NF-κB plays a critical role in the induction and maintenance of innate and adaptive immune transcriptional programs. An associated inhibitor of κB protein (IκB) regulates NF-κB activation and contains a degron motif (DSGΦxS) that undergoes phosphorylation following pathogen recognition or other proinflammatory signals. The E3 ubiquitin ligase SCFβ-TrCP recognizes this phosphodegron through its β-transducin repeat-containing protein (β-TrCP) subunit and induces IκB degradation, allowing NF-κB to translocate to the nucleus and modulate gene expression. Rotavirus (RV), a major cause of pediatric gastroenteritis, can block NF-κB activation through the action of its nonstructural protein NSP1, a putative E3 ubiquitin ligase that mediates the degradation of β-TrCP or other immunomodulatory proteins in a virus strain-specific manner. Here, we show that NSP1 targets β-TrCP by mimicking the IκB phosphodegron. The NSP1 proteins of most human and porcine RV strains conserve a C-terminal phosphodegron-like (PDL) motif, DSGΦS. Deletion of this motif or mutation of its serine residues disrupts NSP1-mediated degradation of β-TrCP and inhibition of NF-κB activation. Additionally, a point mutation within the phosphodegron-binding pocket protects β-TrCP from NSP1-mediated turnover. Fusion of the PDL motif to an NSP1 protein known to target other immunomodulatory proteins generates a chimeric NSP1 protein that can induce β-TrCP degradation and block NF-κB activation. Other viral proteins (Epstein-Barr virus LMP1, HIV-1 Vpu, and vaccinia virus A49) also contain a PDL motif and interact with β-TrCP to inhibit NF-κB activation. Taken together, these data suggest that targeting β-TrCP by molecular mimicry may be a common strategy used by human viruses to evade the host immune response.IMPORTANCE   The transcription factor NF-κB, a central regulator of the host response to infection, is a frequent target of viral antagonism. Pathogen detection activates NF-κB by inducing the phosphorylation of an associated inhibitor protein (IκB), which targets IκB for degradation by the E3 ubiquitin ligase β-TrCP. Rotavirus, a significant cause of childhood gastroenteritis, antagonizes NF-κB through the activity of its NSP1 protein, a putative E3 ubiquitin ligase that mediates β-TrCP turnover. Here, we show that NSP1 functions by mimicking the IκB phosphodegron recognized by β-TrCP. Nearly all human rotavirus strains conserve this motif at the NSP1 C terminus, and its removal disrupts NSP1 antagonist activity. This sequence conserves the biochemical properties of the IκB phosphodegron and can rescue antagonist activity when fused to an NSP1 protein otherwise inactive against β-TrCP. Other viral proteins also mimic IκB to disrupt NF-κB activation, indicating that this is an important immune evasion strategy.

Rotaviruses and orbiviruses are nonturreted Reoviridae members. The rotavirus VP3 protein is a multifunctional capping enzyme and antagonist of the interferon-induced cellular oligoadenylate synthetase-RNase L pathway. Despite mediating important processes, VP3 is the sole protein component of the rotavirus virion whose structure remains unknown. In the current study, we used sequence alignment and homology modeling to identify features common to nonturreted Reoviridae capping enzymes and to predict the domain organization, structure, and active sites of rotavirus VP3. Our results suggest that orbivirus and rotavirus capping enzymes share a domain arrangement similar to that of the bluetongue virus capping enzyme. Sequence alignments revealed conserved motifs and suggested that rotavirus and orbivirus capping enzymes contain a variable N-terminal domain, a central guanine-N7-methyltransferase domain that contains an additional inserted domain, and a C-terminal guanylyltransferase and RNA 5=-triphosphatase domain. Sequence conservation and homology modeling suggested that the insertion in the guanine-N7-methyltransferase domain is a ribose-2=-O-methyltransferase domain for most rotavirus species. Our analyses permitted putative identification of rotavirus VP3 active-site residues, including those that form the ribose-2=-O-methyltransferase catalytic tetrad, interact with S-adenosyl-L-methionine, and contribute to autoguanylation. Previous reports have indicated that group A rotavirus VP3 contains a C-terminal 2H-phosphodiesterase domain that can cleave 2=-5= oligoadenylates, thereby preventing RNase L activation. Our results suggest that a C-terminal phosphodiesterase domain is present in the capping enzymes from two additional rotavirus species. Together, these findings provide insight into a poorly understood area of rotavirus biology and are a springboard for future biochemical and structural studies of VP3. IMPORTANCERotaviruses are an important cause of severe diarrheal disease. The rotavirus VP3 protein caps viral mRNAs and helps combat cellular innate antiviral defenses, but little is known about its structure or enzymatic mechanisms. In this study, we used sequence-and structure-based alignments with related proteins to predict the structure of VP3 and identify enzymatic domains and active sites therein. This work provides insight into the mechanisms of rotavirus transcription and evasion of host innate immune defenses. An improved understanding of these processes may aid our ability to develop rotavirus vaccines and therapeutics.

Rotaviruses (RVs) are 11-segmented, double-stranded RNA viruses that cause severe gastroenteritis in children. In addition to an error-prone genome replication mechanism, RVs can increase their genetic diversity by reassorting genes during host coinfection. Such exchanges allow RVs to acquire advantageous genes and adapt in the face of selective pressures. However, reassortment may also impose fitness costs if it unlinks genes/proteins that have accumulated compensatory, coadaptive mutations and that operate best when kept together. To better understand human RV evolutionary dynamics, we analyzed the genome sequences of 135 strains (genotype G1/G3/G4-P[8]-I1-C1-R1-A1-N1-T1-E1-H1) that were collected at a single location in Washington, DC, during the years 1974 to 1991. Intragenotypic phylogenetic trees were constructed for each viral gene using the nucleotide sequences, thereby defining novel allele level gene constellations (GCs) and illuminating putative reassortment events. The results showed that RVs with distinct GCs cocirculated during the vast majority of the collection years and that some of these GCs persisted in the community unchanged by reassortment. To investigate the influence of protein coadaptation on GC maintenance, we performed a mutual information-based analysis of the concatenated amino acid sequences and identified an extensive covariance network. Unexpectedly, amino acid covariation was highest between VP4 and VP2, which are structural components of the RV virion that are not thought to directly interact. These results suggest that GCs may be influenced by the selective constraints placed on functionally coadapted, albeit noninteracting, viral proteins. This work raises important questions about mutation-reassortment interplay and its impact on human RV evolution. IMPORTANCERotaviruses are devastating human pathogens that cause severe diarrhea and kill >450,000 children each year. The virus can evolve by accumulating mutations and by acquiring new genes from other strains via a process called reassortment. However, little is known about the relationship between mutation accumulation and gene reassortment for rotaviruses and how it impacts viral evolution. In this study, we analyzed the genome sequences of human strains found in clinical fecal specimens that were collected at a single hospital over an 18-year time span. We found that many rotaviruses did not reassort their genes but instead maintained them as specific sets (i.e., constellations). By analyzing the encoded proteins, we discovered concurrent amino acid changes among them, which suggests that they are functionally coadapted to operate best when kept together. This study increases our understanding of how rotaviruses evolve over time in the human population.