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Variola virus (VaV) is the causative agent of smallpox, one of the most devastating diseases encountered by man, that was eradicated in 1980. The deliberate release of VaV would have catastrophic consequences on global public health. However, the mechanisms that contribute to smallpox pathogenesis are poorly understood at the molecular level. The ability of viruses to evade the host defense mechanisms is an important determinant of viral pathogenesis. Here we show that the tumor necrosis factor receptor (TNFR) homologue CrmB encoded by VaV functions not only as a soluble decoy TNFR but also as a highly specific binding protein for several chemokines that mediate recruitment of immune cells to mucosal surfaces and the skin, sites of virus entry and viral replication at late stages of smallpox. CrmB binds chemokines through its C-terminal domain, which is unrelated to TNFR5, was named smallpox virus-encoded chemokine receptor (SECRET) domain and uncovers a family of poxvirus chemokine inhibitors. An active SECRET domain was found in another viral TNFR (CrmD) and three secreted proteins encoded by orthopoxviruses. These findings identify a previously undescribed chemokine-binding and inhibitory domain unrelated to host chemokine receptors and a mechanism of immune modulation in VaV that may influence smallpox pathogenesis. [ABSTRACT FROM AUTHOR]

Sphingomyelin hydrolysis by sphingomyelinase is essential in regulating membrane levels of ceramide, a well-known metabolic signal. Since natural sphingomyelins have a gel-to-fluid transition temperature in the range of the physiological temperatures of mammals and birds, it is important to understand the influence of the physical state of the lipid on the enzyme activity. With that aim, large unilamellar vesicles consisting of pure egg sphingomyelin (gel-to-fluid crystalline transition temperature ca. 39 °C) were treated with sphingomyelinase in the temperature range 10–70 °C. The vesicles were also examined by differential scanning calorimetry (DSC). Shingomyelinase was active on pure sphingomyelin bilayers, leading to concomitant lipid hydrolysis, vesicle aggregation, and leakage of aqueous liposomal contents. Enzyme activity was found to be much higher when the substrate was in the fluid than when it was in the gel state. Sphingomyelinase activity was found to exhibit lag times, followed by bursts of activity. Lag times decreased markedly when the substrate went from the gel to the fluid state. When egg phosphatidylcholine, or egg phosphatidylethanolamine were included in the bilayer composition together with sphingomyelin, sphingomyelinase activity at 37 °C, that was negligible for the pure sphingolipid bilayers, was seen to increase with the proportion of glycerophospholipid, while the latency times became progressively shorter. A DSC study of the mixed-lipid vesicles revealed that both phosphatidylcholine and phosphatidyletanolamine decreased in a dose-dependent way the transition temperature of sphingomyelin. Thus, as those glycerophospholipids were added to the membrane composition, the proportion of sphingomyelin in the fluid state at 37 °C increased accordingly, in this way becoming amenable to rapid hydrolysis by the enzyme. Thus sphingomyelinase requires the substrate in bilayer form to be in the fluid state, irrespective of whether this is achieved through a thermotropic transition or by modulating bilayer composition. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]