteins is leading to new insights in the cellular Bcl-2 network by exploring potential alternate functional modes in the cellular context. We compare the cellular and viral Bcl-2 proteins and discuss how alterations in their structure, sequence and binding specificity lead to differences in behavior, and together with the intrinsic structural plasticity in the Bcl-2 fold enable exquisite control over critical cellular signaling pathways.     

Structure of vaccinia virus A46, an inhibitor of TLR4 signaling pathway,shows the conformation of VIPER motif

Vaccinia virus (VACV) encodes many proteins that interfere with the host immune system. Vaccinia virus A46 protein specifically targets the BB‐loop motif of TIR‐domain‐containing proteins to disrupt receptor:adaptor (e.g., TLR4:MAL and TLR4:TRAM) interactions of the toll‐like receptor signaling. The crystal structure of A46 (75–227) determined at 2.58 Å resolution showed that A46 formed a homodimer and adopted a Bcl‐2‐like fold similar to other VACV proteins such as A52, B14, and K7. Our structure also revealed that VIPER (viral inhibitory peptide of TLR4) motif resides in the α1‐helix and six residues of the VIPER region were exposed to surface for binding to target proteins. In vitro binding assays between wild type and six mutants A46 (75–227) and full‐length MAL identified critical residues in the VIPER motif. Computational modeling of the A46:MAL complex structure showed that the VIPER region of A46 and AB loop of MAL protein formed a major binding interface. In summary, A46 is a homodimer with a Bcl‐2‐like fold and VIPER motif is believed to be involved in the interaction with MAL protein based on our binding assays.     

Identification of hnRNPH1, NF45, and C14orf166 as novel host interacting partners of the mature hepatitis C virus core protein

The hepatitis C virus core protein (HCVc) forms the viral nucleocapsid and is involved in viral persistence and pathogenesis, possibly by interacting with host factors to modulate viral replication and cellular functions. Here, we identified 36 cellular protein candidates by one-dimensional SDS-PAGE and LC-MS/MS-based proteomics after affinity purification with HCVc174, a matured form of HCVc from HCV-1b genotype, tagged with biotin and calmodulin-binding peptide/protein A at N- and C-termini, respectively. By pull-down and confocal imaging techniques, we confirmed that heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1), nuclear factor 45 (NF45), and C14orf166 are novel HCVc174-interacting host proteins, known to participate in mRNA metabolism, gene regulation, and microtubule organization, respectively. Unlike the other 2 proteins, NF45 interacted with HCVc174 in an RNA-dependent manner. These 3 proteins colocalized with ectopic HCVc-1b in both the cytoplasm and nucleus, which demonstrated their spatial interaction with naturally translocated HCVc174 after HCVc biogenesis. Such colocalization, however, shifted to the cytoplasm in cells with replicating virus of 1b or 2a genotype, indicating that active viral replication confined these interacting proteins in the cytoplasm. Collectively, our findings suggest that spatial interactions of hnRNPH1, NF45, and C14orf166 with HCVc174 likely modulate HCV or cellular functions during acute and chronic HCV infection.     

Vaccinia Virus Virion Membrane Biogenesis Protein A11 Associates with Viral Membranes in a Manner That Requires the Expression of Another Membrane Biogenesis Protein,A6

A group of vaccinia virus (VACV) proteins, including A11, L2, and A6, are required for biogenesis of the primary envelope of VACV, specifically, for the acquisition of viral membrane precursors. However, the interconnection among these proteins is unknown and, with the exception of L2, the connection of these proteins with membranes is also unknown. In this study, prompted by the findings that A6 coprecipitated A11 and that the cellular distribution of A11 was dramatically altered by repression of A6 expression, we studied the localization of A11 in cells by using immunofluorescence and cell fractionation analysis. A11 was found to associate with membranes and colocalize with virion membrane proteins in viral replication factories during normal VACV replication. A11 partitioned almost equally between the detergent and aqueous phases upon Triton X-114 phase separation, demonstrating an intrinsic affinity with lipids. However, in the absence of infection or VACV late protein synthesis, A11 did not associate with cellular membranes. Furthermore, when A6 expression was repressed, A11 did not colocalize with any viral membrane proteins or associate with membranes. In contrast, when virion envelope formation was blocked at a later step by repression of A14 expression or by rifampin treatment, A11 colocalized with virion membrane proteins in the factories. Altogether, our data showed that A11 associates with viral membranes during VACV replication, and this association requires A6 expression. This study provides a physical connection between A11 and viral membranes and suggests that A6 regulates A11 membrane association.     

Comparison of the Cowpox Virus and Vaccinia Virus Mature Virion Proteome: Analysis of the Species- and Strain-Specific Proteome

Cowpox virus (CPXV) causes most zoonotic orthopoxvirus (OPV) infections in Europe and Northern as well as Central Asia. The virus has the broadest host range of OPV and is transmitted to humans from rodents and other wild or domestic animals. Increasing numbers of human CPXV infections in a population with declining immunity have raised concerns about the virus’ zoonotic potential. While there have been reports on the proteome of other human-pathogenic OPV, namely vaccinia virus (VACV) and monkeypox virus (MPXV), the protein composition of the CPXV mature virion (MV) is unknown. This study focused on the comparative analysis of the VACV and CPXV MV proteome by label-free single-run proteomics using nano liquid chromatography and high-resolution tandem mass spectrometry (nLC-MS/MS). The presented data reveal that the common VACV and CPXV MV proteome contains most of the known conserved and essential OPV proteins and is associated with cellular proteins known to be essential for viral replication. While the species-specific proteome could be linked mainly to less genetically-conserved gene products, the strain-specific protein abundance was found to be of high variance in proteins associated with entry, host-virus interaction and protein processing.     

Sheeppox Virus SPPV14 Encodes a Bcl-2-Like Cell Death Inhibitor That Counters a Distinct Set of Mammalian Proapoptotic Proteins

Many viruses express inhibitors of programmed cell death (apoptosis), thereby countering host defenses that would otherwise rapidly clear infected cells. To counter this, viruses such as adenoviruses and herpesviruses express recognizable homologs of the mammalian prosurvival protein Bcl-2. In contrast, the majority of poxviruses lack viral Bcl-2 (vBcl-2) homologs that are readily identified by sequence similarities. One such virus, myxoma virus, which is the causative agent of myxomatosis, expresses a virulence factor that is a potent inhibitor of apoptosis. In spite of the scant sequence similarity to Bcl-2, myxoma virus M11L adopts an almost identical 3-dimensional fold. We used M11L as bait in a sequence similarity search for other Bcl-2-like proteins and identified six putative vBcl-2 proteins from poxviruses. Some are potent inhibitors of apoptosis, in particular sheeppox virus SPPV14, which inhibited cell death induced by multiple agents. Importantly, SPPV14 compensated for the loss of antiapoptotic F1L in vaccinia virus and acts to directly counter the cell death mediators Bax and Bak. SPPV14 also engages a unique subset of the death-promoting BH3-only ligands, including Bim, Puma, Bmf, and Hrk. This suggests that SPPV14 may have been selected for specific biological roles as a virulence factor for sheeppox virus.     

Structure of M11L: A myxoma virus structural homolog of the apoptosis inhibitor, Bcl-2

Apoptosis of virally infected cells is an innate host mechanism used to prevent viral spread. However, viruses have evolved a number of proteins that function to modulate the apoptotic cascades and thereby favor productive viral replication. One such antiapoptotic protein, myxoma virus M11L, has been shown to inhibit mitochondrial-dependent apoptosis by binding to and blocking the two executioner proteins Bak and Bax. Since M11L has no obvious sequence homology with Bcl-2 or Bcl-x(L), the normal cellular inhibitors for Bak and Bax, and the structure of M11L has not been solved, the mode of binding to Bak and Bax is not known. In order to understand how M11L functions, the crystal structure of M11L was solved to 2.91 A. Despite the lack of sequence similarity, M11L is a structural homolog of Bcl-2. Studies using a peptide derived from Bak indicate that M11L binds to Bak with a similar affinity (4.9 +/- 0.3 microM) to the published binding affinities of Bcl-2 and Bcl-x(L) to the same peptide (12.7 microM and 0.5 microM, respectively), indicating that M11L inhibits apoptosis by mimicking and competing with host proteins for the binding of Bak and Bax. The structure provides important insight into how myxoma virus and other poxviruses facilitate viral dissemination by inhibiting mitochondrial dependent apoptosis.     

Viral Bcl-2 homologs and their role in virus replication and associated diseases

Cellular Bcl-2 family proteins regulate a critical step in the mammalian programmed cell death pathway by modulating mitochondrial permeability and function. Bcl-2 family proteins are also encoded by several large DNA viruses, including all known gamma herpesviruses, adenoviruses, and several other unrelated viruses. Viral Bcl-2 proteins can prevent cell death but often escape cellular regulatory mechanisms that govern their cellular counterparts. By evading the "altruistic" suicide of infected cells, viruses can ensure replication and propagation in the infected host, but sometimes in surprising ways. Many human cancers and other disorders are associated with viruses that encode Bcl-2 homologs. Here we consider the available mechanistic data for viral compared to cellular Bcl-2 protein function along with relevance to the virus life cycle and human disease states.     

The actin nucleator Spir-1 is a virus restriction factor that promotes innate immune signalling

Cellular proteins often have multiple and diverse functions. This is illustrated with protein Spir-1 that is an actin nucleator, but, as shown here, also functions to enhance innate immune signalling downstream of RNA sensing by RIG-I/MDA-5. In human and mouse cells lacking Spir-1, IRF3 and NF-κB-dependent gene activation is impaired, whereas Spir-1 overexpression enhanced IRF3 activation. Furthermore, the infectious virus titres and sizes of plaques formed by two viruses that are sensed by RIG-I, vaccinia virus (VACV) and Zika virus, are increased in Spir-1 KO cells. These observations demonstrate the biological importance of Spir-1 in the response to virus infection. Like cellular proteins, viral proteins also have multiple and diverse functions. Here, we also show that VACV virulence factor K7 binds directly to Spir-1 and that a diphenylalanine motif of Spir-1 is needed for this interaction and for Spir-1-mediated enhancement of IRF3 activation. Thus, Spir-1 is a new virus restriction factor and is targeted directly by an immunomodulatory viral protein that enhances virus virulence and diminishes the host antiviral responses.     

The orf virus inhibitor of apoptosis functions in a Bcl-2-like manner, binding and neutralizing a set of BH3-only proteins and active Bax

We have previously shown that the Orf virus protein, ORFV125, is a potent inhibitor of the mitochondrial pathway of apoptosis and displays rudimentary sequence similarities to cellular anti-apoptotic Bcl-2 proteins. Here we investigate the proposal that ORFV125 acts in a Bcl-2-like manner to inhibit apoptosis. We show that the viral protein interacted with a range of BH3-only proteins (Bik, Puma, DP5, Noxa and all 3 isoforms of Bim) and neutralized their pro-apoptotic activity. In addition, ORFV125 bound to the active, but not the inactive, form of Bax, and reduced the formation of Bax dimers. Mutation of specific amino acids in ORFV125 that are conserved and functionally important in mammalian Bcl-2 family proteins led to loss of both binding and inhibitory functions. We conclude that ORFV125’s mechanism of action is Bcl-2-like and propose that the viral protein’s combined ability to bind to a range of BH3-only proteins as well as the active form of Bax provides significant protection against apoptosis. Furthermore, we demonstrate that the binding profile of ORFV125 is distinct to that of other poxviral Bcl-2-like proteins.     

Role of sulfatide in vaccinia virus infection

Background information. Vaccinia virus (VACV) was used as a surrogate of variola virus (genus Orthopoxvirus), the causative agent of smallpox, to study orthopoxvirus infection. VACV infects cells via attachment and fusion of the viral membrane with the host cell membrane. Glycosphingolipids, expressed in multiple organs, are major components of lipid rafts and have been associated with the infectious route of several pathogens. Results. We demonstrate that the VACV‐WR (VACV Western‐Reserve strain) displays no binding to Cer (ceramide) or to Gal‐Cer (galactosylceramide), but binds to a natural sulfated derivative of these molecules: the Sulf (sulfatide) 3′ sulfogalactosylceramide. The interaction between Sulf and VACV‐WR resulted in a time‐dependent inhibition of virus infection. Virus cell attachment was the crucial step inhibited by Sulf. Electron microscopy showed that SUVs (small unilamellar vesicles) enriched in Sulf bound to VACV particles. Both the A27 and L5 viral membrane proteins were shown to interact with Sulf, indicating that they could be the major viral ligands for Sulf. Soluble Sulf was successful in preventing mortality, but not morbidity, in a lethal mouse model infection with VACV‐WR. Conclusions. Together the results suggest that Sulf could play a role as an alternate receptor for VACV‐WR and probably other Orthopoxviruses.     

Genome of deerpox virus

Deerpox virus (DPV), an uncharacterized and unclassified member of the Poxviridae, has been isolated from North American free-ranging mule deer (Odocoileus hemionus) exhibiting mucocutaneous disease. Here we report the genomic sequence and comparative analysis of two pathogenic DPV isolates, W-848-83 (W83) and W-1170-84 (W84). The W83 and W84 genomes are 166 and 170 kbp, containing 169 and 170 putative genes, respectively. Nucleotide identity between DPVs is 95% over the central 157 kbp. W83 and W84 share similar gene orders and code for similar replicative, structural, virulence, and host range functions. DPV open reading frames (ORFs) with putative virulence and host range functions include those similar to cytokine receptors (R), including gamma interferon receptor (IFN-gammaR), interleukin 1 receptor (IL-1R), and type 8 CC-chemokine receptors; cytokine binding proteins (BP), including IL-18BP, IFN-alpha/betaBP, and tumor necrosis factor binding protein (TNFBP); serpins; and homologues of vaccinia virus (VACV) E3L, K3L, and A52R proteins. DPVs also encode distinct forms of major histocompatibility complex class I, C-type lectin-like protein, and transforming growth factor beta1 (TGF-beta1), a protein not previously described in a mammalian chordopoxvirus. Notably, DPV encodes homologues of cellular endothelin 2 and IL-1R antagonist, novel poxviral genes also likely involved in the manipulation of host responses. W83 and W84 differ from each other by the presence or absence of five ORFs. Specifically, homologues of a CD30 TNFR family protein, swinepox virus SPV019, and VACV E11L core protein are absent in W83, and homologues of TGF-beta1 and lumpy skin disease virus LSDV023 are absent in W84. Phylogenetic analysis indicates that DPVs are genetically distinct from viruses of other characterized poxviral genera and that they likely comprise a new genus within the subfamily Chordopoxvirinae.