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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.

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.

IFN regulatory factor 3 (IRF3), a constitutively expressed protein localizing largely to the cytoplasm, is a primary effector of the innate immune response. Infection can trigger the phosphorylation, dimerization, and nuclear translocation of IRF3, where the factor stimulates the expression and release of IFN. In this study, we determined that the rotavirus gene 5 product, nonstructural protein 1 (NSP1), interacts with IRF3 in the infected cell. To understand the importance of the interaction, we compared IRF3 activation by rotaviruses expressing wild-type and C-truncated forms of NSP1. The analysis showed that IRF3 underwent dimerization and nuclear translocation and stimulated IFN promoter activity in infected cells expressing truncated NSP1. In contrast, infected cells expressing wild-type NSP1 were characterized by the rapid degradation of IRF3 during the replication cycle, severe decreases in IRF3 dimerization and nuclear translocation, and lack of IFN promoter activity. The implication of these results, that wild-type NSP1 is an antagonist of the IFN-signaling pathway, was confirmed in transient expression assays, which showed that wild-type NSP1, but not the C-truncated protein, induced the degradation of IRF3 fusion proteins. Related experiments indicated that NSP1 mediates IRF3 degradation through a proteasome-dependent pathway. The critical role of NSP1 in promoting cell-to-cell spread of rotavirus was demonstrated by using gene 5-specific short interfering RNAs in plaque assays. Although several viruses have been described that subvert the innate immune response by preventing IRF3 activation, rotavirus is identified as one that accomplishes this task by inducing the degradation of IRF3.

Rotavirus, a cause of severe gastroenteritis, contains a segmented double-stranded (ds)RNA genome that replicates using viral mRNAs as templates. The highly conserved 3'-consensus sequence (3'CS), UGUGACC, of the mRNAs promotes dsRNA synthesis and enhances translation. We have found that the 3'CS of the gene (g5) encoding NSP1, an antagonist of interferon signaling, undergoes rapid mutation when rhesus rotavirus (RRV) is serially passaged at high multiplicity of infection (MOI) in cells permitting high titer growth. These mutations increase the promoter activity of the g5 3'-sequence, but decrease its activity as a translation enhancer. The location of the mutations defines the minimal essential promoter for dsRNA synthesis as URN0-5CC. Under passage conditions where cell-to-cell spread of the virus is required to complete infection (low MOI), the 3'CS is retained due to the need for NSP1 to be expressed at levels sufficient to prevent establishment of the antiviral state. These data demonstrate that host cell type and propagation conditions affect the capacity of RRV to produce the virulence gene product NSP1, an important consideration in producing RRV-based vaccines.