Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.
We previously showed that an envelope A27L protein of intracellular mature virions (IMV) of vaccinia virus binds to cell surface heparan sulfate during virus infection. In the present study we identified another viral envelope protein, D8L, that binds to chondroitin sulfate on cells. Soluble D8L protein interferes with the adsorption of wild-type vaccinia virions to cells, indicating a role in virus entry. To explore the interaction of cell surface glycosaminoglycans and vaccinia virus, we generated mutant viruses from a control virus, WR32-7/Ind14K (A27L+ D8L+) to be defective in expression of either the A27L or the D8L gene (A27L+D8L− or A27L− D8L+) or both (A27L− D8L−). The A27L+D8L+ and A27L− D8L+ mutants grew well in BSC40 cells, consistent with previous observations. However, the IMV titers of A27L+ D8L− and A27L− D8L− viruses in BSC40 cells were reduced, reaching only 10% of the level for the control virus. The data suggested an important role for D8L protein in WR32-7/Ind14K virus growth in cell cultures. A27L protein, on the other hand, could not complement the functions of D8L protein. The low titers of the A27L+ D8L− and A27L−D8L− mutant viruses were not due to defects in the morphogenesis of IMV, and the mutant virions demonstrated a brick shape similar to that of the control virions. Furthermore, the infectivities of the A27L+ D8L− and A27L−D8L− mutant virions were 6 to 10% of that of the A27L+ D8L+ control virus. Virion binding assays revealed that A27L+ D8L− and A27L− D8L− mutant virions bound less well to BSC40 cells, indicating that binding of viral D8L protein to cell surface chondroitin sulfate could be important for vaccinia virus entry.
Tumor necrosis factor alpha (TNF-␣) activates the nuclear factor B (NF-B) signaling pathway that regulates expression of many cellular factors playing important roles in innate immune responses and inflammation in infected hosts. Poxviruses employ many strategies to inhibit NF-B activation in cells. In this report, we describe a poxvirus host range protein, CP77, which blocked NF-B activation by TNF-␣. Immunofluorescence analyses revealed that nuclear translocation of NF-B subunit p65 protein in TNF-␣-treated HeLa cells was blocked by CP77. CP77 did so without blocking IB␣ phosphorylation, suggesting that upstream kinase activation was not affected by CP77. Using GST pull-down, we showed that CP77 bound to the NF-B subunit p65 through the N-terminal six-ankyrin-repeat region in vitro. CP77 also bound to Cullin-1 and Skp1 of the SCF complex through a C-terminal 13-amino-acid F-box-like sequence. Both regions of CP77 are required to block NF-B activation. We thus propose a model in which poxvirus CP77 suppresses NF-B activation by two interactions: the C-terminal F-box of CP77 binding to the SCF complex and the N-terminal six ankyrins binding to the NF-B subunit p65. In this way, CP77 attenuates innate immune response signaling in cells. Finally, we expressed CP77 or a CP77 F-box deletion protein from a vaccinia virus host range mutant (VV-hr-GFP) and showed that either protein was able to rescue the host range defect, illustrating that the F-box region, which is important for NF-B modulation and binding to SCF complex, is not required for CP77's host range function. Consistently, knocking down the protein level of NF-B did not relieve the growth restriction of VV-hr-GFP in HeLa cells.Vaccinia virus, the prototype of the poxvirus family, infects a wide range of cells in vitro and animal species in vivo (14). Vaccinia virus has a double-stranded DNA genome that encodes 263 open reading frames (ORFs). Vaccinia virus expresses different classes of viral genes in a cascade-regulated manner and completes the virus life cycle in the cytoplasm of infected cells (11).To replicate successfully in infected hosts, poxviruses have evolved various strategies to overcome cellular immune responses (20, 39). Viral infections activate cellular antiviral signaling and inflammatory responses (49), such as NF-B, which plays a critical role in inflammatory signaling and immune activation (23). NF-B contains five different members, NF-B1 (p50/p105), NF-B2 (p52/p100), RelA (p65), RelB, and c-Rel, all of which share a Rel homology domain for DNA binding, dimerization, and interaction with IB (22, 23). The most abundant activated form consists of a p50 or p52 subunit and a p65 subunit (16,26). In the inactive state, dimerized NF-B (such as p65/p50) is bound by IB␣, and the crystal structure of the IB␣/p65/p50 complex shows multiple contact sites between the ankyrin repeats of IB␣ and NF-B (29). In well-characterized canonical NF-B signaling, such as tumor necrosis factor alpha (TNF-␣) treatment, receptor activation sends intracellular sig...
We previously showed that vaccinia virus infection of BSC40 cells was blocked by soluble heparin, suggesting that cell surface heparan sulfate mediates vaccinia virus binding (C.-S. Chung, J.-C. Hsiao, Y.-S. Chang, and W. Chang, J. Virol. 72:1577–1585, 1998). In this study, we extended our previous work and demonstrated that soluble A27L protein bound to heparan sulfate on cells and interfered with vaccinia virus infection at a postbinding step. In addition, we investigated the structure of A27L protein that provides for its binding to heparan sulfate on cells. A mutant of A27L protein, named D-A27L, devoid of a cluster of 12 amino acids rich in basic residues, was constructed. In contrast to the soluble A27L protein, purified D-A27L protein was inactive in all of our assays, including binding to heparin in vitro, binding to heparan sulfate on cells, and the ability to block virus infection. These data demonstrated that the N-terminal region acts as a glycosaminoglycan (GAG)-binding domain critical for A27L protein binding to cells. Previously A27L protein was thought to be involved in fusion of virus-infected cells induced by acid treatment. When we investigated whether cell surface GAGs also participate in A27L-dependent fusion, our results indicated that soluble A27L protein blocked cell fusion, whereas D-A27L protein did not. Taken together, the results therefore demonstrated that A27L-mediated cell fusion is triggered by its interaction with cell surface GAGs through the N-terminal domain.
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