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Rotavirus nonstructural protein NSP1 can inhibit expression of interferon (IFN) and IFN-stimulated gene products by inducing proteasome-mediated degradation of IFN-regulatory factors (IRFs), including IRF3, IRF5, and IRF7. All IRF proteins share an N-terminal DNA-binding domain (DBD), and IRF3, IRF5, and IRF7 contain a similar C-proximal IRF association domain (IAD) that mediates IRF dimerization. An autoinhibitory domain (ID) at the extreme C terminus interacts with the IAD, burying residues necessary for IRF dimerization. Phosphorylation of serine/threonine residues in the ID induces charge repulsions that unmask the IAD, enabling IRF dimerization and subsequent nuclear translocation. To define the region of IRF proteins targeted for degradation by NSP1, we generated IRF3 and IRF7 truncation mutants and transiently expressed each with simian SA11-4F NSP1. These assays indicated that the IAD represented a necessary and sufficient target for degradation. Because NSP1 did not mediate degradation of truncated forms of the IAD, NSP1 likely requires a structurally intact IAD for recognition and targeting of IRF proteins. IRF9, which contains an IAD-like region that directs interactions with signal inducer and activator of transcription (STAT) proteins, was also targeted for degradation by NSP1, while IRF1, which lacks an IAD, was not. Analysis of mutant forms of IRF3 unable to undergo dimerization or that were constitutively dimeric showed that both were targeted for degradation by NSP1. These results indicate that SA11-4F NSP1 can induce degradation of inactive and activated forms of IAD-containing IRF proteins (IRF3 to IRF9), allowing a multipronged attack on IFN-based pathways that promote antiviral innate and adaptive immune responses.

Group A human rotaviruses (HRVs) are the major cause of severe viral gastroenteritis in infants and young children. To gain insight into the level of genetic variation among HRVs, we determined the genome sequences for 10 strains belonging to different VP7 serotypes (G types). The HRVs chosen for this study, D, DS-1, P, ST3, IAL28, Se584, 69M, WI61, A64, and L26, were isolated from infected persons and adapted to cell culture to use as serotype references. Our sequencing results revealed that most of the individual proteins from each HRV belong to one of three genotypes (1, 2, or 3) based on their similarities to proteins of genogroup strains (Wa, DS-1, or AU-1, respectively). Strains D, P, ST3, IAL28, and WI61 encode genotype 1 (Wa-like) proteins, whereas strains DS-1 and 69M encode genotype 2 (DS-1-like) proteins. Of the 10 HRVs sequenced, 3 of them (Se584, A64, and L26) encode proteins belonging to more than one genotype, indicating that they are intergenogroup reassortants. We used amino acid sequence alignments to identify residues that distinguish proteins belonging to HRV genotype 1, 2, or 3. These genotype-specific changes cluster in definitive regions within each viral protein, many of which are sites of known protein-protein interactions. For the intermediate viral capsid protein (VP6), the changes map onto the atomic structure at the VP2-VP6, VP4-VP6, and VP7-VP6 interfaces. The results of this study provide evidence that group A HRV gene constellations exist and may be influenced by interactions among viral proteins during replication.

Serial undiluted passage of a porcine rotavirus in MA104 cells yielded three distinct virus populations, each of which bore different rearranged genes. Sequencing revealed that each of two populations bore a distinct intragenic recombinant NSP3 gene consisting of a partial duplication in a head-to-tail orientation without altering the NSP3 open reading frame and the third population carried both an intragenic recombinant NSP3 gene and an intergenic recombinant gene (1,647 nucleotides in length) which contained a truncated NSP2 gene inserted into the NSP5 gene at residue 332. The former two populations were viable, whereas the latter population was defective and interfering.

Secretion of interferon (IFN) by virus-infected cells is essential for activating autocrine and paracrine pathways that promote cellular transition to an antiviral state. In most mammalian cells, IFN production is initiated by the activation of constitutively expressed IFN regulatory factor 3, IRF3, which in turn leads to the induction of IRF7, the "master regulator" of IFN type I synthesis (alpha/beta IFN). Previous studies established that rotavirus NSP1 antagonizes IFN signaling by inducing IRF3 degradation. In the present study, we have determined that, in comparison to wild-type rotaviruses, rotaviruses encoding defective NSP1 grow to lower titers in some cell lines and that this poor growth phenotype is due to their failure to suppress IFN expression. Furthermore, we provide evidence that rotaviruses encoding wild-type NSP1 subvert IFN signaling by inducing the degradation of not only IRF3, but also IRF7, with both events occurring through proteasome-dependent processes that proceed with similar efficiencies. The capacity of NSP1 to induce IRF7 degradation may allow rotavirus to move across the gut barrier by enabling the virus to replicate in specialized trafficking cells (dendritic cells and macrophages) that constitutively express IRF7. Along with IRF3 and IRF7, NSP1 was found to induce the degradation of IRF5, a factor that upregulates IFN expression and that is involved in triggering apoptosis during viral infection. Our analysis suggests that NSP1 mediates the degradation of IRF3, IRF5, and IRF7 by recognizing a common element of IRF proteins, thereby allowing NSP1 to act as a broad-spectrum antagonist of IRF function.