CrmA orthologs from diverse poxviruses potently inhibit caspases-1 and -8, yet cleavage site mutagenesis frequently produces caspase-1-specific variants.

Poxviruses encode many proteins that enable them to evade host anti-viral defense mechanisms. Spi-2 proteins, including Cowpox virus CrmA, suppress anti-viral immune responses and contribute to poxviral pathogenesis and lethality. These proteins are 'serpin' protease inhibitors, which function via a pseudosubstrate mechanism involving initial interactions between the protease and a cleavage site within the serpin. A conformational change within the serpin interrupts the cleavage reaction, deforming the protease active site and preventing dissociation. Spi-2 proteins like CrmA potently inhibit caspases-1, -4 and -5, which produce proinflammatory cytokines, and caspase-8, which facilitates cytotoxic lymphocyte-mediated target cell death. It is not clear whether both of these functions are equally perilous for the virus, or whether only one must be suppressed for poxviral infectivity and spread but the other is coincidently inhibited merely because these caspases are biochemically similar. We compared the caspase specificity of CrmA to three orthologs from orthopoxviruses and four from more distant chordopoxviruses. All potently blocked caspases-1, -4, -5 and -8 activity but exhibited negligible inhibition of caspases-2, -3 and -6. The orthologs differed markedly in their propensity to inhibit non-mammalian caspases. We determined the specificity of CrmA mutants bearing various residues in positions P4, P3 and P2 of the cleavage site. Almost all variants retained the ability to inhibit caspase-1, but many lacked caspase-8 inhibitory activity. The retention of Spi-2 proteins' caspase-8 specificity during chordopoxvirus evolution, despite this function being readily lost through cleavage site mutagenesis, suggests that caspase-8 inhibition is crucial for poxviral pathogenesis and spread.

Combined Proteomics/Genomics Approach Reveals Proteomic Changes of Mature Virions as a Novel Poxvirus Adaptation Mechanism.

DNA viruses, like poxviruses, possess a highly stable genome, suggesting that adaptation of virus particles to specific cell types is not restricted to genomic changes. Cowpox viruses are zoonotic poxviruses with an extraordinarily broad host range, demonstrating their adaptive potential in vivo. To elucidate adaptation mechanisms of poxviruses, we isolated cowpox virus particles from a rat and passaged them five times in a human and a rat cell line. Subsequently, we analyzed the proteome and genome of the non-passaged virions and each passage. While the overall viral genome sequence was stable during passaging, proteomics revealed multiple changes in the virion composition. Interestingly, an increased viral fitness in human cells was observed in the presence of increased immunomodulatory protein amounts. As the only minor variant with increasing frequency during passaging was located in a viral RNA polymerase subunit and, moreover, most minor variants were found in transcription-associated genes, protein amounts were presumably regulated at transcription level. This study is the first comparative proteome analysis of virus particles before and after cell culture propagation, revealing proteomic changes as a novel poxvirus adaptation mechanism.

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.

Characterization of a large, proteolytically processed cowpox virus membrane glycoprotein conserved in most chordopoxviruses.

Most poxvirus proteins are either highly conserved and essential for basic steps in replication or less conserved and involved in host interactions. Homologs of the CPXV219 protein, encoded by cowpox virus, are present in nearly all chordopoxvirus genera and some species have multiple copies. The CPXV219 homologs have estimated masses of greater than 200 kDa, making them the largest known poxvirus proteins. We showed that CPXV219 was expressed early in infection and cleaved into N- and C-terminal fragments that remained associated. The protein has a signal peptide and transited the secretory pathway where extensive glycosylation and proteolytic cleavage occurred. CPXV219 was located by immunofluorescence microscopy in association with the endoplasmic reticulum, Golgi apparatus and plasma membrane. In non-permeabilized cells, CPXV219 was accessible to external antibody and biotinylation. Mutants that did not express CPXV219 replicated normally in cell culture and retained virulence in a mouse respiratory infection model.

Comparative Biochemical and Functional Analysis of Viral and Human Secreted Tumor Necrosis Factor (TNF) Decoy Receptors.

The blockade of tumor necrosis factor (TNF) by etanercept, a soluble version of the human TNF receptor 2 (hTNFR2), is a well established strategy to inhibit adverse TNF-mediated inflammatory responses in the clinic. A similar strategy is employed by poxviruses, encoding four viral TNF decoy receptor homologues (vTNFRs) named cytokine response modifier B (CrmB), CrmC, CrmD, and CrmE. These vTNFRs are differentially expressed by poxviral species, suggesting distinct immunomodulatory properties. Whereas the human variola virus and mouse ectromelia virus encode one vTNFR, the broad host range cowpox virus encodes all vTNFRs. We report the first comprehensive study of the functional and binding properties of these four vTNFRs, providing an explanation for their expression profile among different poxviruses. In addition, the vTNFRs activities were compared with the hTNFR2 used in the clinic. Interestingly, CrmB from variola virus, the causative agent of smallpox, is the most potent TNFR of those tested here including hTNFR2. Furthermore, we demonstrate a new immunomodulatory activity of vTNFRs, showing that CrmB and CrmD also inhibit the activity of lymphotoxin ß. Similarly, we report for the first time that the hTNFR2 blocks the biological activity of lymphotoxin ß. The characterization of vTNFRs optimized during virus-host evolution to modulate the host immune response provides relevant information about their potential role in pathogenesis and may be used to improve anti-inflammatory therapies based on soluble decoy TNFRs.

Crystal structure of the cowpox virus-encoded NKG2D ligand OMCP.

The NKG2D receptor is expressed on the surface of NK, T, and macrophage lineage cells and plays an important role in antiviral and antitumor immunity. To evade NKG2D recognition, herpesviruses block the expression of NKG2D ligands on the surface of infected cells using a diverse repertoire of sabotage methods. Cowpox and monkeypox viruses have taken an alternate approach by encoding a soluble NKG2D ligand, the orthopoxvirus major histocompatibility complex (MHC) class I-like protein (OMCP), which can block NKG2D-mediated cytotoxicity. This approach has the advantage of targeting a single conserved receptor instead of numerous host ligands that exhibit significant sequence diversity. Here, we show that OMCP binds the NKG2D homodimer as a monomer and competitively blocks host ligand engagement. We have also determined the 2.25-Å-resolution crystal structure of OMCP from the cowpox virus Brighton Red strain, revealing a truncated MHC class I-like platform domain consisting of a beta sheet flanked with two antiparallel alpha helices. OMCP is generally similar in structure to known host NKG2D ligands but has notable variations in regions typically used to engage NKG2D. Additionally, the determinants responsible for the 14-fold-higher affinity of OMCP for human than for murine NKG2D were mapped to a single loop in the NKG2D ligand-binding pocket.

Structural mechanism of ER retrieval of MHC class I by cowpox.

One of the hallmarks of viral immune evasion is the capacity to disrupt major histocompatibility complex class I (MHCI) antigen presentation to evade T-cell detection. Cowpox virus encoded protein CPXV203 blocks MHCI surface expression by exploiting the KDEL-receptor recycling pathway, and here we show that CPXV203 directly binds a wide array of fully assembled MHCI proteins, both classical and non-classical. Further, the stability of CPXV203/MHCI complexes is highly pH dependent, with dramatically increased affinities at the lower pH of the Golgi relative to the endoplasmic reticulum (ER). Crystallographic studies reveal that CPXV203 adopts a beta-sandwich fold similar to poxvirus chemokine binding proteins, and binds the same highly conserved MHCI determinants located under the peptide-binding platform that tapasin, CD8, and natural killer (NK)-receptors engage. Mutagenesis of the CPXV203/MHCI interface identified the importance of two CPXV203 His residues that confer low pH stabilization of the complex and are critical to ER retrieval of MHCI. These studies clarify mechanistically how CPXV203 coordinates with other cowpox proteins to thwart antigen presentation.

Cowpox virus expresses a novel ankyrin repeat NF-kappaB inhibitor that controls inflammatory cell influx into virus-infected tissues and is critical for virus pathogenesis.

Many pathogenic orthopoxviruses like variola virus, monkeypox virus, and cowpox virus (CPXV), but not vaccinia virus, encode a unique family of ankyrin (ANK) repeat-containing proteins that interact directly with NF-kappaB1/p105 and inhibit the NF-kappaB signaling pathway. Here, we present the in vitro and in vivo characterization of the targeted gene knockout of this novel NF-kappaB inhibitor in CPXV. Our results demonstrate that the vCpx-006KO uniquely induces a variety of NF-kappaB-controlled proinflammatory cytokines from infected myeloid cells, accompanied by a rapid phosphorylation of the IkappaB kinase complex and subsequent degradation of the NF-kappaB cellular inhibitors IkappaBalpha and NF-kappaB1/p105. Moreover, the vCpx-006KO virus was attenuated for virulence in mice and induced a significantly elevated cellular inflammatory process at tissue sites of virus replication in the lung. These results indicate that members of this ANK repeat family are utilized specifically by pathogenic orthopoxviruses to repress the NF-kappaB signaling pathway at tissue sites of virus replication in situ.

The vaccinia virus soluble interferon-gamma receptor is a homodimer.

The vaccinia virus (VV) interferon (IFN)-gamma receptor (IFN-gammaR) is a 43 kDa soluble glycoprotein that is secreted from infected cells early during infection. Here we demonstrate that the IFN-gammaR from VV, cowpox virus and camelpox virus exists naturally as a homodimer, whereas the cellular IFN-gammaR dimerizes only upon binding the homodimeric IFN-gamma. The existence of the virus protein as a dimer in the absence of ligand may provide an advantage to the virus in efficient binding and inhibition of IFN-gamma in solution.

Crystal structure of the apoptotic suppressor CrmA in its cleaved form.

BACKGROUND: Cowpox virus expresses the serpin CrmA (cytokine response modifier A) in order to avoid inflammatory and apoptotic responses of infected host cells. The targets of CrmA are members of the caspase family of proteases that either initiate the extrinsic pathway of apoptosis (caspases 8 and 10) or trigger activation of the pro-inflammatory cytokines interleukin-1beta and interleukin-18 (caspase 1). RESULTS: We have determined the structure of a cleaved form of CrmA to 2.26 A resolution. CrmA has the typical fold of a cleaved serpin, even though it lacks the N-terminal half of the A helix, the entire D helix, and a portion of the E helix that are present in all other known serpins. The reactive-site loop of CrmA was mutated to contain the optimal substrate recognition sequence for caspase 3; however, the mutation only marginally increased the ability of CrmA to inhibit caspase 3. Superposition of the reactive-site loop of alpha1-proteinase inhibitor on the cleaved CrmA structure provides a model for virgin CrmA that can be docked to caspase 1, but not to caspase 3. CONCLUSIONS: CrmA exemplifies viral economy, selective pressure having resulted in a 'minimal' serpin that lacks the regions not needed for structural integrity or inhibitory activity. The docking model provides an explanation for the selectivity of CrmA. Our demonstration that engineering optimal substrate recognition sequences into the CrmA reactive-site loop fails to generate a good caspase 3 inhibitor is consistent with the docking model.

Structure of a soluble secreted chemokine inhibitor vCCI (p35) from cowpox virus.

Most poxviruses, including variola, the causative agent of smallpox, express a secreted protein of 35 kDa, vCCI, which binds CC-chemokines with high affinity. This viral protein competes with the host cellular CC-chemokine receptors (CCRs), reducing inflammation and interfering with the host immune response. Such proteins or derivatives may have therapeutic uses as anti-inflammatory agents. We have determined the crystal structure to 1.85-A resolution of vCCI from cowpox virus, the prototype of this poxvirus virulence factor. The molecule is a beta-sandwich of topology not previously described. A patch of conserved residues on the exposed face of a beta-sheet that is strongly negatively charged might have a role in binding of CC-chemokines, which are positively charged.