Vincent P. Smith

Ectromelia, vaccinia and cowpox viruses encode secreted interleukin-18-binding proteins.

Interleukin-18 (IL-18) is a proinflammatory cytokine that plays a key role in the activation of natural killer and T helper 1 cell responses principally by inducing interferon-gamma (IFN-gamma). Human and mouse secreted IL-18-binding proteins (IL-18BPs) have recently been described which block IL-18 activity but have no sequence similarity to membrane IL-18 receptors. Several poxvirus genes encode proteins with sequence similarity to IL-18BPs. Here we show that vaccinia, ectromelia and cowpox viruses secrete from infected cells a soluble IL-18BP (vIL-18BP) that may modulate the host antiviral response. The ectromelia virus protein was found to block NF-kappaB activation and induction of IFN-gamma in response to IL-18. The highly attenuated vaccinia virus modified virus Ankara encodes IL-18-binding activity, and thus deletion of the vIL-18BP may improve further the safety and immunogenicity of this promising human vaccine candidate. We confirm that molluscum contagiosum virus, a molluscipoxvirus that produces small skin tumours in immunocompetent individuals and opportunistic infections in immunodeficient AIDS patients, also encodes a related, larger vIL-18BP (gene MC54L). This protein may contribute to the lack of inflammatory response characteristic of molluscum contagiosum virus lesions. The expression of vIL-18BPs by distinct poxvirus genera that cause local or general viral dissemination, or persistent or acute infections in the host, emphasizes the importance of IL-18 in response to viral infections.

Expression of Secreted Cytokine and Chemokine Inhibitors by Ectromelia Virus

ABSTRACT The production of secreted proteins that bind cytokines and block their activity has been well characterized as an immune evasion strategy of the orthopoxviruses vaccinia virus (VV) and cowpox virus (CPV). However, very limited information is available on the expression of similar cytokine inhibitors by ectromelia virus (EV), a virulent natural mouse pathogen that causes mousepox. We have characterized the expression and binding properties of three major secreted immunomodulatory activities in 12 EV strains and isolates. Eleven of the 12 EVs expressed a soluble, secreted 35-kDa viral chemokine binding protein with properties similar to those of homologous proteins from VV and CPV. All of the EVs expressed soluble, secreted receptors that bound to mouse, human, and rat tumor necrosis factor alpha. We also detected the expression of a soluble, secreted interleukin-1β (IL-1β) receptor (vIL-1βR) by all of the EVs. EV differed from VV and CPV in that binding of human 125I-IL-1β to the EV vIL-1βR could not be detected. Nevertheless, the EV vIL-1βR prevented the interaction of human and mouse IL-1β with cellular receptors. There are significant differences in amino acid sequence between the EV vIL-1βR and its VV and CPV homologs which may account for the results of the binding studies. The conservation of these activities in EV suggests evolutionary pressure to maintain them in a natural poxvirus infection. Mousepox represents a useful model for the study of poxvirus pathogenesis and immune evasion. These findings will facilitate future study of the role of EV immunomodulatory factors in the pathogenesis of mousepox.

Inhibition of Interferons by Ectromelia Virus

ABSTRACT Ectromelia virus (EV) is an orthopoxvirus (OPV) that causes mousepox, a severe disease of laboratory mice. Mousepox is a useful model of OPV infection because EV is likely to be a natural mouse pathogen, unlike its close relatives vaccinia virus (VV) and variola virus. Several studies have highlighted the importance of mouse interferons (IFNs) in resistance to and recovery from EV infection, but little is known of the anti-IFN strategies encoded by the virus itself. We have determined that 12 distinct strains and isolates of EV encode soluble, secreted receptors for IFN-γ (vIFN-γR) and IFN-α/β (vIFN-α/βR) that are homologous to those identified in other OPVs. We demonstrate for the first time that the EV vIFN-γR has the unique ability to inhibit the biological activity of mouse IFN-γ. The EV vIFN-α/βR was a potent inhibitor of human and mouse IFN-α and human IFN-β but, surprisingly, was unable to inhibit mouse IFN-β. The replication of all of the EVs included in our study and of cowpox virus was more resistant than VV to the antiviral effects induced in mouse L-929 cells by IFN-α/β and IFN-γ. Sequencing studies showed that this EV resistance is likely to be partly mediated by the double-stranded-RNA-binding protein encoded by an intact EV homolog of the VV E3L gene. The absence of a functional K3L gene, which encodes a viral eIF-2α homolog, in EV suggests that the virus encodes a novel mechanism to counteract the IFN response. These findings will facilitate future studies of the role of viral anti-IFN strategies in mousepox pathogenesis. Their significance in the light of earlier data on the role of IFNs in mousepox is discussed.

An Ectromelia Virus Protein That Interacts with Chemokines through Their Glycosaminoglycan Binding Domain

ABSTRACT Poxviruses encode a number of secreted virulence factors that modulate the host immune response. The vaccinia virus A41 protein is an immunomodulatory protein with amino acid sequence similarity to the 35-kDa chemokine binding protein, but the host immune molecules targeted by A41 have not been identified. We report here that the vaccinia virus A41 ortholog encoded by ectromelia virus, a poxvirus pathogen of mice, named E163 in the ectromelia virus Naval strain, is a secreted 31-kDa glycoprotein that selectively binds a limited number of CC and CXC chemokines with high affinity. A detailed characterization of the interaction of ectromelia virus E163 with mutant forms of the chemokines CXCL10 and CXCL12α indicated that E163 binds to the glycosaminoglycan binding site of the chemokines. This suggests that E163 inhibits the interaction of chemokines with glycosaminoglycans and provides a mechanism by which E163 prevents chemokine-induced leukocyte migration to the sites of infection. In addition to interacting with chemokines, E163 can interact with high affinity with glycosaminoglycan molecules, enabling E163 to attach to cell surfaces and to remain in the vicinity of the sites of viral infection. These findings identify E163 as a new chemokine binding protein in poxviruses and provide a molecular mechanism for the immunomodulatory activity previously reported for the vaccinia virus A41 ortholog. The results reported here also suggest that the cell surface and extracellular matrix are important targeting sites for secreted poxvirus immune modulators.

The gammaherpesvirus chemokine binding protein can inhibit the interaction of chemokines with glycosaminoglycans

Chemokines are small glycosaminoglycan (GAG) binding proteins that direct the migration of leukocytes by signaling through G protein coupled receptors (GPCR). Many viruses encode proteins that disrupt chemokine responses. The murine gammaherpesvirus‐68 gene M3 encodes a chemokine binding protein (vCKBP‐3), which has no sequence similarity to chemokine receptors. Initial characterization of vCKBP‐3 showed that it inhibits receptor binding and chemokine‐induced calcium influx. The structural requirements for the chemokines CXCL8 and CCL2 to bind to vCKBP‐3 have been determined. Both chemokines bind to vCKBP‐3 via their N‐loop, a site that can participate in GAG binding for some chemokines. We have investigated the effect of vCKBP‐3 on the interaction of chemokines with GAGs. We found that vCKBP‐3 can prevent a range of chemokines from binding to GAGs. Moreover, we also found that vCKBP‐3 can displace chemokines from a heparin‐coated surface. Together, these data imply that vCKBP‐3 can inhibit chemokine activity at two distinct levels. First, it inhibits chemokines from binding to their GPCR. Second, it inhibits their GAG binding and disrupts pre‐formed chemokine gradients. This dual ability of vCKBP‐3 makes it a more effective inhibitor of chemokine activity.