title: The poxvirus p28 virulence factor is an E3 ubiquitin ligase.
abstract: A majority of the orthopoxviruses, including the variola virus that causes the dreaded smallpox disease, encode a highly conserved 28-kDa protein with a classic RING finger sequence motif (C(3)HC(4)) at their carboxyl-terminal domains. The RING domain of p28 has been shown to be a critical determinant of viral virulence for the ectromelia virus (mousepox virus) in a murine infection model (Senkevich, T. G., Koonin, E. V., and Buller, R. M. (1994) Virology 198, 118-128). Here, we demonstrate that the p28 proteins encoded by the ectromelia virus and the variola virus possess E3 ubiquitin ligase activity in biochemical assays as well as in cultured mammalian cells. Point mutations disrupting the RING finger domain of p28 completely abolish its E3 ligase activity. In addition, p28 functions cooperatively with Ubc4 and UbcH5c, the E2 conjugating enzymes involved in 26 S proteasome degradation of protein targets. Moreover, p28 catalyzes the formation of Lys-63-linked polyubiquitin chains in the presence of Ubc13/Uev1A, a heterodimeric E2 conjugating enzyme, indicating that p28 may regulate the biological activity of its cognate viral and/or host cell target(s) by Lys-63-linked ubiquitin multimers. We thus conclude that the poxvirus p28 virulence factor is a new member of the RING finger E3 ubiquitin ligase family and has a unique polyubiquitylation activity. We propose that the E3 ligase activity of the p28 virulence factor may be targeted for therapeutic intervention against infections by the variola virus and other poxviruses.
authors.affiliation: Rigel Pharmaceuticals, Inc., South San Francisco, California 94080, USA.
authors.forename: Jianing
authors.initials: J
authors.lastname: Huang
authors.forename: Qi
authors.initials: Q
authors.lastname: Huang
authors.forename: Xiulan
authors.initials: X
authors.lastname: Zhou
authors.forename: Mary M
authors.initials: MM
authors.lastname: Shen
authors.forename: Ann
authors.initials: A
authors.lastname: Yen
authors.forename: Simon X
authors.initials: SX
authors.lastname: Yu
authors.forename: GuoQiang
authors.initials: G
authors.lastname: Dong
authors.forename: Kunbin
authors.initials: K
authors.lastname: Qu
authors.forename: Peiyong
authors.initials: P
authors.lastname: Huang
authors.forename: Emily M
authors.initials: EM
authors.lastname: Anderson
authors.forename: Sarkiz
authors.initials: S
authors.lastname: Daniel-Issakani
authors.forename: R Mark L
authors.initials: RM
authors.lastname: Buller
authors.forename: Donald G
authors.initials: DG
authors.lastname: Payan
authors.forename: H Henry
authors.initials: HH
authors.lastname: Lu
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010802:Phylogeny; D017354:Point Mutation; D011212:Poxviridae / Q000737:chemistry*; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012901:Variola virus / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; D014764:Viral Proteins; C407905:virulence factor p28, Ectromelia virus; D044767:Ubiquitin-Protein Ligases