Proteomic screening of variola virus reveals a unique NF-κB inhibitor that is highly conserved among pathogenic orthopoxviruses

Abstract

Identification of the binary interactions between viral and host proteins has become a valuable tool for investigating viral tropism and pathogenesis. Here, we present the first systematic protein interaction screening of the unique variola virus proteome by using yeast 2-hybrid screening against a variety of human cDNA libraries. Several protein-protein interactions were identified, including an interaction between variola G1R, an ankryin/F-box containing protein, and human nuclear factor kappa-B1 (NF-B1)/p105. This represents the first direct interaction between a pathogen-encoded protein and NF-B1/p105. Orthologs of G1R are present in a variety of pathogenic orthopoxviruses, but not in vaccinia virus, and expression of any one of these viral proteins blocks NF-B signaling in human cells. Thus, proteomic screening of variola virus has the potential to uncover modulators of the human innate antiviral responses.ankyrin repeats ͉ Skp1 ͉ yeast 2-hybrid

Cowpox Virus Expresses a Novel Ankyrin Repeat NF-κB Inhibitor That Controls Inflammatory Cell Influx into Virus-Infected Tissues and Is Critical for Virus Pathogenesis

“…Members of this VARV G1R family, including versions from ECTV, MPXV, and CPXV, were shown to be capable of interacting with NF-B1/p105 and inhibited the NF-B signaling pathway in transfected cells (27). In order to evaluate the physiological relevance of these proteins during viral infection, we were interested in assessing the impact of knocking out this novel NF-B inhibitor on viral replication both in vitro and in vivo.…”

“…Recently, following a yeast two-hybrid screen of the unique proteins encoded by the genome of VARV against several human cDNA libraries, we demonstrated that a novel ankyrin (ANK) repeat-containing protein encoded by VARV, called G1R, could bind NF-B1/p105 and was a bona fide inhibitor of the NF-B signaling pathway in cultured cells (27). Functional orthologs of VARV G1R also exist for other pathogenic orthopoxviruses, including ECTV, CPXV, and MPXV, but the gene is conspicuously fragmented in VACV.…”

Orf Virus ORFV121 Encodes a Novel Inhibitor of NF-κB That Contributes to Virus Virulence

“…An exception here is ORFV-encoded ORFV002, which binds to and decreases the acetylation of NF-B-p65 in the nucleus without significantly affecting virus virulence in the natural host (12). It would be interesting to determine if other poxviral encoded proteins that target NF-B subunits (28,29) have a similar effect on virus virulence and disease pathogenesis in natural hosts in vivo. Poxviruses have evolved a striking variety of mechanisms to inhibit the NF-B signaling pathway, targeting ligand-receptor interactions on the cell surface or intracellular cytoplasmic events of the pathway (28).…”

“…However, selected inhibitors encoded by these viruses have been shown to function downstream in the pathway directly on NF-B subunits (5,29,30). For example, myxoma virus (MYXV) M013, variola virus (VARV) G1R and its orthologs in monkeypox virus (MPXV MPXV003), ectromelia virus (ECTV ECTV002), and cowpox virus (CPXV) (CPXV006) were shown to interact with the NF-B subunit NF-B-p105, consequently inhibiting the nuclear translocation of NF-B-p50/NF-B-p65 (29,30). Additionally, CPXV-encoded CPXV077 was shown to interact with and inhibit the nuclear translocation of the NF-B transactivating subunit NF-B-p65 (5).…”

Innate immune activation of NFκB and its antagonism by poxviruses

“…CP77 might also target p65 for proteasomal degradation through these binding activities [30]. Other poxviral proteins demonstrated or suspected to directly interact with NFkB include: MYXV protein M150 which co-localises with NFkB and supresses inflammation [31]; VARV protein G1 which contains ankyrin repeats, is conserved in a number of orthopoxviruses (CPV, MYXV and ECTV) and has been shown to associate with p105 and block NFkB nuclear translocation [32]; MYXV protein M013 which also binds p105 to prevent its processing and subsequent NFkB activation [33].…”