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Human rotavirus (HRV) is a leading cause of viral gastroenteritis in children across the globe. The virus has long been established as a pathogen of the gastrointestinal tract, targeting small intestine epithelial cells and leading to diarrhea, nausea, and vomiting. Recently, this classical infection pathway was challenged by the findings that murine strains of rotavirus can infect the salivary glands of pups and dams and transmit via saliva from pups to dams during suckling. Here, we aimed to determine if HRV was also capable of infecting salivary glands and spreading in saliva using a gnotobiotic (Gn) pig model of HRV infection and disease. Gn pigs were orally inoculated with various strains of HRV, and virus shedding was monitored for several days post-inoculation. HRV was shed nasally and in feces in all inoculated pigs. Infectious HRV was detected in the saliva of four piglets. Structural and non-structural HRV proteins, as well as the HRV genome, were detected in the intestinal and facial tissues of inoculated pigs. The pigs developed high IgM antibody responses in serum and small intestinal contents at 10 days post-inoculation. Additionally, inoculated pigs had HRV-specific IgM antibody-secreting cells present in the ileum, tonsils, and facial lymphoid tissues. Taken together, these findings indicate that HRV can replicate in salivary tissues and prime immune responses in both intestinal and facial lymphoid tissues of Gn pigs.

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.

Derivatives of the rotavirus SA11-H96 strain, isolated in 1958 from an overtly healthy vervet monkey, have been used extensively to probe the viral life cycle. To gain insight into the phenotypic and genotypic differences among SA11 isolates, we sequenced the segmented double-stranded RNA genomes of SA11-H96 (P5B[2]:G3), two SA11-4F-like viruses (P6[1]:G3), two SA11-4F-like viruses with gene 5 rearrangements, and relevant segments of SA11 temperature-sensitive mutants and the "O" (Offal) agent (P6[1]:G8), a rotavirus isolated in 1965 from abattoir waste. This analysis indicates that the only complete genomic sequence previously reported for SA11 (Both) is instead that of a reassortant, originating like the SA11-4F-like viruses, from the introduction of an "O" agent gene into the SA11 genetic background. These results, combined with identification of mutations that correlate with altered growth properties and ts phenotype, emphasize the importance of considering segment origin and sequence variation in interpreting experimental outcomes with SA11 strains.

Rotaviruses are the major cause of acute gastroenteritis in infants world-wide. The genome consists of eleven double stranded RNA segments. The major segment encodes the structural protein VP1, the viral RNA-dependent RNA polymerase (RdRp), which is a minor component of the viral inner core. This study is a detailed bioinformatic assessment of the VP1 sequence. Using various methods we have identified canonical motifs within the VP1 sequence which correspond to motifs previously identified within RdRps of other positive strand, double-strand RNA viruses. The study also predicts an overall structural conservation in the middle region that may correspond to the palm subdomain and part of the fingers and thumb subdomains, which comprise the polymerase core of the protein. Based on this analysis, we suggest that the rotavirus replicase has the minimal elements to function as an RNA-dependent RNA polymerase. VP1, besides having common RdRp features, also contains large unique regions that might be responsible for characteristic features observed in the Reoviridae family.

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.

Rotavirus genome synthesis is a two-step process; the synthesis of the eleven viral mRNAs and the production of the complementary strand (minus strand synthesis) yielding the double stranded genomic RNA (dsRNA). Purified rotavirus open cores are a helpful tool to study this replication event. These open cores can use viral dsRNA and mRNAs as template for RNA synthesis. In the present communication it was demonstrated that open cores prefers to use viral mRNA as template instead of the genomic dsRNA. The use by the open cores of both templates can be blocked through two independent strategies. In the presence of ssRNAs molecules, the use of the viral dsRNA template is completely blocked without affecting the minus strand synthesis. Nevertheless, an antisense oligonucleotide strategy previously reported to block the minus strand synthesis of the target gene do not affect the use of the dsRNA template. These results suggest the recognition of different signals in each template by the open core protein complex.

The eleven rotavirus mRNAs contain 5'-cap structures and most end with the 3'-consensus sequence 5'-UGACC-3'. The UGACC functions as a common translation enhancer (3'-TE-con) that upregulates viral protein expression through a process mediated by the nonstructural protein NSP3. To address the possibility that gene-specific enhancers are also contained in the untranslated regions (UTRs) of the rotavirus mRNAs, we used rabbit reticulocyte lysates to investigate the translation efficiencies of analog RNAs containing viral-specific 5'-and 3'-UTRs and the open reading frame for chloramphenicol acetyltransferase. These experiments combined with the analysis of full-length viral RNAs and RNAs containing 3'-truncations showed that a highly active enhancer was present near the 5'-end of the 139-nucleotide 3'-UTR of the gene 6 mRNA (3'-TEg6). The 3'-TEg6 represents a functionally independent enhancer, as no other portion of the gene 6 mRNA was required for its activity. The 3'-TEg6 differs significantly from the 3'-TE-con in that the gene 6-specific enhancer does not require viral protein for activity and is formed by a sequence unique to only one of the eleven viral mRNAs. Together, our findings suggest that the 3'-UTR of the gene 6 mRNA contains two TEs, one is gene-specific (3'-TEg6) and the other is common to nearly all rotavirus genes (3'-TE-con). The activity of the 3'-TEg6 is likely important for directing the efficient translation of the gene 6 mRNA at levels sufficient to provide the 780 copies of VP6 necessary for the assembly of each progeny virion.

Elucidation of the function of the non-structural rotavirus proteins during infection is difficult in the absence of a reverse genetic system. To study the role of NSP5, nonstructural phosphoprotein NSP5, we constructed a reassortant strain (SACC11) in the SA11 background that harbours a heterologous segment 11 encoding a variant protein (h-NSP5). Cells infected by SACC11 produced viral polypeptides at earlier times than SA11 infected cells while showing less accumulation of genomic dsRNA. These changes suggested that NSP5 might direct viral messenger RNA to protein synthesis or genome replication. Distinct patterns of proteins were shown to form complexes with NSP5 in co-immunoprecipitation studies with SA11 and SACC11 infected cells. Recombinant h-NSP5 from either bacteria or eucaryotic cells migrated faster in PAGE suggesting that it was hypophosphorylated. Indeed, the kinase inhibitor H-7 enhanced translation of viral proteins in SA11 but not SACC11 infected cells suggesting that NSP5 function in the regulation of the fate of viral positive strand RNA is mediated by phosphorylation.

The core of the rotavirion consists of three proteins, including the viral RNA polymerase, and 11 segments of double-stranded (ds)RNA. The RNA polymerase of disrupted (open) cores is able to catalyze the synthesis of dsRNA from exogenous viral mRNAs in vitro. In this study, we have identified sequences in exogenous viral mRNAs important for RNA replication using antisense oligonucleotides. The results showed that oligonucleotides complementary to the highly conserved 3'-terminal sequence of rotavirus mRNAs prevented all but basal levels of dsRNA synthesis. Notably, we observed that the addition of oligonucleotides which were complementary to nonconserved sequences present either at the 5'- or 3'-end of a viral mRNA effectively inhibited its replication without interfering with the replication of other viral mRNAs present in the same replication assay. Thus, the nonconserved sequences in rotavirus mRNAs contain gene-specific information that promotes RNA replication. The fact that antisense oligonucleotides inhibited dsRNA synthesis indicates that the strandedness (single- versus double-stranded) and secondary structure of the viral mRNA template are factors that affect the efficiency of minus strand synthesis., (Copyright 2001 Academic Press.)