The predicted stem-loop structure in the 3'-end of the human norovirus antigenomic sequence is required for its genomic RNA synthesis by its RdRp.

The norovirus genome consists of a single positive-stranded RNA. The mechanism by which this single-stranded RNA genome is replicated is not well understood. To reveal the mechanism underlying the initiation of the norovirus genomic RNA synthesis by its RNA-dependent RNA polymerase (RdRp), we used an in vitro assay to detect the complementary RNA synthesis activity. Results showed that the purified recombinant RdRp was able to synthesize the complementary positive-sense RNA from a 100-nt template corresponding to the 3'-end of the viral antisense genome sequence, but that the RdRp could not synthesize the antisense genomic RNA from the template corresponding to the 5'-end of the positive-sense genome sequence. We also predicted that the 31 nt region at the 3'-end of the RNA antisense template forms a stem-loop structure. Deletion of this sequence resulted in the loss of complementary RNA synthesis by the RdRp, and connection of the 31 nt to the 3'-end of the inactive positive-sense RNA template resulted in the gain of complementary RNA synthesis by the RdRp. Similarly, an electrophoretic mobility shift assay further revealed that the RdRp bound to the antisense RNA specifically, but was dependent on the 31 nt at the 3'-end. Therefore, based on this observation and further deletion and mutation analyses, we concluded that the predicted stem-loop structure in the 31 nt end and the region close to the antisense viral genomic stem sequences are both important for initiating the positive-sense human norovirus genomic RNA synthesis by its RdRp.

Human sapovirus propagation in human cell lines supplemented with bile acids.

Human sapoviruses (HuSaVs) cause acute gastroenteritis similar to human noroviruses. Although HuSaVs were discovered four decades ago, no HuSaV has been grown in vitro, which has significantly impeded the understanding of viral biology and the development of antiviral strategies. In this study, we identified two susceptible human cell lines, that originated from testis and duodenum, that support HuSaV replication and found that replication requires bile acids. HuSaVs replicated more efficiently in the duodenum cell line, and viral RNA levels increased up to ∼6 log10-fold. We also detected double-stranded RNA, viral nonstructural and structural proteins in the cell cultures, and intact HuSaV particles. We confirmed the infectivity of progeny viruses released into the cell culture supernatants by passaging. These results indicate the successful growth of HuSaVs in vitro. Additionally, we determined the minimum infectious dose and tested the sensitivities of HuSaV GI.1 and GII.3 to heat and ultraviolet treatments. This system is inexpensive, scalable, and reproducible in different laboratories, and can be used to investigate mechanisms of HuSaV replication and to evaluate antivirals and/or disinfection methods for HuSaVs.

Activation of protein kinase R by hepatitis C virus RNA-dependent RNA polymerase.

Hepatitis C virus (HCV) was shown to activate protein kinase R (PKR), which inhibits expression of interferon (IFN) and IFN-stimulated genes by controlling the translation of newly transcribed mRNAs. However, it is unknown exactly how HCV activates PKR. To address the molecular mechanism(s) of PKR activation mediated by HCV infection, we examined the effects of viral proteins on PKR activation. Here, we show that expression of HCV NS5B strongly induced PKR and eIF2α phosphorylation, and attenuated MHC class I expression. In contrast, expression of Japanese encephalitis virus RNA-dependent RNA polymerase did not induce phosphorylation of PKR. Co-immunoprecipitation analyses showed that HCV NS5B interacted with PKR. Furthermore, expression of NS5B with polymerase activity-deficient mutation failed to phosphorylate PKR, suggesting that RNA polymerase activity is required for PKR activation. These results suggest that HCV activates PKR by association with NS5B, resulting in translational suppression of MHC class I to establish chronic infection.

Evaluation of the use of various rat strains for immunogenic potency tests of Sabin-derived inactivated polio vaccines.

Slc:Wistar rats have been the only strain used in Japan for purpose of evaluating a national reference vaccine for the Sabin-derived inactivated polio vaccine (sIPV) and the immunogenicity of sIPV-containing products. However, following the discovery that the Slc:Wistar strain was genetically related to the Fischer 344 strain, other "real" Wistar strains, such as Crlj:WI, that are available worldwide were tested in terms of their usefulness in evaluating the immunogenicity of the past and current lots of a national reference vaccine. The response of the Crlj:WI rats against the serotype 1 of sIPV was comparable to that of the Slc:Wistar rats, while the Crlj:WI rats exhibited a higher level of response against the serotypes 2 and 3. The immunogenic potency units of a national reference vaccine determined using the Slc:Wistar rats were reproduced on tests using the Crlj:WI rats. These results indicate that a titer of the neutralizing antibody obtained in response to a given dose of sIPV cannot be directly compared between these two rat strains, but that, more importantly, the potency units are almost equivalent for the two rat strains.

First complete genome sequences of genogroup V, genotype 3 porcine sapoviruses: common 5'-terminal genomic feature of sapoviruses.

Sapoviruses (SaVs) are enteric viruses and have been detected in various mammals. They are divided into multiple genogroups and genotypes based on the entire major capsid protein (VP1) encoding region sequences. In this study, we determined the first complete genome sequences of two genogroup V, genotype 3 (GV.3) SaV strains detected from swine fecal samples, in combination with Illumina MiSeq sequencing of the libraries prepared from viral RNA and PCR products. The lengths of the viral genome (7494 nucleotides [nt] excluding polyA tail) and short 5'-untranslated region (14 nt) as well as two predicted open reading frames are similar to those of other SaVs. The amino acid differences between the two porcine SaVs are most frequent in the central region of the VP1-encoding region. A stem-loop structure which was predicted in the first 41 nt of the 5'-terminal region of GV.3 SaVs and the other available complete genome sequences of SaVs may have a critical role in viral genome replication. Our study provides complete genome sequences of rarely reported GV.3 SaV strains and highlights the common 5'-terminal genomic feature of SaVs detected from different mammalian species.

Phenotypes of Aging Postovulatory Oocytes After Somatic Cell Nuclear Transfer in Mice.

Oocytes rapidly lose their developmental potential after ovulation, termed postovulatory oocyte aging, and often exhibit characteristic phenotypes, such as cytofragmentation, abnormal spindle shapes, and chromosome misalignments. Here, we reconstructed mouse oocytes using somatic cell nuclear transfer (SCNT) to reveal the effect of somatic cell-derived nuclei on oocyte physiology during aging. Normal oocytes started undergoing cytofragmentation 24 hours after oocyte collection; however, this occurred earlier in SCNT oocytes and was more severe at 48 hours, suggesting that the transferred somatic cell nuclei affected oocyte physiology. We found no difference in the status of acetylated α-tubulin (Ac-Tub) and α-tubulin (Tub) between normal and SCNT aging oocytes, but unlike normal oocytes, aging SCNT oocytes did not have astral microtubules. Interestingly, aging SCNT oocytes displayed more severely scattered chromosomes or irregularly shaped spindles. Observations of the microfilaments showed that, in normal oocytes, there was a clear actin ring beneath the plasma membrane and condensed microfilaments around the spindle (the actin cap) at 0 hours, and the actin filaments started degenerating at 1 hour, becoming completely disrupted and distributed to the cytoplasm at 24 hours. By contrast, in SCNT oocytes, an actin cap formed around the transplanted nuclei within 1 hour of SCNT, which was still present at 24 hours. Thus, SCNT oocytes age in a similar but distinct way, suggesting that they not only contain nuclei with abnormal epigenetics but are also physiologically different.

Norovirus binding to intestinal epithelial cells is independent of histo-blood group antigens.

Human noroviruses (NoVs) are a major cause of non-bacterial gastroenteritis. Although histo-blood group antigens (HBGAs) have been implicated in the initial binding of NoV, the mechanism of that binding before internalization is not clear. To determine the involvement of NoVs and HBGAs in cell binding, we examined the localization of NoV virus-like particles (VLPs) and HBGAs in a human intestinal cell line and the human ileum biopsy specimens by immunofluorescence microscopy. The localizations of Ueno 7k VLPs (genogroup II.6) and each HBGA (type H1-, H2- and Le(b)-HBGAs) on the human intestinal cell line, Caco-2, were examined by confocal laser-scanning microscopy. To explore any interactions of NoVs and HBGAs in vivo, fresh biopsy specimens from human ileum were directly incubated with NoV VLPs and examined by immunofluorescence microscopy. We found that VLP binding depended on the state of cell differentiation, but not on the presence of HBGAs. In differentiated Caco-2 cells, we detected no type H1 HBGAs, but VLPs bound to the cells anyway. We incubated fresh biopsies of human ileum directly with VLPs, a model that better replicates the in vivo environment. VLPs mainly bound epithelial cells and goblet cells. Although the incubations were performed at 4°C to hinder internalization, VLPs were still detected inside cells. Our results suggest that VLPs utilize molecule(s) other than HBGAs during binding and internalization into cells.

Amplification of all 11 RNA segments of group A rotaviruses based on reverse transcription polymerase chain reaction.

Group A rotaviruses (RVA) are a major cause of acute infantile gastroenteritis. The viral genome comprises 11 double-stranded RNA segments and the respective gene segments are classified into more than eight genotypes, according to the nucleotide sequence similarities. So far, it has been difficult to amplify full-length sequences of long RNA segments of rotaviruses by one-time only RT-PCR (especially in the genes for the viral proteins VP1, VP2, VP3 and VP4). In this study, a set of universal primers to amplify all 11 segments of RVA was designed by aligning the nucleotide sequences of the typical rotavirus strains. Using these primers and a high-fidelity and rapid DNA polymerase in a one-step reverse transcription polymerase chain reaction, almost the entire length of all 11 segments of the seven rotavirus strains Wa, DS-1, Hochi, 69M, WI61, M37 and SA11-S1 were accurately and rapidly amplified. In addition, all 11 segments of rotavirus obtained from a fecal specimen were successfully amplified. In conclusion, the method described here will be useful as an RVA detection system and protocol for complete analysis of the 11 genome sequences.

Structural basis for norovirus inhibition and fucose mimicry by citrate.

Human noroviruses bind with their capsid-protruding domains to histo-blood-group antigens (HBGAs), an interaction thought to direct their entry into cells. Although human noroviruses are the major cause of gastroenteritis outbreaks, development of antivirals has been lacking, mainly because human noroviruses cannot be cultivated. Here we use X-ray crystallography and saturation transfer difference nuclear magnetic resonance (STD NMR) to analyze the interaction of citrate with genogroup II (GII) noroviruses. Crystals of citrate in complex with the protruding domain from norovirus GII.10 Vietnam026 diffracted to 1.4 Å and showed a single citrate bound at the site of HBGA interaction. The citrate interaction was coordinated with a set of capsid interactions almost identical to that involved in recognizing the terminal HBGA fucose, the saccharide which forms the primary conserved interaction between HBGAs and GII noroviruses. Citrate and a water molecule formed a ring-like structure that mimicked the pyranoside ring of fucose. STD NMR showed the protruding domain to have weak affinity for citrate (460 µM). This affinity, however, was similar to the affinities of the protruding domain for fucose (460 µM) and H type 2 trisaccharide (390 µM), an HBGA shown previously to be specifically recognized by human noroviruses. Importantly, competition STD NMR showed that citrate could compete with HBGA for norovirus binding. Together, the results suggest that citrate and other glycomimetics have the potential to block human noroviruses from binding to HBGAs.

Translational insensitivity to potent activation of PKR by HCV IRES RNA.

Translation of hepatitis C virus (HCV) is initiated at an internal ribosome entry site (IRES) located at the 5'end of its RNA genome. The HCV IRES is highly structured and greater than 50% of its nucleotides form based-paired helices. We report here that the HCV IRES is an activator of PKR, an interferon-induced enzyme that participates in host cell defense against viral infection. Binding of HCV IRES RNA to PKR leads to a greatly increased (20-fold) rate and level (4.5-fold) of PKR autophosphorylation compared to previously studied dsRNA activators. We have mapped the domains in the IRES required for PKR activation to domains III-IV and demonstrate that the N-terminal double-stranded RNA binding domains of PKR bind to the IRES in a similar manner to other RNA activators. Addition of HCV IRES RNA inhibits cap-dependent translation in lysates via phosphorylation of PKR and eIF2alpha. However, HCV IRES-mediated translation is not inhibited by the phosphorylation of PKR and eIF2alpha. The results presented here suggest that hydrolysis of GTP by eIF2 is not an essential step in IRES-mediated translation. Thus, HCV can use structured RNAs to its advantage in translation, while avoiding the deleterious effects of PKR activation.

Biophysical and biochemical investigations of dsRNA-activated kinase PKR.

Protein kinase RNA-activated (PKR) is a serine/threonine kinase that contains an N-terminal RNA-binding domain (dsRNA) and a C-terminal kinase domain. On binding viral dsRNA molecules, PKR can become activated and phosphorylate cellular targets, such as eukaryotic translation initiation factor 2alpha (eIF-2alpha). Phosphorylation of eIF-2alpha results in attenuation of protein translation initiation. Therefore, PKR plays an integral role in the antiviral response to cellular infection. Here we provide a methodological framework for probing PKR function by use of assays for phosphorylation, RNA-protein stability, PKR dimerization, and in vitro translation. These methods are complemented by nuclear magnetic resonance approaches for probing structural features of PKR activation. Considerations required for both PKR and dsRNA sample preparation are also discussed.

Viral dsRNA inhibitors prevent self-association and autophosphorylation of PKR.

Host response to viral RNA genomes and replication products represents an effective strategy to combat viral invasion. PKR is a Ser/Thr protein kinase that binds to double-stranded (ds)RNA, autophosphorylates its kinase domain, and subsequently phosphorylates eukaryotic initiation factor 2alpha (eIF2alpha). This results in attenuation of protein translation, preventing synthesis of necessary viral proteins. In certain DNA viruses, PKR function can be evaded by transcription of highly structured virus-encoded dsRNA inhibitors that bind to and inactivate PKR. We probe here the mechanism of PKR inhibition by two viral inhibitor RNAs, EBER(I) (from Epstein-Barr) and VA(I) (from human adenovirus). Native gel shift mobility assays and isothermal titration calorimetry experiments confirmed that the RNA-binding domains of PKR are sufficient and necessary for the interaction with dsRNA inhibitors. Both EBER(I) and VA(I) are effective inhibitors of PKR activation by preventing trans-autophosphorylation between two PKR molecules. The RNA inhibitors prevent self-association of PKR molecules, providing a mechanistic basis for kinase inhibition. A variety of approaches indicated that dsRNA inhibitors remain associated with PKR under activating conditions, as opposed to activator dsRNA molecules that dissociate due to reduced affinity for the phosphorylated form of PKR. Finally, we show using a HeLa cell extract system that inhibitors of PKR result in translational recovery by the protein synthesis machinery. These data indicate that inhibitory dsRNAs bind preferentially to the latent, dephosphorylated form of PKR and prevent dimerization that is required for trans-autophosphorylation.

Down-regulation of the internal ribosome entry site (IRES)-mediated translation of the hepatitis C virus: critical role of binding of the stem-loop IIId domain of IRES and the viral core protein.

In a previous study, we observed that hepatitis C virus (HCV) core protein specifically inhibits translation initiated by an HCV internal ribosome entry site (IRES). To investigate the mechanism by which down-regulation of HCV translation occurs, a series of mutations were introduced into the IRES element, as well as the core protein, and their effect on IRES activity examined in this study. We found that expression of the core protein inhibits HCV translation possibly by binding to a stem-loop IIId domain, particularly a GGG triplet within the hairpin loop structure of the domain, within the IRES. Basic-residue clusters located at the N-terminus of the core protein have an inhibitory effect on HCV translation, and at least one of three known clusters is required for inhibition. We propose a model in which competitive binding of the core protein for the IRES and 40S ribosomal subunit regulates HCV translation.

Molecular determinants for subcellular localization of hepatitis C virus core protein.

Hepatitis C virus (HCV) core protein is a putative nucleocapsid protein with a number of regulatory functions. In tissue culture cells, HCV core protein is mainly located at the endoplasmic reticulum as well as mitochondria and lipid droplets within the cytoplasm. However, it is also detected in the nucleus in some cells. To elucidate the mechanisms by which cellular trafficking of the protein is controlled, we performed subcellular fractionation experiments and used confocal microscopy to examine the distribution of heterologously expressed fusion proteins involving various deletions and point mutations of the HCV core combined with green fluorescent proteins. We demonstrated that a region spanning amino acids 112 to 152 can mediate association of the core protein not only with the ER but also with the mitochondrial outer membrane. This region contains an 18-amino-acid motif which is predicted to form an amphipathic alpha-helix structure. With regard to the nuclear targeting of the core protein, we identified a novel bipartite nuclear localization signal, which requires two out of three basic-residue clusters for efficient nuclear translocation, possibly by occupying binding sites on importin-alpha. Differences in the cellular trafficking of HCV core protein, achieved and maintained by multiple targeting functions as mentioned above, may in part regulate the diverse range of biological roles of the core protein.

Interaction of hepatitis C virus core protein with retinoid X receptor alpha modulates its transcriptional activity.

Hepatic steatosis and hepatocellular carcinoma (HCC) are common and serious features of hepatitis C virus (HCV) infection, and the core protein has been shown to play distinct roles in the pathogenesis. Here we report the direct interaction of HCV core protein with retinoid X receptor alpha (RXRalpha), a transcriptional regulator that controls many aspects of cell proliferation, differentiation, and lipid metabolism. The core protein binds to the DNA-binding domain of RXRalpha, leading to increase the DNA binding of RXRalpha to its responsive element. In addition, RXRalpha is activated in cells expressing the core protein as well as in the livers of the core-transgenic mice that would develop hepatic steatosis and HCC later in their lives. Using promoter genes of cellular retinol binding protein II (CRBPII) and acyl-CoA oxidase as reporters, we also show that the expression of the core protein enhances the transcriptional activity regulated by the RXRalpha homodimer as well as by the heterodimer with peroxisome proliferator activated receptor alpha. Furthermore, expression of the CRBPII gene is also up-regulated in the livers of HCV core-transgenic mice. In conclusion, these results suggest that modulation of RXRalpha-controlled gene expression via interaction with the core protein contributes to the pathogenesis of HCV infection.