Molecular determinants for CC-chemokine recognition by a poxvirus CC-chemokine inhibitor.

Poxviruses express a family of secreted proteins that bind with high affinity to chemokines and antagonize the interaction with their cognate G protein-coupled receptors (GPCRs). These viral inhibitors are novel in structure and, unlike cellular chemokine receptors, are able to specifically interact with most, if not all, CC-chemokines. We therefore sought to define the structural features of CC-chemokines that facilitate this broad-spectrum interaction. Here, we identify the residues present on human monocyte chemoattractant protein-1 (MCP-1) that are required for high-affinity interaction with the vaccinia virus 35-kDa CC-chemokine binding protein (VV-35kDa). Not only do these residues correspond to those required for interaction with the cognate receptor CCR2b but they are also conserved among many CC-chemokines. Thus, the results provide a structural basis for the ability of VV-35kDa to promiscuously recognize CC-chemokines and block binding to their receptors.

Glycosaminoglycan binding properties of the myxoma virus CC-chemokine inhibitor, M-T1.

Poxviruses encode a number of secreted virulence factors that function to mitigate or modulate the host immune response. M-T1 is a secreted 43-kDa glycoprotein produced by the myxoma virus, a poxvirus pathogen of rabbits, that binds CC-chemokines with high affinity, blocks binding to their cognate G-protein coupled receptors, and thereby inhibits chemokine-induced leukocyte chemotaxis. The present study indicates that M-T1, but not the related vaccinia virus 35-kDa CC-chemokine-binding protein, can localize to cell surfaces through an interaction with glycosaminoglycan molecules. In addition to biochemically characterizing the nature of this interaction, we demonstrate that M-T1 can also simultaneously interact with CC-chemokines while bound to heparin, suggesting that the binding sites on M-T1 for chemokines and heparin are distinct. Furthermore, using recombinant baculovirus-expressed M-T1 truncation and internal deletion mutants, we localize the heparin-binding region of M-T1 to the C terminus of the protein, a region that contains a high abundance of basic residues and includes two clusters of basic amino acid residues that resemble Cardin and Weintraub heparin-binding consensus sequences. The ability of M-T1 to simultaneously interact with chemokines and glycosaminoglycans may enable M-T1 to tether to endothelial surfaces or extracellular matrix and capture host chemokines that are expressed close to sites of virus infection.

Regulation of nitric oxide synthase 2 in rabbit corneal cells.

PURPOSE: The purpose of these studies was to investigate the role of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), and transforming growth factor-beta (TGF-beta) in the regulation of inducible nitric oxide synthase (NOS2) activity in rabbit corneal cells. METHODS: Rabbit corneal epithelial, stromal, and endothelial cells were grown in culture and treated with cytokines and growth factors, alone or in combination. NOS activity was measured at times up to 72 hours after treatment by assaying the culture medium for nitrite using the Griess reaction. Cell lysates were analyzed by Western blot analysis for NOS2 protein. RNA was isolated and amplified with NOS1-, NOS2-, and NOS3-specific primers by RT-PCR. RESULTS: NOS2 expression was induced by combined cytokine treatment from nondetectable levels to abundant levels in low passage (<4) stromal cells and to low levels in corneal endothelial cells but not in corneal epithelial cells. In the absence of IFN-gamma, little or no nitrite accumulation was induced by TNF-alpha, IL-1beta, or lipopolysaccharide (LPS) treatment. The inductive effects of IFN-gamma were antagonized in a dose-dependent manner by the myxoma virus rabbit IFN-gamma receptor homolog, M-T7. rRaIFN-gamma, in combination with IL-1beta and TNF-alpha, induced the appearance of NOS2 mRNA within 24 hours but detectable nitrite did not accumulate in large amounts (>10 microM) until after 24 hours postinduction. NOS2 was identified as a 130 kDa protein on Western blot analysis using monoclonal antibody against murine NOS2. TGF-beta(1) and beta(2) inhibited the accumulation of cytokine-induced nitrite in a dose-dependent manner while not significantly reducing the steady state level of NOS2 mRNA. The activity of the induced NOS was inhibited by 1400W, a NOS2-selective inhibitor, but not 7-nitroindazole, a NOS1-selective inhibitor. CONCLUSIONS: In cultured corneal stromal cells, NOS2 expression was upregulated by IFN-gamma in combination with IL-1beta and TNF-alpha but not by any of these cytokines alone, while TGF-beta downregulated the activity. Cultures of corneal epithelial cells could not be induced to express NOS2, yet cultures of endothelial cells produced low amounts of NO in response to cytokines. The NOS1 and NOS3 isoforms were not detected in any of these corneal cells.

Post-translational modification of the myxoma-virus anti-inflammatory serpin SERP-1 by a virally encoded sialyltransferase.

SERP-1 is a secreted serpin (serine-proteinase inhibitor) encoded by myxoma virus, a poxvirus pathogen of rabbits. SERP-1 is required for myxoma-virus virulence, and the purified protein has been shown to possess independent anti-inflammatory activity in animal models of restenosis and antigen-induced arthritis. As an inhibitor of serine proteinases, SERP-1 acts against tissue-type plasminogen activator, urokinase-type plasminogen activator, plasmin, thrombin and Factor Xa. In the present study, examination of SERP-1 glycosylation-site mutants showed that the N-linked glycosylation of Asn(172) was essential for SERP-1 secretion, whereas mutation of Asn(99) decreased secretion efficiency, indicating that N-linked glycosylation plays an essential role in the processing and trafficking of SERP-1. Furthermore, comparison of SERP-1 from wild-type myxoma virus and a virus containing a targeted disruption of the MST3N sialyltransferase locus demonstrated that SERP-1 is specifically modified by this myxoma-virus-encoded sialyltransferase, and is thus the first reported viral protein shown to by modified by a virally encoded glycosyltransferase. Sialylation of SERP-1 by the MST3N gene product creates a uniquely charged species of secreted SERP-1 that is distinct from SERP-1 produced from other eukaryotic expression systems, though this has no apparent effect upon the kinetics of in vitro proteinase inhibition. Rather, the role of viral sialylation of SERP-1 likely relates to masking antigenicity or targeting SERP-1 to specific sites of action in vivo.

Immunomodulation by viruses: the myxoma virus story.

Myxoma virus is a poxvirus pathogen of rabbits that has evolved to replicate successfully in the presence of an active immune response by an infected host. To accomplish this, the virus has developed a variety of strategies to avoid detection by or obstruct specific aspects of the antiviral response whose consolidated action is antagonistic to virus survival. We describe two distinct viral strategies carried out by viral proteins with which myxoma virus subverts the host immune response. The first strategy is the production of virus-encoded proteins known as viroceptors or virokines that mimic host receptors or cytokines. These seek to actively block extracellular immune signals required for effective virus clearance and produce a local environment in the infected tissue that is "virus friendly". The second strategy, carried out by intracellular viral proteins, seeks to retard the innate antiviral responses such as apoptosis, and hinder attempts by the infected cell to communicate with the cellular arm of the immune system. By studying these viral strategies of immune evasion, the myxoma system can provide insights into virus-host interactions and also provide new insights into the complex immune system.

Functional comparisons among members of the poxvirus T1/35kDa family of soluble CC-chemokine inhibitor glycoproteins.

Many poxviruses express a 35-40-kDa secreted protein, termed "T1" (for leporipoxviruses) or "35kDa" (for orthopoxviruses), that binds CC-chemokines with high affinity but is unrelated to any known cellular proteins. Many previously identified poxvirus cytokine-binding proteins display strict species ligand-binding specificity. Because the T1 and 35kDa proteins share only 40% amino acid identity, we compared the abilities of purified myxoma virus-T1 (M-T1) and vaccinia virus (strain Lister)- and rabbitpox virus-35kDa proteins to inhibit human CC-chemokines in vitro. All three proteins were equally effective in preventing several human CC-chemokines from binding to target chemokine receptors and blocking subsequent intracellular calcium release. The inhibitory affinities were comparable (Ki = 0.07-1.02 nM). These proteins also displayed similar abilities to inhibit (IC50 = 6.3-10.5 nM) human macrophage inflammatory protein-1alpha-mediated chemotaxis of human monocytes. None of the viral proteins blocked interleukin-8-mediated calcium flux or chemotaxis of human neutrophils, confirming that the biological specificity of the T1/35kDa family is targeted inhibition of CC-chemokines. Despite the significant sequence divergence between the leporipoxvirus T1 and orthopoxvirus 35kDa proteins, our data suggest that their CC-chemokine binding and inhibitory properties appear to be species nonspecific and that the critical motifs most likely reside within the limited regions of conservation.