A structural viral mimic of prosurvival Bcl-2: a pivotal role for sequestering proapoptotic Bax and Bak

Many viruses express antiapoptotic proteins to counter host defense mechanisms that would otherwise trigger the rapid clearance of infected cells. For example, adenoviruses and some gamma-herpesviruses express homologs of prosurvival Bcl-2 to subvert the host's apoptotic machinery. Myxoma virus, a double-stranded DNA virus of the pox family, harbors antiapoptotic M11L, its virulence factor. Analysis of its three-dimensional structure reveals that despite lacking any primary sequence similarity to Bcl-2, it adopts a virtually identical protein fold. This allows it to associate with BH3 domains, especially those of Bax and Bak. We found that M11L acts primarily by sequestering Bax and Bak, thereby blocking the killing action of these essential cell-death mediators. These findings expand the family of protein sequences that act like Bcl-2 to block apoptosis and support the conclusion that the prosurvival action of these proteins critically depends on their ability to bind and antagonize Bax and/or Bak.     

Epstein-Barr virus provides a new paradigm: a requirement for the immediate inhibition of apoptosis

DNA viruses such as herpesviruses are known to encode homologs of cellular antiapoptotic viral Bcl-2 proteins (vBcl-2s), which protect the virus from apoptosis in its host cell during virus synthesis. Epstein-Barr virus (EBV), a human tumor virus and a prominent member of γ-herpesviruses, infects primary resting B lymphocytes to establish a latent infection and yield proliferating, growth-transformed B cells in vitro. In these cells, 11 viral genes that contribute to cellular transformation are consistently expressed. EBV also encodes two vBcl-2 genes whose roles are unclear. Here we show that the genetic inactivation of both vBcl-2 genes disabled EBV's ability to transform primary resting B lymphocytes. Primary B cells infected with a vBcl-2-negative virus did not enter the cell cycle and died of immediate apoptosis. Apoptosis was abrogated in infected cells in which vBcl-2 genes were maximally expressed within the first 24 h postinfection. During latent infection, however, the expression of vBcl-2 genes became undetectable. Thus, both vBcl-2 homologs are essential for initial cellular transformation but become dispensable once a latent infection is established. Because long-lived, latently infected memory B cells and EBV-associated B-cell lymphomas are derived from EBV-infected proapoptotic germinal center B cells, we conclude that vBcl-2 genes are essential for the initial evasion of apoptosis in cells in vivo in which the virus establishes a latent infection or causes cellular transformation or both.     

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.     

The vaccinia virus-encoded Bcl-2 homologues do not act as direct Bax inhibitors

Many viruses, including members of several poxvirus genera, encode inhibitors that block apoptosis by simultaneously binding the proapoptotic Bcl-2 proteins Bak and Bax. The Orthopoxvirus vaccinia virus encodes the Bcl-2-like F1 protein, which sequesters Bak but not Bax. However, N1, a potent virulence factor, is reported to be antiapoptotic and to interact with Bax. Here we investigated whether vaccinia virus inhibits Bak/Bax-dependent apoptosis via the cooperative action of F1 and N1. We found that Western Reserve (WR) and ΔN1L viruses inhibited drug- and infection-induced apoptosis equally. Meanwhile, infections with ΔF1L or ΔN1L/F1L virus resulted in similar levels of Bax activation and apoptosis. Outside the context of infection, N1 did not block drug- or Bax-induced cell death or interact with Bax. In addition to F1 and N1, vaccinia virus encodes further structural homologs of Bcl-2 proteins that are conserved in orthopoxviruses, including A46, A52, B14, C1, C6, C16/B22, K7, and N2. However, we found that these do not associate with Bax or inhibit drug-induced cell death. Based on our findings that N1 is not an antiapoptotic protein, we propose that the F1 orthologs represent the only orthopoxvirus Bcl-2 homolog to directly inhibit the Bak/Bax checkpoint.     

A new example of viral intein in Mimivirus

Background

Poxviruses encode a range of immunomodulatory genes to subvert or evade the challenges posed by the innate and adaptive immune responses. However, the inactivated poxviruses possessed immunostimulating capacity and were used as a prophylactic or metaphylactic application that efficiently reduced susceptibility to infectious diseases in different species. This fact is intensively studied in different genera of poxviruses. However, little is known about the basic mechanisms adopted by sheeppox virus (SPPV). SPPV causes an acute disease of sheep that recently, has been observed to reinfect its host in spite of vaccination.

Results

By injecting inactivated or attenuated sheeppox virus SPPV vaccine in adult male Swiss mice, SPPV was found to reduce macrophages' functions in a local event that occurs at the site of application 12 h after vaccine administration as indicated by increased level of IL-10 and decreased level of SOD from cultured peritoneal macrophages. In contrast increased levels of IL-12, and SOD activity from cultured splenic macrophages, lymphocyte response to PHA-P, and in-vivo response to T-dependant Ag were detected. These effects were observed in both attenuated and inactivated SPPV, but more prominent in attenuated one.

Conclusion

The results of this study help to elucidate, the phenomenon of existence natural SPPV infections in sheep instead of vaccination and the basic mechanisms responsible for the immunostimulating capacity of sheeppox virus. Locally, SPPV shows evidence for an immune escape mechanism that alleviates the host's immune response. Later and systemically, the virus protects the host from any fatal consequences of the immune system suppression.     

Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands

Apoptosis is an important part of the host's defense mechanism for eliminating invading pathogens. Some viruses express proteins homologous in sequence and function to mammalian pro-survival Bcl-2 proteins. Anti-apoptotic F1L expressed by vaccinia virus is essential for survival of infected cells, but it bears no discernable sequence homology to proteins other than its immediate orthologues in related pox viruses. Here we report that the crystal structure of F1L reveals a Bcl-2-like fold with an unusual N-terminal extension. The protein forms a novel domain-swapped dimer in which the alpha1 helix is the exchanged domain. Binding studies reveal an atypical BH3-binding profile, with sub-micromolar affinity only for the BH3 peptide of pro-apoptotic Bim and low micromolar affinity for the BH3 peptides of Bak and Bax. This binding interaction is sensitive to F1L mutations within the predicted canonical BH3-binding groove, suggesting parallels between how vaccinia virus F1L and myxoma virus M11L bind BH3 domains. Structural comparison of F1L with other Bcl-2 family members reveals a novel sequence signature that redefines the BH4 domain as a structural motif present in both pro- and anti-apoptotic Bcl-2 members, including viral Bcl-2-like proteins.     

Differentiation of sheeppox and goatpox viruses by polymerase Chain reaction-restriction fragment length polymorphism

In the present study, the partial gene sequences of P32 protein, an immunogenic envelope protein of Capripoxviruses (CaPV), were analyzed to assess the genetic relationship among sheeppox and goatpox virus isolates, and restriction enzyme specific PCR-RFLP was developed to differentiate CaPV strains. A total of six goatpox virus (GTPV) and nine sheeppox virus (SPPV) isolates of Indian origin were included in the sequence analysis of the attachment gene. The sequence analysis revealed a high degree of sequence identity among all the Indian SPPV and GTPV isolates at both nucleotide and amino acid levels. Phylogenetic analysis showed three distinct clusters of SPPV, GTPV and Lumpy skin disease virus (LSDV) isolates. Further, multiple sequence alignment revealed a unique change at G120A in all GTPV isolates resulting in the formation of Dra I restriction site in lieu of EcoR I, which is present in SPPV isolates studied. This change was unique and exploited to develop restriction enzyme specific PCR-RFLP for detection and differentiation of SPPV and GTPV strains. The optimized PCR-RFLP was validated using a total of fourteen (n=14) cell culture isolates and twenty two (n=22) known clinical samples of CaPV. The Restriction Enzyme specific PCR-RFLP to differentiate both species will allow a rapid differential diagnosis during CaPV outbreaks particularly in mixed flocks of sheep and goats and could be an adjunct/supportive tool for complete gene or virus genome sequencing methods.     

Antiapoptotic herpesvirus Bcl-2 homologs escape caspase-mediated conversion to proapoptotic proteins

The antiapoptotic Bcl-2 and Bcl-x(L) proteins of mammals are converted into potent proapoptotic factors when they are cleaved by caspases, a family of apoptosis-inducing proteases (E. H.-Y. Cheng, D. G. Kirsch, R. J. Clem, R. Ravi, M. B. Kastan, A. Bedi, K. Ueno, and J. M. Hardwick, Science 278:1966-1968, 1997; R. J. Clem, E. H.-Y. Cheng, C. L. Karp, D. G. Kirsch, K. Ueno, A. Takahashi, M. B. Kastan, D. E. Griffin, W. C. Earnshaw, M. A. Veliuona, and J. M. Hardwick, Proc. Natl. Acad. Sci. USA 95:554-559, 1998). Gamma herpesviruses also encode homologs of the Bcl-2 family. All tested herpesvirus Bcl-2 homologs possess antiapoptotic activity, including the more distantly related homologs encoded by murine gammaherpesvirus 68 (gammaHV68) and bovine herpesvirus 4 (BHV4), as described here. To determine if viral Bcl-2 proteins can be converted into death factors, similar to their cellular counterparts, five herpesvirus Bcl-2 homologs from five different viruses were tested for their susceptibility to caspases. Only the viral Bcl-2 protein encoded by gammaHV68 was susceptible to caspase digestion. However, unlike the caspase cleavage products of cellular Bcl-2, Bcl-x(L), and Bid, which are potent inducers of apoptosis, the cleavage product of gammaHV68 Bcl-2 lacked proapoptotic activity. KSBcl-2, encoded by the Kaposi's sarcoma-associated herpesvirus, was the only viral Bcl-2 homolog that was capable of killing cells when expressed as an N-terminal truncation. However, because KSBcl-2 was not cleavable by caspases, the latent proapoptotic activity of KSBcl-2 apparently cannot be released. The Bcl-2 homologs encoded by herpesvirus saimiri, Epstein-Barr virus, and BHV4 were not cleaved by apoptotic cell extracts and did not possess latent proapoptotic activities. Thus, herpesvirus Bcl-2 homologs escape negative regulation by retaining their antiapoptotic activities and/or failing to be converted into proapoptotic proteins by caspases during programmed cell death.     

The genomes of sheeppox and goatpox viruses

Sheeppox virus (SPPV) and goatpox virus (GTPV), members of the Capripoxvirus genus of the Poxviridae, are etiologic agents of important diseases of sheep and goats in northern and central Africa, southwest and central Asia, and the Indian subcontinent. Here we report the genomic sequence and comparative analysis of five SPPV and GTPV isolates, including three pathogenic field isolates and two attenuated vaccine viruses. SPPV and GTPV genomes are approximately 150 kbp and are strikingly similar to each other, exhibiting 96% nucleotide identity over their entire length. Wild-type genomes share at least 147 putative genes, including conserved poxvirus replicative and structural genes and genes likely involved in virulence and host range. SPPV and GTPV genomes are very similar to that of lumpy skin disease virus (LSDV), sharing 97% nucleotide identity. All SPPV and GTPV genes are present in LSDV. Notably in both SPPV and GTPV genomes, nine LSDV genes with likely virulence and host range functions are disrupted, including a gene unique to LSDV (LSDV132) and genes similar to those coding for interleukin-1 receptor, myxoma virus M003.2 and M004.1 genes (two copies each), and vaccinia virus F11L, N2L, and K7L genes. The absence of these genes in SPPV and GTPV suggests a significant role for them in the bovine host range. SPPV and GTPV genomes contain specific nucleotide differences, suggesting they are phylogenetically distinct. Relatively few genomic changes in SPPV and GTPV vaccine viruses account for viral attenuation, because they contain 71 and 7 genomic changes compared to their respective field strains. Notable genetic changes include mutation or disruption of genes with predicted functions involving virulence and host range, including two ankyrin repeat proteins in SPPV and three kelch-like proteins in GTPV. These comparative genomic data indicate the close genetic relationship among capripoxviruses, and they suggest that SPPV and GTPV are distinct and likely derived from an LSDV-like ancestor.     

Fowlpox virus encodes a Bcl-2 homologue that protects cells from apoptotic death through interaction with the proapoptotic protein Bak

Poxviruses are renowned for encoding numerous immunomodulatory proteins capable of undermining potent immune defenses. One effective barrier against infection is apoptosis, a process controlled at the mitochondria by pro- and antiapoptotic members of the highly conserved Bcl-2 family of proteins. Although poxviruses are known to encode an array of effective inhibitors of apoptosis, members of the Avipoxvirus genus, which includes fowlpox virus, encode proteins with Bcl-2 homology. Here, we show that FPV039, a fowlpox virus protein with limited Bcl-2 homology, inhibited apoptosis in response to a variety of cytotoxic stimuli, including virus infection itself. Similar to other antiapoptotic Bcl-2 proteins, FPV039 localized predominantly to the mitochondria in both human and chicken cells and protected human cells from tumor necrosis factor alpha-induced loss of the mitochondrial membrane potential. In addition, coimmunoprecipitation revealed that FPV039 interacted constitutively with the proapoptotic Bcl-2 protein, Bak, in both human and chicken cells. Concordantly, FPV039 also inhibited apoptosis induced by the transient overexpression of Bak. To confirm these results in the context of virus infection, we generated a recombinant vaccinia virus lacking F1L, the endogenous apoptotic inhibitor in vaccinia virus, and expressing FPV039. In the context of vaccinia virus infection, FPV039 retained the ability to localize to the mitochondria and interacted with Bak. Moreover, FPV039 prevented the activation of Bak and protected infected cells from apoptosis induced by staurosporine and virus infection. Together, our data indicate that FPV039 is a functional Bcl-2 homologue that inhibits apoptosis by neutralizing the proapoptotic Bcl-2 family member Bak.     

Sheeppox virus kelch-like gene SPPV-019 affects virus virulence

Sheeppox virus (SPPV), a member of the Capripoxvirus genus of the Poxviridae, is the etiologic agent of a significant disease of sheep in the developing world. Genomic analysis of pathogenic and vaccine capripoxviruses identified genes with potential roles in virulence and host range, including three genes with similarity to kelch-like genes of other poxviruses and eukaryotes. Here, a mutant SPPV with a deletion in the SPPV-019 kelch-like gene, DeltaKLP, was derived from the pathogenic strain SPPV-SA. DeltaKLP exhibited in vitro growth characteristics similar to those of SPPV-SA and revertant virus (RvKLP). DeltaKLP-infected cells exhibited a reduction in Ca(2+)-independent cell adhesion, suggesting that SPPV-019 may modulate cellular adhesion. When inoculated in sheep by the intranasal or intradermal routes, DeltaKLP was markedly attenuated, since all DeltaKLP-infected lambs survived infection. In contrast, SPPV-SA and RvKLP induced mortality approaching 100%. Lambs inoculated with DeltaKLP exhibited marked reduction or delay in fever response, gross lesions, viremia, and virus shedding compared to parental and revertant viruses. Together, these findings indicate that SPPV-019 is a significant SPPV virulence determinant in sheep.     

Adventures with poxviruses of vertebrates

Because they were the largest of all viruses and could be visualised with a light microscope, the poxviruses were the first viruses to be intensively studied in the laboratory. It was clear from an early date that they caused important diseases of humans and their domestic animals, such as smallpox, cowpox, camelpox, sheeppox, fowlpox and goatpox. This essay recounts some of the early history of their recognition and classification and then expands on aspects of research on poxviruses in which the author has been involved. Studies on the best-known genus, Orthopoxvirus, relate to the use of infectious ectromelia of mice as a model for smallpox, embracing both experimental epidemiology and pathogenesis, studies on the genetics of vaccinia virus and the problem of non-genetic reactivation (previously termed 'transformation') and the campaign for the global eradication of smallpox. The other group of poxviruses described here, the genus Leporipoxvirus, came to prominence when the myxoma virus was used for the biological control of Australian wild rabbits. This provided a unique natural experiment on the coevolution of a virus and its host. Future research will include further studies of the many immunomodulatory genes found in all poxviruses of vertebrates, since these provide clues about the workings of the immune system and how viruses have evolved to evade it. Some of the many recombinant poxvirus constructs currently being studied may come into use as vaccines or for immunocontraception. A field that warrants study but will probably remain neglected is the natural history of skunkpox, raccoonpox, taterapox, yabapox, tanapox and other little-known poxviruses. A dismal prospect is the possible use of smallpox virus for bioterrorism.     

Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-kappaB 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-kappaB, thereby blocking the production of pro-inflammatory cytokines. We have solved the crystal structures of A52 and B14 at 1.9 A and 2.7 A 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.     

C7L family of poxvirus host range genes inhibits antiviral activities induced by type I interferons and interferon regulatory factor 1

Vaccinia virus (VACV) K1L and C7L function equivalently in many mammalian cells to support VACV replication and antagonize antiviral activities induced by type I interferons (IFNs). While K1L is limited to orthopoxviruses, genes that are homologous to C7L are found in diverse mammalian poxviruses. In this study, we showed that the C7L homologues from sheeppox virus and swinepox virus could rescue the replication defect of a VACV mutant deleted of both K1L and C7L (vK1L(-)C7L(-)). Interestingly, the sheeppox virus C7L homologue could rescue the replication of vK1L(-)C7L(-) in human HeLa cells but not in murine 3T3 and LA-4 cells, in contrast to all other C7L homologues. Replacing amino acids 134 and 135 of the sheeppox virus C7L homologue, however, made it functional in the two murine cell lines, suggesting that these two residues are critical for antagonizing a putative host restriction factor which has some subtle sequence variation in human and murine cells. Furthermore, the C7L family of host range genes from diverse mammalian poxviruses were all capable of antagonizing type I IFN-induced antiviral activities against VACV. Screening of a library of more than 350 IFN-stimulated genes (ISGs) identified interferon-regulated factor 1 (IRF1) as an inhibitor of vK1L(-)C7L(-) but not wild-type VACV. Expression of either K1L or C7L, however, rendered vK1L(-)C7L(-) resistant to IRF1-induced antiviral activities. Altogether, our data show that K1L and C7L antagonize IRF1-induced antiviral activities and that the host modulation function of C7L is evolutionally conserved in all poxviruses that can readily replicate in tissue-cultured mammalian cells.     

Comparative analysis of poxvirus orthologues of the vaccinia virus E3 protein: modulation of protein kinase R activity, cytokine responses, and virus pathogenicity

Poxviruses are important human and animal pathogens that have evolved elaborate strategies for antagonizing host innate and adaptive immunity. The E3 protein of vaccinia virus, the prototypic member of the orthopoxviruses, functions as an inhibitor of innate immune signaling and is essential for vaccinia virus replication in vivo and in many human cell culture systems. However, the function of orthologues of E3 expressed by poxviruses of other genera with different host specificity remains largely unknown. In the present study, we characterized the E3 orthologues from sheeppox virus, yaba monkey tumor virus, swinepox virus, and myxoma virus for their ability to modulate protein kinase R (PKR) function, cytokine responses and virus pathogenicity. We found that the E3 orthologues of myxoma virus and swinepox virus could suppress PKR activation and interferon (IFN)-induced antiviral activities and restore the host range function of E3 in HeLa cells. In contrast, the E3 orthologues from sheeppox virus and yaba monkey tumor virus were unable to inhibit PKR activation. While the sheeppox orthologue was unable to restore the host range function of E3, the yaba monkey tumor virus orthologue partially restored E3-deficient vaccinia virus replication in HeLa cells, correlated with its ability to suppress IFN-induced antiviral activities. Moreover, poxvirus E3 orthologues show varying ability to inhibit the induction of antiviral and proinflammatory cytokines. Despite these in vitro results, none of the E3 orthologues tested was capable of restoring pathogenicity to E3-deficient vaccinia virus in vivo.     

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.     

How Poxviruses Oppose Apoptosis

Poxviruses express a variety of proteins that are able to modulate the innate cellular apoptotic response triggered by virus infection. Poxviruses are the only DNA viruses to replicate exclusively in the cytoplasm of infected cells, and to date, members of this family have been shown to encode a wide variety of proteins that block or delay apoptosis, including caspase inhibitors, other serpins, death domain effectors, bcl-2/CED-9 homologs, modulators of the FAS/TNF pathway, and inhibitors of PKR. It is predicted that this list of poxvirus apoptosis modulators will continue to grow in the coming years and should provide an increasingly rich and diverse family of apoptosis regulators.     

Tumorigenic poxviruses up-regulate intracellular superoxide to inhibit apoptosis and promote cell proliferation

Tumorigenic leporipoxviruses encode catalytically inactive homologs of cellular Cu-Zn superoxide dismutase (SOD1). The function of the orthologous myxoma virus M131R and Shope fibroma virus S131R gene products is uncertain, but they inhibit SOD1 activity by a process linked to binding its copper chaperone. Using a superoxide-sensitive dye (hydroethidine), we observed that virus infection increased intracellular superoxide levels in an M/S131R-dependent manner. To see whether this effect promotes infection, we deleted the Shope fibroma virus S131R gene and compared the clinical manifestations of wild-type and mutant virus infections in rabbits. S131RDelta virus produced significantly smaller fibroxanthosarcoma-like growths in vivo and, at a point where these growths were already receding, wild-type infections still showed extensive leukocyte infiltration, necrosis, and fibromatous cell proliferation. Coincidentally, whereas Jurkat cells are protected from mitochondria- and Fas-mediated apoptosis by wild-type myxoma virus in vitro, M131RDelta virus could not block Fas-initiated apoptosis as judged by DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end labeling, and caspase 3 cleavage assays. These data suggest that tumorigenic poxviruses can modulate intracellular redox status to their advantage to stimulate infected cell growth and inhibit programmed cell death.     

Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus counterpart inhibits apoptosis independent of Bim

Subversion of host cell apoptosis is an important survival strategy for viruses to ensure their own proliferation and survival. Certain viruses express proteins homologous in sequence, structure and function to mammalian pro-survival B-cell lymphoma 2 (Bcl-2) proteins, which prevent rapid clearance of infected host cells. In vaccinia virus (VV), the virulence factor F1L was shown to be a potent inhibitor of apoptosis that functions primarily be engaging pro-apoptotic Bim. Variola virus (VAR), the causative agent of smallpox, harbors a homolog of F1L of unknown function. We show that VAR F1L is a potent inhibitor of apoptosis, and unlike all other characterized anti-apoptotic Bcl-2 family members lacks affinity for the Bim Bcl-2 homology 3 (BH3) domain. Instead, VAR F1L engages Bid BH3 as well as Bak and Bax BH3 domains. Unlike its VV homolog, variola F1L only protects against Bax-mediated apoptosis in cellular assays. Crystal structures of variola F1L bound to Bid and Bak BH3 domains reveal that variola F1L forms a domain-swapped Bcl-2 fold, which accommodates Bid and Bak BH3 in the canonical Bcl-2-binding groove, in a manner similar to VV F1L. Despite the observed conservation of structure and sequence, variola F1L inhibits apoptosis using a startlingly different mechanism compared with its VV counterpart. Our results suggest that unlike during VV infection, Bim neutralization may not be required during VAR infection. As molecular determinants for the human-specific tropism of VAR remain essentially unknown, identification of a different mechanism of action and utilization of host factors used by a VAR virulence factor compared with its VV homolog suggest that studying VAR directly may be essential to understand its unique tropism.Variola virus (VAR), the causative agent of smallpox, is a member of the poxvirus family and belongs to the orthopoxviridae. Despite its successful eradication nearly 30 years ago, VAR remains an ongoing concern because of its potential use as a bioterrorism agent.¹ The threat of intentional use of VAR coupled with the absence of an FDA-approved drug for the prevention or treatment of smallpox infection is cause for considerable interest in the development of small-molecule therapeutics against VAR. Current strategies for dealing with smallpox are based on vaccination using live vaccinia virus (VV),^{2, 3} a closely related member of the orthopoxvirus genus, which shares >90% sequence identity with VAR. Vaccination using live VV, however, can cause serious complications,⁴ underscoring the need for effective anti-viral treatments, particularly since anti-viral treatment may be a more efficacious strategy compared with vaccination.⁵ Recent strategies to target VAR for small-molecule therapeutics included the use of polymerase inhibitors,⁶ notably Cidofovir, inhibitors of extracellular virus formation⁷ and tyrosine kinase inhibitors including Gleevec.^{8, 9} Cidofovir is currently the only approved antiviral drug for the treatment of orthopoxviruses, although it is not approved for smallpox treatment. Other host–virus interactions have been identified that may be suitable drug targets^{10, 11} but currently require further investigation.Several poxvirus members other than VAR have been shown to rely on virulence factors that prevent premature host cell demise via programmed cell death or apoptosis,^{12, 13, 14, 15, 16} thus ensuring survival and proliferation. The B-cell lymphoma 2 (Bcl-2) protein family is a key mediator for maintaining cell survival or to drive apoptosis, thereby removing infected, damaged or unwanted cells,¹⁷ and sequence, structural and functional orthologs of Bcl-2 have been found in a number of poxviruses.¹⁸ Certain viral Bcl-2-like proteins were only identified as family members after their 3D structures were determined, owing to their complete lack of sequence identity to mammalian Bcl-2 proteins. This group of proteins include the myxoma virus M11L¹² and VV F1L¹⁵ and N1L.¹⁹ Myxoma virus M11L was shown to adopt the classical Bcl-2 fold^{20, 21} that utilizes the canonical Bcl-2 homology 3 (BH3)-binding groove to engage BH3 ligands to exert its pro-survival effect. VV F1L also adopts a Bcl-2 fold, but unlike M11L it exists as a domain-swapped dimer,^{22, 23} whereas N1L also adopted a dimeric Bcl-2 fold but with a different dimeric arrangement.^{24, 25}Although F1L from VAR has not previously been investigated, the VV homolog is well characterized. VV F1L has been shown to inhibit the mitochondrial pathway of apoptosis by replacing Mcl-1²⁶ and interacts with the isolated BH3 domains of Bim, Bax and Bak,²³ which are bound in the canonical Bcl-2-binding groove.²² Furthermore, an F1L-deficient VV potently causes Bak/Bax-mediated apoptosis.^{15, 27} Functionally, VV F1L appears to rely primarily on neutralization of Bim in the context of a viral infection.²² Given the close similarity between VAR and VV, VAR may also rely on inhibition of host cell apoptosis for successful infection and proliferation. Disruption of VAR ability to inhibit apoptosis thus may constitute an attractive strategy for small-molecule-based intervention. To investigate this possibility, we performed a biochemical, structural and functional characterization of VAR F1L. Here we report that despite possessing a nearly identical 3D structure and sequence, VAR F1L inhibits apoptosis via a different mechanism compared with its homolog in VV.     

Downregulation of autophagy by herpesvirus Bcl-2 homologs

The critical role of the cellular autophagy pathway in viral infection and pathogenesis has become increasingly apparent. Mounting evidences suggest that viruses have developed different strategies to meticulously modulate intracellular autophagy for their own benefits, thereby either promoting efficient viral replication or facilitating viral persistence. While our understanding of these strategies is still in its incipient stage, recent advances demonstrate that gamma herpesvirus Bcl-2 homolog (vBcl-2), which protects virus-infected cells from apoptosis, also suppresses cellular autophagy pathway through its direct interaction with the autophagy protein Beclin1. Interestingly, vBcl-2 has evolved to harbor the enhanced anti-autophagic activity compared to its host counterpart, suggesting an important role of cellular autophagy in response to viral infection and virus-associated pathogenesis. Thus, a detailed study of vBcl-2-mediated regulation of autophagy signal transduction pathway may lead to a better understanding of not only how virus escapes from host innate immunity but also how autophagy regulates viral infection and environmental stresses.