Interferon Regulatory Factor 3 Is a Cellular Partner of Rotavirus NSP1

Rotaviruses are the most important cause of severe, often life-threatening gastroenteritis in infants and children under 2 years of age (33). These viruses are ubiquitous in nature and are also responsible for a significant proportion of neonatal diarrheal illness in domestic animals, particularly in bovine and porcine species (18, 40). Substantial research efforts have thus focused on understanding the correlates of a protective immune response to rotavirus infection and the molecular mechanisms of virus replication so that efficacious vaccines can be developed. The rotavirus segmented double-stranded RNA genome encodes six structural proteins (VP) and six nonstructural proteins (NSP) (reviewed in reference 17). The structural proteins VP1, VP2, VP3, VP4, VP6, and VP7 are well characterized in terms of their antigenic, structural, and biochemical properties. The functions of the rotavirus nonstructural proteins NSP1 to NSP6 are less well defined with regard to the roles that these proteins play in the rotavirus replication cycle. Intriguing functions have recently been described for some. NSP3 binds the 3′ consensus sequence of viral mRNAs (37) and acts as a functional analog of poly(A) binding protein through its interaction with eIF4GI (36). NSP4 is both an intracellular glycoprotein receptor for maturating rotavirus particles that bud through the endoplasmic reticulum (3, 32) and a viral enterotoxin that induces diarrhea in mice in the absence of any other viral protein (5). Functions of the remaining nonstructural proteins, NSP1, NSP2, NSP5, and NSP6, have been proposed based predominately on biochemical properties and activities of recombinant proteins (reviewed in reference 17). NSP1 displays several interesting properties that warrant investigation. NSP1 has a calculated molecular weight of approximately 54,000 and is the least conserved protein encoded by the rotavirus genome when NSP1s of different strains are compared (23, 34). The N terminus contains a conserved zinc finger motif that binds zinc and viral mRNA in vitro (10, 22). Immunofluorescent staining showed NSP1 to be localized throughout the cytoplasm, in contrast to most other rotavirus proteins, which concentrate in viroplasms (24). NSP1 is also found associated with the cytoskeleton when analyzed by subcellular fractionation (24). NSP1 apparently is not required for rotavirus replication because strains with rearrangements in gene 5 that result in the synthesis of truncated NSP1 have been isolated from animals and from both immune-deficient and immune-competent children (1, 7, 15, 25, 35, 44, 45). Each of the described strains replicates in cell culture to titers close to those of their wild-type counterparts, but they yield small- to minute-plaque phenotypes. These observations suggest that NSP1 plays a role in regulating the efficiency of viral gene expression or in modulating host cell responses. We addressed the second possibility by constructing an MA104 cell cDNA library into the activation domain vector of the Matchmaker 3 yeast two-hybrid interaction trap (Clontech). We screened the library with NSP1 as bait to identify candidate partners of NSP1 that would provide clues to its function in rotavirus-infected cells. Polyadenylated mRNA was isolated from MA104 cells in exponential growth phase with TriZol reagent (Life Technologies) and purified by column chromatography with the mRNA purification system from Amersham Pharmacia. cDNA was synthesized according to the protocol supplied with reagents from Clontech, with modifications. Briefly, 5 μg of mRNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase and an oligo(dT)-XhoI linker primer. Double-stranded cDNA was generated with DNA polymerase I, and the ends were blunted with Pfu DNA polymerase (Stratagene). Double-stranded cDNA fragments ligated to EcoRI adapters were size fractionated with ChromaSpin 400 columns (Clontech) and ligated into the Matchmaker pGADT7 vector prepared by EcoRI-XhoI digestion. Transformations that were sufficient to obtain 2.5 × 106 CFU were performed. The colonies were collected into Luria broth, and DNA was purified by two sequential centrifugations through CsCl density gradients. The library was quantified by measuring the absorbance at 260 nm, aliquoted, and stored at −80°C. Gene 5 encoding NSP1 of bovine rotavirus strain B641 was cloned by reverse transcriptase PCR (unpublished data). The nucleotide sequence of B641 gene 5 is 98% identical to that of gene 5 of bovine strain RF (9). A plasmid containing gene 5 of murine strain EW was a generous gift of Harry Greenberg, Stanford University School of Medicine. Gene 5 cDNAs from B641 and EW were cloned into pGBKT7 by standard cloning techniques to generate pGBK-bNSP1 (bovine) and pGBK-mNSP1 (murine), respectively. All yeast transformations followed the lithium acetate-polyethylene glycol (PEG) method described by Gietz and Woods (19). The DNA binding domain vector pGBKT7 carries the TRP1 nutritional selection marker, and colonies containing bait plasmids were isolated by culture on synthetic complete medium lacking tryptophan (SC−W medium). The Matchmaker activation domain vector pGADT7 carries the LEU2 nutritional marker for selection of yeast. Positive two-hybrid interactions were scored by determining colony growth on SC medium without leucine and tryptophan (SC−L−W medium) and activation of the reporter genes HIS3, ADE2, and MEL1. Reporter gene activation was indicated by growth in the absence of histidine and adenine and by the ability to metabolize the chromogenic substrate X-α-Gal(α-5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; ICN).pGBK-bNSP1 yeast cells (6 × 109) cultured in SC−W liquid medium were transformed with 120 μg of the cDNA library by the lithium acetate-PEG procedure. Transformants were plated on SC medium without leucine, tryptophan, histidine, and adenine (SC−L−W−H−A medium) and cultured for 2 to 4 days at 30°C. Colonies measuring 2 to 4 mm in diameter were restreaked on SC−L−W−H−A plus X-α-Gal medium and again incubated for 2 to 4 days at 30°C. Plasmid DNA was isolated by electroporation into DH10B cells (Life Technologies) and cultured on antibiotic selection media.