Vaccinia virus intracellular mature virus (IMV) binds to glycosaminoglycans (GAGs) on cells via three virion proteins, H3L, A27L, and D8L. In this study, we demonstrated that binding of IMV to BSC40 cells was competitively inhibited by soluble laminin but not by fibronectin or collagen V, suggesting that this cell surface extracellular matrix (ECM) protein may play a role in vaccinia virus entry. Moreover, IMV infection of GAG ؊ sog9 cells was also inhibited by laminin, demonstrating that virion binding to laminin does not involve a prior interaction with GAGs. Furthermore, comparative envelope protein analyses of wild-type vaccinia virus strain Western Reserve, which binds to laminin, and of a mutant virus, IA27L, which does not, showed that the A26L open reading frame (ORF), encoding an envelope protein, was mutated in IA27L, resulting in A26L being absent from the IMV. Expression of the wild-type A26L ORF in IA27L resulted in laminin binding activity. Moreover, recombinant A26L protein bound to laminin in vitro with a high affinity, providing direct evidence that A26L is the laminin binding protein on IMV. In summary, these results reveal a novel role for the vaccinia viral envelope protein A26L in binding to the ECM protein laminin, an association that is proposed to facilitate IMV 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...