Rotavirus Replication Research Articles

Investigation of Stilbenoids as Potential Therapeutic Agents for Rotavirus Gastroenteritis.

Rotavirus (RV) infections cause severe diarrhea in infants and young children worldwide. Vaccines are available but cost prohibitive for many countries and only reduce severe symptoms. Vaccinated infants continue to shed infectious particles, and studies show decreased efficacy of the RV vaccines in tropical and subtropical countries where they are needed most. Continuing surveillance for new RV strains, assessment of vaccine efficacy, and development of cost effective antiviral drugs remain an important aspect of RV studies. This study was to determine the efficacy of antioxidant and anti-inflammatory stilbenoids to inhibit RV replication. Peanut (A. hypogaea) hairy root cultures were induced to produce stilbenoids, which were purified by high performance countercurrent chromatography (HPCCC) and analyzed by HPLC. HT29.f8 cells were infected with RV in the presence stilbenoids. Cell viability counts showed no cytotoxic effects on HT29.f8 cells. Viral infectivity titers were calculated and comparatively assessed to determine the effects of stilbenoid treatments. Two stilbenoids, trans-arachidin-1 and trans-arachidin-3, show a significant decrease in RV infectivity titers. Western blot analyses performed on the infected cell lysates complemented the infectivity titrations and indicated a significant decrease in viral replication. These studies show the therapeutic potential of the stilbenoids against RV replication.

22 T-cell Receptor β (TCR β) Complementarity-Determining Region 3 (CDR3) Sequencing Provides a Distinct Profile Defining IBD Patients and Identifies Unique TCR β CDR3 Regions Significantly Associated With Crohn's Disease or Ulcerative Colitis

Background: Rotavirus (RV), the world's leading cause of severe viral gastroenteritis amongst children, primarily infects small intestinal epithelial cells (IECs) and is responsible for over 500,000 deaths and millions of physician visits/hospitalizations per year. While successful resolution of infection and protection against future infection is mediated by adaptive immunity, particularly mucosal IgA, relatively little is known how innate immunity contains acute infection and drives the adaptive immune response to this virus. In light of the broad role of MyD88 in signaling by TLRs and inflammasome cytokine receptors signaling, our goal was examine the consequence of absence of MyD88 in a mouse model of RV infection as means to mechanistically explore innate immunity to this virus. Methods: Suckling and adult mice (WT, MyD88KO, Caspase-1KO, and various bone-marrow irradiation chimeras) mice were inoculated with mouse-passaged RVEC strain. Infection was assessed by ELISA and qRT-PCR. RV replication was assessed by quantitating + to strand ratios. Diarrhea was scored visually. RV-specific immune responses were measured by fecal/serum ELISA. Results: Loss of MyD88 markedly impaired innate immunity to RV resulting in higher levels of virus in feces, intestinal lysates, and peripheral blood cells as well as increased RV replicative ability. Such increased RV levels correlated with a marked increase in the incidence and duration of diarrhea in neonatal mice. These results were fully phenocopied by loss of Caspase-1 and partially mimicked by loss of IL-1 and IL-18 suggesting a role for inflammasome signaling in innate control of RV infection. Such inability to control RV was not observed in irradiated WTmice reconstituted with MyD88KO or Caspase-1KO bone marrow nor could WT bone marrow restore innate immunity to RV in MyD88KO or Caspase-1KO mice suggesting a prominent role for inflammasome signaling in IEC in limiting RV infection/ pathogenesis. Loss of MyD88 also profoundly impaired adaptive immunity to RV. Specifically, MyD88KOmice produced less serum RV-specific IgG and IgA and have Th2 skewed antibody responses indicative of improper Th responses and isotype class switching. In contrast to the role of MyD88 in innate immunity, MyD88-mediated adaptive immunity was independent of Caspase-1, IL-1, and IL-18 and was mediated by MyD88 in bone marrow derived cells. Conclusion: Inflammasome signaling, in IECs, plays a major role in limiting RV infection and, consequently, the diarrheal disease that the great mortality/morbidity caused by this pathogen. TLR signaling in bone-marrow derived cells is important for driving the humoral immune responses that provide lasting protection against this pathogen.

21 MyD88 Is Important for Rotavirus Infection Containment and Specific Adaptive Responses

Background: Rotavirus (RV), the world's leading cause of severe viral gastroenteritis amongst children, primarily infects small intestinal epithelial cells (IECs) and is responsible for over 500,000 deaths and millions of physician visits/hospitalizations per year. While successful resolution of infection and protection against future infection is mediated by adaptive immunity, particularly mucosal IgA, relatively little is known how innate immunity contains acute infection and drives the adaptive immune response to this virus. In light of the broad role of MyD88 in signaling by TLRs and inflammasome cytokine receptors signaling, our goal was examine the consequence of absence of MyD88 in a mouse model of RV infection as means to mechanistically explore innate immunity to this virus. Methods: Suckling and adult mice (WT, MyD88KO, Caspase-1KO, and various bone-marrow irradiation chimeras) mice were inoculated with mouse-passaged RVEC strain. Infection was assessed by ELISA and qRT-PCR. RV replication was assessed by quantitating + to strand ratios. Diarrhea was scored visually. RV-specific immune responses were measured by fecal/serum ELISA. Results: Loss of MyD88 markedly impaired innate immunity to RV resulting in higher levels of virus in feces, intestinal lysates, and peripheral blood cells as well as increased RV replicative ability. Such increased RV levels correlated with a marked increase in the incidence and duration of diarrhea in neonatal mice. These results were fully phenocopied by loss of Caspase-1 and partially mimicked by loss of IL-1 and IL-18 suggesting a role for inflammasome signaling in innate control of RV infection. Such inability to control RV was not observed in irradiated WTmice reconstituted with MyD88KO or Caspase-1KO bone marrow nor could WT bone marrow restore innate immunity to RV in MyD88KO or Caspase-1KO mice suggesting a prominent role for inflammasome signaling in IEC in limiting RV infection/ pathogenesis. Loss of MyD88 also profoundly impaired adaptive immunity to RV. Specifically, MyD88KOmice produced less serum RV-specific IgG and IgA and have Th2 skewed antibody responses indicative of improper Th responses and isotype class switching. In contrast to the role of MyD88 in innate immunity, MyD88-mediated adaptive immunity was independent of Caspase-1, IL-1, and IL-18 and was mediated by MyD88 in bone marrow derived cells. Conclusion: Inflammasome signaling, in IECs, plays a major role in limiting RV infection and, consequently, the diarrheal disease that the great mortality/morbidity caused by this pathogen. TLR signaling in bone-marrow derived cells is important for driving the humoral immune responses that provide lasting protection against this pathogen.

Immune responses elicited against rotavirus middle layer protein VP6 inhibit viral replication in vitro and in vivo

Rotavirus (RV) is a common cause of severe gastroenteritis (GE) in children worldwide. Live oral RV vaccines protect against severe RVGE, but the immune correlates of protection are not yet clearly defined. Inner capsid VP6 protein is a highly conserved, abundant, and immunogenic RV protein, and VP6-specific mucosal antibodies, especially IgA, have been implicated to protect against viral challenge in mice. In the present study systemic and mucosal IgG and IgA responses were induced by immunizing BALB/c mice intranasally with a combination of recombinant RV VP6 protein (subgroup II [SGII]) and norovirus (NoV) virus-like particles (VLPs) used in a candidate vaccine. Following immunization mice were challenged orally with murine RV strain EDIMwt (SG non-I-non-II, G3P10[16]). In order to determine neutralizing activity of fecal samples, sera, and vaginal washes (VW) against human Wa RV (SGII, G1P1A[8]) and rhesus RV (SGI, G3P5B[3]), the RV antigen production was measured with an ELISA-based antigen reduction neutralization assay. Only VWs of immunized mice inhibited replication of both RVs, indicating heterotypic protection of induced antibodies. IgA antibody depletion and blocking experiments using recombinant VP6 confirmed that neutralization was mediated by anti-VP6 IgA antibodies. Most importantly, after the RV challenge significant reduction in viral shedding was observed in feces of immunized mice. These results suggest a significant role for mucosal RV VP6-specific IgA for the inhibition of RV replication in vitro and in vivo. In addition, these results underline the importance of non-serotype-specific immunity induced by the conserved subgroup-specific RV antigen VP6 in clearance of RV infection.

Rotavirus increases levels of lipidated LC3 supporting accumulation of infectious progeny virus without inducing autophagosome formation.

Replication of many RNA viruses benefits from subversion of the autophagic pathway through many different mechanisms. Rotavirus, the main etiologic agent of pediatric gastroenteritis worldwide, has been recently described to induce accumulation of autophagosomes as a mean for targeting viral proteins to the sites of viral replication. Here we show that the viral-induced increase of the lipidated form of LC3 does not correlate with an augmented formation of autophagosomes, as detected by immunofluorescence and electron microscopy. The LC3-II accumulation was found to be dependent on active rotavirus replication through the use of antigenically intact inactivated viral particles and of siRNAs targeting viral genes that are essential for viral replication. Silencing expression of LC3 or of Atg7, a protein involved in LC3 lipidation, resulted in a significant impairment of viral titers, indicating that these elements of the autophagic pathway are required at late stages of the viral cycle.

Immunogenicity and protective efficacy of yeast extracts containing rotavirus-like particles: A potential veterinary vaccine

Rotavirus is the most common cause of severe diarrhea in many animal species of economic interest. A simple, safe and cost-effective vaccine is required for the control and prevention of rotavirus in animals. In this study, we evaluated the use of Saccharomyces cerevisiae extracts containing rotavirus-like particles (RLP) as a vaccine candidate in an adult mice model. Two doses of 1mg of yeast extract containing rotavirus proteins (between 0.3 and 3μg) resulted in an immunological response capable of reducing the replication of rotavirus after infection. Viral shedding in all mice groups diminished in comparison with the control group when challenged with 100 50% diarrhea doses (DD50) of murine rotavirus strain EDIM. Interestingly, when immunizing intranasally protection against rotavirus infection was observed even when no increase in rotavirus-specific antibody titers was evident, suggesting that cellular responses were responsible of protection. Our results indicate that raw yeast extracts containing rotavirus proteins and RLP are a simple, cost-effective alternative for veterinary vaccines against rotavirus.

Antibiotic Treatment Suppresses Rotavirus Infection and Enhances Specific Humoral Immunity

Rotavirus causes 500 000 deaths and millions of physician visits and hospitalizations per year, with worse outcomes and reduced vaccine efficacy in developing countries. We hypothesized that the gut microbiota might modulate rotavirus infection and/or antibody response and thus potentially play a role in such regional differences. The microbiota was ablated via germ-free or antibiotic approaches. Enhanced exposure to microbiota was achieved via low-dose dextran sodium sulfate (DSS) treatment. Rotavirus infection and replication was assessed by enzyme-linked immunosorbent assay (ELISA) and quantitative reverse-transcription polymerase chain reaction. Diarrhea was scored visually. Humoral responses to rotavirus were measured by ELISA and enzyme-linked immunosorbent spot assay. Microbiota elimination delayed infection and reduced infectivity by 42%. Antibiotics did not alter ratios of positive-sense to negative-sense strands, suggesting that entry rather than replication was influenced. Antibiotics reduced the diarrhea incidence and duration, indicating that the reduction in the level of rotavirus antigen was biologically significant. Despite lowered antigen level, antibiotics resulted in a more durable rotavirus mucosal/systemic humoral response. Increased rotavirus antibody response durability correlated with increased small intestinal rotavirus-specific, immunoglobulin A-producing antibody-secreting cell concentration in antibiotic-treated mice. Conversely, DSS treatment impaired generation of rotavirus-specific antibodies. Microbiota ablation resulted in reduced rotavirus infection/diarrhea and a more durable rotavirus antibody response, suggesting that antibiotic administration before rotavirus vaccination could raise low seroconversion rates that correlate with the vaccine's inefficacy in developing regions.

Rotaviruses

Rotavirus, a member of the family Reoviridae, was identified as the leading etiological agent of severe gastroenteritis in infants and young children in 1973. The rotavirus genome is composed of 11 gene segments of double-stranded (ds)RNA. During the last 40 years, a large amount of basic research on rotavirus structure, genome, antigen, replication, pathogenesis, epidemiology, immune responses, and evolution has been accumulated. This article reviews the fundamental aspects of rotavirology including recent important achievements in research.

Erratum: Structural insights into the coupling of virion assembly and rotavirus replication

Erratum: Structural insights into the coupling of virion assembly and rotavirus replication

Inhibition of rotavirus replication by downregulation of fatty acid synthesis

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Sequestration Strikes Again: Rotavirus-Induced Accumulation of Cellular Transcripts in the Nucleus Inhibits Host Protein Translation

Evaluation of: Rubio RM, Mora SI, Romero P, Arias CF, López S. Rotavirus prevents the expression of host responses by blocking the nucleocytoplasmic transport of polyadenylated mRNAs. J. Virol. 87(11), 6336–6345 (2013). Rotaviruses are a leading cause of pediatric diarrhea worldwide and are estimated to be responsible for approximately 450,000 deaths each year. Although the incidence of severe diarrhea induced by rotavirus has fallen sharply in developed countries due to the introduction of vaccines, it remains a leading cause of diarrhea leading to death in low-income countries. A key feature of rotavirus replication is the robust production of viral proteins and simultaneous inhibition of host protein synthesis in infected cells. Studies have shown an accumulation of cytoplasmic poly(A)-binding protein (PABPC) in the nucleus of rotavirus-infected cells, but the consequences of this were unknown. Here, Rubio and colleagues show that the nuclear accumulation of PABPC inhibits the export of polyadenylated host mRNAs to the cytoplasm, thereby preventing their translation. The viral nonstructural protein NSP3 is responsible for the observed increase of PABPC and poly(A)-containing mRNAs in the nucleus of infected cells. If introduced directly into the cytoplasm of infected cells, polyadenylated mRNAs are efficiently translated, suggesting there is not a preferential translation of viral mRNAs over cellular mRNAs once they are in the cytoplasm. The results presented demonstrate that the prevention of polyadenylated mRNA export from the nucleus of infected cells is an important strategy to inhibit host translation, and probably works in concert with other viral strategies to prevent the translation of host proteins.

Cyclosporin A Inhibits Rotavirus Replication and Restores Interferon-Beta Signaling Pathway In Vitro and In Vivo

Rotavirus (RV) is the most common cause of severe diarrhea among infants and young children. Currently, there is no specific drug available against rotavirus, largely due to the lack of an ideal target molecule which has hampered drug development. Our previous studies have revealed that cyclosporin A (CsA) might be potentially useful as an anti-RV drug. We therefore used both cellular and mouse models to study the immunological safety and effectiveness of CsA as an anti-RV drug. We found that CsA treatment of HT-29 cells before, during, and after viral infection efficiently inhibited Wa strain RV replication and restored IFN-β expression in a HT-29 cell line model. Exploring the underlying mechanisms showed that CsA promoted Interferon Regulatory Factor-5 (IRF-5) expression (a key positive regulator of the type I IFN signaling pathway), but not IRF-1, IRF-3, or IRF-7. Additionally, CsA inhibited SOCS-1 expression (the key negative regulator of IFN-α/β), but not SOCS-2 or SOCS-3. The antiviral effect of CsA was confirmed in an RV-infected neonatal mouse model by evaluation of antigen clearance and assessment of changes in intestinal tissue pathology. Also, no differences in T cell frequency or proliferation between the CsA- and vehicle-treated groups were observed. Thus, both our in vitro and in vivo findings suggest that CsA, through modulating the expression of key regulators in IFN signaling pathway, promote type I IFN-based intracellular innate immunity in RV host cells. These findings suggest that CsA may be a useful candidate to develop a new anti-RV strategy, although further evaluation and characterization of CsA on RV-induced diarrhea are warranted.

232: Inhibition of IFN-mediated STAT1 activation in intestinal epithelial cells by NSP1 – The rotavirus-encoded inhibitor of IFN induction

Rotavirus (RV) replication in intestinal epithelial cells (IECs) in vivo is robust despite the vigorous activation of local early host interferon (IFN) responses. Using single cell microfluidics qRT-PCR coupled with flow sorting, we recently demonstrated that homologous RV efficiently inhibits IFN induction in single infected and bystander villous IECs in vivo and, using genetic approaches, associated this function with the RV NSP1 protein. Paradoxically, RV also induces robust type I IFNs in the intestinal hemaotopoietic compartment in a relatively replication-independent manner raising the possibility that RV replication and spread in IECs occurs despite exogenous stimulation of the STAT1-mediated IFN signaling pathway. However, viral factor(s) responsible for the possible inhibition of STAT1 function are unknown. Here we report the ability of RV to mediate direct and remote inhibition of IFN-mediated STAT1 activation in IECs and identify the RV NSP1 protein as a direct inhibitor of the pathway. Infection of human IECs with RV strains that inhibit IFN induction by two distinct strategies targeting either NF-kB or IRF3 both resulted in effective regulation of IFN secretion. At early times during infection, neither type of RV strain could effectively inhibit the activation of STAT1 in response to exogenously added IFN. However, at later times in infection, both types of RV could inhibit IFN-mediated STAT1 activation, and by single cell analysis this blockage occurred in both infected and bystander cell populations. We found that the rotavirus NSP1 protein, when expressed by itself, is an efficient inhibitor of STAT1 activation. Analysis of NSP1 determinants in the inhibition of IFN induction and signaling pathways revealed that these properties are encoded on discrete domains. These findings explain previously observed remote regulation of the host IFN response in the intestine and uncover a novel function of NSP1 as a STAT1 antagonist.

Thiazolides, a New Class of Antiviral Agents Effective against Rotavirus Infection, Target Viral Morphogenesis, Inhibiting Viroplasm Formation

Rotaviruses, nonenveloped viruses presenting a distinctive triple-layered particle architecture enclosing a segmented double-stranded RNA genome, exhibit a unique morphogenetic pathway requiring the formation of cytoplasmic inclusion bodies called viroplasms in a process involving the nonstructural viral proteins NSP5 and NSP2. In these structures the concerted packaging and replication of the 11 positive-polarity single-stranded RNAs take place to generate the viral double-stranded RNA (dsRNA) genomic segments. Rotavirus infection is a leading cause of gastroenteritis-associated severe morbidity and mortality in young children, but no effective antiviral therapy exists. Herein we investigate the antirotaviral activity of the thiazolide anti-infective nitazoxanide and reveal a novel mechanism by which thiazolides act against rotaviruses. Nitazoxanide and its active circulating metabolite, tizoxanide, inhibit simian A/SA11-G3P[2] and human Wa-G1P[8] rotavirus replication in different types of cells with 50% effective concentrations (EC50s) ranging from 0.3 to 2 μg/ml and 50% cytotoxic concentrations (CC50s) higher than 50 μg/ml. Thiazolides do not affect virus infectivity, binding, or entry into target cells and do not cause a general inhibition of viral protein expression, whereas they reduce the size and alter the architecture of viroplasms, decreasing rotavirus dsRNA formation. As revealed by protein/protein interaction analysis, confocal immunofluorescence microscopy, and viroplasm-like structure formation analysis, thiazolides act by hindering the interaction between the nonstructural proteins NSP5 and NSP2. Altogether the results indicate that thiazolides inhibit rotavirus replication by interfering with viral morphogenesis and may represent a novel class of antiviral drugs effective against rotavirus gastroenteritis.

Characterization of Rotavirus RNAs That Activate Innate Immune Signaling through the RIG-I-Like Receptors

In mammalian cells, the first line of defense against viral pathogens is the innate immune response, which is characterized by induction of type I interferons (IFN) and other pro-inflammatory cytokines that establish an antiviral milieu both in infected cells and in neighboring uninfected cells. Rotavirus, a double-stranded RNA virus of the Reoviridae family, is the primary etiological agent of severe diarrhea in young children worldwide. Previous studies demonstrated that rotavirus replication induces a MAVS-dependent type I IFN response that involves both RIG-I and MDA5, two cytoplasmic viral RNA sensors. This study reports the isolation and characterization of rotavirus RNAs that activate IFN signaling. Using an in vitro approach with purified rotavirus double-layer particles, nascent single-stranded RNA (ssRNA) transcripts (termed in vitro ssRNA) were found to be potent IFN inducers. In addition, large RNAs isolated from rotavirus-infected cells six hours post-infection (termed in vivo 6 hr large RNAs), also activated IFN signaling, whereas a comparable large RNA fraction isolated from cells infected for only one hour lacked this stimulatory activity. Experiments using knockout murine embryonic fibroblasts showed that RIG-I is required for and MDA5 partly contributes to innate immune signaling by both in vitro ssRNA and in vivo 6 hr large RNAs. Enzymatic studies demonstrated that in vitro ssRNA and in vivo 6 hr large RNA samples contain uncapped RNAs with exposed 5’ phosphate groups. RNAs lacking 2’-O-methylated 5’ cap structures were also detected in the in vivo 6 hr large RNA sample. Taken together, our data provide strong evidence that the rotavirus VP3 enzyme, which encodes both guanylyltransferase and methyltransferase activities, is not completely efficient at either 5’ capping or 2’-O-methylation of the 5’ cap structures of viral transcripts, and in this way produces RNA patterns that activate innate immune signaling through the RIG-I-like receptors.

Rotavirus Replication in the Cholangiocyte Mediates the Temporal Dependence of Murine Biliary Atresia

Biliary atresia (BA) is a neonatal disease that results in obliteration of the biliary tree. The murine model of BA, which mirrors the human disease, is based upon infection of newborn mice with rhesus rotavirus (RRV), leading to an obstructive cholangiopathy. The purpose of this study was to characterize the temporal relationship between viral infection and the induction of this model. BALB/c mice were infected with RRV on day of life (DOL) 0, 3, 5, and 7. Groups were characterized as early-infection (infection by DOL 3) or late-infection (infection after DOL 5). Early RRV infection induced symptoms in 95% of pups with a mortality rate of 80%. In contrast, late infection caused symptoms in only 50% of mice, and 100% of pups survived. The clinical findings correlated with histological analysis of extrahepatic biliary trees, cytokine expression, and viral titers. Primary murine cholangiocytes isolated, cultured, and infected with RRV yielded higher titers of infectious virus in those harvested from DOL 2 versus DOL 9 mice. Less interferon alpha and beta was produced in DOL 2 versus DOL 9 RRV infected primary cholangiocytes. Injection of BALB/c interferon alpha/beta receptor knockout (IFN-αβR−/−) pups at DOL 7 showed increased symptoms (79%) and mortality (46%) when compared to late infected wild type mice. In conclusion, the degree of injury sustained by relatively immature cholangiocytes due to more robust RRV replication correlated with more severe clinical manifestations of cholangiopathy and higher mortality. Interferon alpha production by cholangiocytes appears to play a regulatory role. These findings confirm a temporal dependence of RRV infection in murine BA and begin to define a pathophysiologic role of the maturing cholangiocyte.

The Role of Cellular Lipid Droplets in Rotavirus Replication

Viroplasms, cytoplasmic inclusion bodies in rotavirus (RV)-infected cells in which viral RNA replication and early morphogenesis take place, were found to be associated with the cellular organelles lipid droplets (LDs). Compounds affecting LD homoeostasis, including agents causing lipolysis and others that inhibit fatty acid biosynthesis, decrease RV replication. Gradient ultracentrifugation of infected cell extracts shows LD components cosedimenting with viroplasms in low-density fractions. Disturbance of fatty acid biosynthesis decreases the production of both double-layered and triple-layered (infectious) RV particles. Future studies should explore the LD components important for RV replication, and the potential of chemical compounds interfering with lipid metabolism for treatment of RV disease.

Innate cellular responses to rotavirus infection

Rotavirus is a leading cause of severe dehydrating diarrhoea in infants and young children. Following rotavirus infection in the intestine an innate immune response is rapidly triggered. This response leads to the induction of type I and type III interferons (IFNs) and other cytokines, resulting in a reduction in viral replication. Here we review the current literature describing the detection of rotavirus infection by pattern recognition receptors within host cells, the subsequent molecular mechanisms leading to IFN and cytokine production, and the processes leading to reduced rotavirus replication and the development of protective immunity. Rotavirus countermeasures against innate responses, and their roles in modulating rotavirus replication in mice, also are discussed. By linking these different aspects of innate immunity, we provide a comprehensive overview of the host's first line of defence against rotavirus infection. Understanding these processes is expected to be of benefit in improving strategies to combat rotavirus disease.

The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells

A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.

The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells.

A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.