VP4 differentially regulates TRAF2 signaling, disengaging JNK activation while directing NF-kappa B to effect rotavirus-specific cellular responses.
Rotaviruses rapidly activate NF-kappaB and induce the secretion of selected chemokines after infection. The ability of rotavirus particles lacking genomic RNA to activate NF-kappaB suggested that rotavirus proteins direct cell signaling responses. We identified conserved TNFR-associated factor (TRAF) binding motifs within the rotavirus capsid protein VP4 and its N-terminal VP8* cleavage product. TRAFs (-1, -2, and -3) are bound by the rhesus rotavirus VP8* protein through three discrete TRAF binding domains. Expression of VP4 or VP8* from rhesus or human rotaviruses induced a 5-7-fold increase in NF-kappaB activity and synergistically enhanced TRAF2-mediated NF-kappaB activation. Mutagenesis of VP8* TRAF binding motifs abolished VP8* binding to TRAFs and the ability of the protein to activate NF-kappaB. Expression of pathway-specific dominant negative (DN) inhibitors DN-TRAF2 or DN-NF-kappaB-inducing kinase also abolished VP8*-, VP4-, or rotavirus-mediated NF-kappaB activation. These findings demonstrate that rotavirus primarily activates NF-kappaB through a TRAF2-NF-kappaB-inducing kinase signaling pathway and that VP4 and VP8* proteins direct pathway activation through interactions with cellular TRAFs. In contrast, transcriptional responses from AP-1 reporters were inhibited 5-fold by VP8* and were not activated by rotavirus infection, suggesting the differential regulation of TRAF2 signaling responses by VP8*. VP8* blocked JNK activation directed by TRAF2 or TRAF5 but had no effect on JNK activation directed by TRAF6 or MEKK1. This establishes that fully cytoplasmic rotaviruses selectively engage signaling pathways, which regulate cellular transcriptional responses. These findings also demonstrate that TRAF2 interactions can disengage JNK signaling from NF-kappaB activation and thereby provide a new means for TRAF2 interactions to determine pathway-specific responses.
LaMonica
R
R
Department of Medicine, Department of Molecular Genetics and Microbiology, and Molecular Cell Biology Program, State University of New York, Stony Brook, New York 11794, USA.
Kocer
S S
SS
Nazarova
J
J
Dowling
W
W
Geimonen
E
E
Shaw
R D
RD
Mackow
E R
ER
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000502:physiology*; D036022:Capsid Proteins; D017931:DNA Primers; D004789:Enzyme Activation; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012401:Rotavirus / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D047992:TNF Receptor-Associated Factor 2; D020798:Two-Hybrid System Techniques
D036022:Capsid Proteins; D017931:DNA Primers; D016328:NF-kappa B; D011506:Proteins; D047992:TNF Receptor-Associated Factor 2; C060339:VP4 protein, Rotavirus; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases