Rotavirus NSP1 is an interferon system antagonist

Abstract

Rotaviruses cause severe gastroenteritis in mammals, including humans and livestock. Most rotavirus proteins have known functions, either as a mediator of virus replication or as a component of the infectious virus particle. The function of nonstructural protein 1 (NSP1) was unknown. However, it has been suggested that the function of NSP1 involved interactions with cellular proteins. Using the NSP1 encoded by a bovine rotavirus as the bait protein of a yeast-two hybrid interaction trap, interferon regulatory factor 3 (IRF3) was identified as an NSP1-interacting protein. Due to the importance of IRF3 in initiating an interferon response, we hypothesized that NSP1 acts to antagonize the interferon system. A comprehensive set of experiments yielded the following observations. Interferon-beta (IFNbeta) induction was blocked in wild-type, but not NSP1 null, infected cells. Expression of NSP1 in the absence of infection resulted in proteasome-mediated degradation of IRF3. A cysteine-rich zinc-binding region near the amino-terminus of all known NSP1 sequences resembles a domain found in hundreds of E3 ubiquitin ligases. Mutational analysis of the zinc-binding domain was consistent with NSP1 acting as an E3 enzyme. The NSP1 of a murine strain of rotavirus also interacted with IRF3. The NSP1 proteins encoded by the bovine and murine rotavirus strains showed low sequence homology (37% identity), which indicated that directing degradation of IRF3 may be a common property of NSP1 proteins from many different rotavirus strains. Screening of simian, bovine, and porcine rotavirus strains indicated that the porcine strain OSU was the only wild-type strain that was unable to direct IRF3 degradation. Characterization of OSU showed that this strain was able to block IFNbeta induction by antagonizing NFKB signaling, rather than IRF3 signaling. While analyzing NFKB signaling in rotavirus infected cells, we found that the F box protein, betaTrCP, of the Skp1/Cul1/F box multi-subunit E3 enzyme was targeted for degradation by OSU NSP1 and the NSP1 of a bovine rotavirus strain. Together, the results presented in this dissertation have determined that rotavirus NSP1 functions as an interferon antagonist by directing the proteasome-mediated degradation of IFNbeta induction signaling components, most likely by acting as an E3 ubiquitin ligase.