Norwalk virus RNA is infectious in mammalian cells

Human noroviruses are positive-sense RNA viruses and are the leading cause of epidemic acute viral gastroenteritis in developed countries. The absence of an in vitro cell culture model for human norovirus infection has limited the development of effective antivirals and vaccines. Human histo-blood group antigens have been regarded as receptors for norovirus infection, and expression of the α(1,2) fucosyltransferase gene (FUT2) responsible for the secretor phenotype is required for susceptibility to Norwalk virus (NV) infection. We report for the first time that transfection of NV RNA, isolated from stool samples from human volunteers, into human hepatoma Huh-7 cells leads to viral replication, with expression of viral antigens, RNA replication, and release of viral particles into the medium. Prior treatment of the RNA with proteinase K completely abolishes RNA infectivity, suggesting a key role of an RNA-protein complex. Although overexpression of the human FUT2 gene enhances virus binding to cells, it is not sufficient to allow a complete viral infection, and viral spread from NV-transfected cells to naïve cells does not occur. Finally, no differences in NV RNA replication are observed between Huh-7 and Huh-7.5.1 cells, which contain an inactivating mutation in retinoic acid-inducible gene I (RIG-I), suggesting that the RIG-I pathway does not play a role in limiting NV replication. Our results strongly suggest that the block(s) to NV replication in vitro is at the stage of receptor and/or coreceptor binding and/or uncoating, either because cells lack some specific factor or activation of cellular antiviral responses independent of RIG-I inhibits virus replication.The human pathogen Norwalk virus (NV) is the prototype strain of the Norovirus genus in the family Caliciviridae. Noroviruses are responsible for the majority of outbreaks of nonbacterial gastroenteritis in developed countries, and it is estimated that they have a significant impact in developing countries as well. Although human noroviruses were originally identified more than 30 years ago, our understanding of their replication cycle and mechanisms of pathogenicity has been limited because these viruses are noncultivatable in established cell lines and a small animal model to study viral infection is not available. Only recently, it has been reported that both genogroup I (GI) and GII strains of human noroviruses can be passaged several times with limited replication in a differentiated three-dimensional cell culture system derived from a human small intestinal cell line (40). In addition, gnotobiotic pigs can support replication of a human norovirus GII strain, with occurrence of mild diarrhea and virus shedding and immunofluorescent detection of both structural and nonstructural proteins in enterocytes (10). Although these results are promising, it remains unclear whether these systems are robust enough to be widely used to efficiently propagate human noroviruses in vitro, and the factors responsible for the block(s) of viral replication using standard cell culture systems remain unknown.The NV genome is a positive-sense, polyadenylated, single-stranded RNA molecule of 7.7 kb and contains three open reading frames (ORFs): ORF1 encodes a nonstructural polyprotein, and ORF2 and ORF3 encode the major and minor capsid proteins, VP1 and VP2, respectively (14, 24). Due to the lack of an in vitro system to propagate human noroviruses, features of their life cycle have been inferred from studies using other animal caliciviruses that can grow in mammalian cell cultures. A 3′ coterminal polyadenylated subgenomic RNA is produced within infected cells, and it is believed that both genomic and subgenomic RNAs are covalently linked to the nonstructural protein VPg at their 5′ ends. Upon infection of cells, nonstructural proteins are expressed from genomic RNA and form an RNA replication complex, which generates new genomic RNA molecules as well as subgenomic RNAs encoding VP1 and VP2. After expression of the structural proteins from subgenomic RNA molecules, the capsid is assembled and viral RNA encapsidated prior to progeny release. Some of these features have been confirmed using recombinant systems to express the native NV genome in mammalian cells by using vaccinia virus expression systems (2, 25).Studies with human volunteers have shown that some individuals are either repeatedly susceptible or resistant to NV infection (36) and led to the identification of a genetically determined factor that predicts a person''s susceptibility to infection and disease (19, 30). Binding experiments using recombinant NV virus-like particles (VLPs) demonstrated attachment of VLPs to surface epithelial cells of the gastroduodenal junction on biopsies from secretors but not to cells from nonsecretors, showing that the expression pattern of ABH histo-blood group antigens may influence susceptibility to NV (32). The gene responsible for the secretor phenotype encodes an α(1,2)fucosyltransferase (FUT2) that produces H antigens on the surface of epithelial cells and in mucosal secretions (27). Since it was observed that transfection of the FUT2 gene into nonpermissive cells enhances NV binding (31), it has been hypothesized that H antigens or related blood group antigens may function as a receptor for NV.The main goal of our study was to understand the molecular basis of the restricted growth of NV in cultured cells by transfecting wild-type NV RNA into human cells. Our studies show for the first time that transfection of wild-type NV RNA isolated from human stool samples can lead to the production of viral particles, indicating that wild-type NV RNA is infectious and replicates. However, a block to NV spread to other cells in the culture remains, indicating that the block(s) exists at the cell entry and/or uncoating steps.     

Attachment of mesophilic aeromonads to cultured mammalian cells

The interaction between mesophilic aeromonads and cultured mouse adrenal cells was examined. Preliminary experiments indicated that aeromonad attachment was dependent upon inoculum size, incubation time, and incubation temperature. Optimal attachment was observed after 30 min of incubation at 37°C with an inoculum size of 1×10⁷ CFU. Heat-killed and formalin-treated organisms did not attach to the cultured cell system. The attachment of aeromonads to the mammalian cell surface was confirmed by light and scanning electron microscopy. Aeromonad attachment correlated both with the presence of pili and the specific aeromonad species, but not with hydrophobicity or the ability to autoagglutinate. Piliated strains were more likely to show high or moderate attachment.Aeromonas sobria, A. hydrophila, andA. veronii showed a greater ability to bind adrenal cells than didA. caviae. Removal of the pili from twoA. sobria isolates markedly reduced their attachment. In contrast, oneA. hydrophila isolate was strongly adherent after the removal of pili. The hemagglutination patterns produced byA. sobria and the other aeromonad species were distinctly different, but potentially predictive of the ability of aeromonads to attach to cultured mouse adrenal cells. These studies indicate that multiple mechanisms are important for the attachment of mesophilic aeromonads to mammalian cells. This model may prove useful for studying the pathogenesis of aeromonad infections.     

The response of mammalian cells to double-stranded RNA

Double-stranded RNA (dsRNA) has long been recognized as a central component of the interferon (IFN) system. It was originally characterized as a key mediator of IFN induction in response to virus infection. Subsequently, it was identified as a prime activator of the antiviral response. In recent years the discovery of the RNA interference (RNAi) pathway in mammals has renewed interest in dsRNA-mediated cellular responses. This has coincided with the identification of key components of the IFN induction pathway. Here, we present an overview of the current knowledge of dsRNA-mediated pathways in mammalian cells and introduce a link between these pathways and application of RNAi.     

Bacterial delivery of functional messenger RNA to mammalian cells

The limited access to the nuclear compartment may constitute one of the major barriers after bacteria-mediated expression plasmid DNA delivery to eukaryotic cells. Alternatively, a self-destructing Listeria monocytogenes strain was used to release translation-competent mRNA directly into the cytosol of epithelial cells, macrophages and human dendritic cells. Enhanced green fluorescent protein (EGFP)-encoding mRNA, adapted for translation in mammalian cells by linking an IRES element to the 5'-end of the egfp coding sequence, was produced by T7 RNA polymerase in the carrier bacteria upon entry into the cytosol where the mRNA is efficiently released from the lysed bacteria and immediately translated in eukaryotic host cells. Besides the much earlier expression of EGFP being detectable already 4 h after infection, the number of EGFP expressing mammalian cells obtained with this novel RNA delivery technique is comparable to or - especially in phagocytic cells - even higher than that obtained with the expression plasmid DNA delivery strategy. Accordingly, bacteria-mediated delivery of ovalbumin-encoding mRNA to macrophages resulted in efficient antigen processing and presentation in vitro indicating that this approach may also be adapted for the in vivo delivery of antigen-encoding mRNA leading to a more efficient immune response when applied to vaccine development.     

Sensitivity to UV radiation of small nuclear RNA synthesis in mammalian cells.

It was demonstrated previously that the synthesis of small nuclear RNA (snRNA) species U1 and U2 in human cells is very sensitive to UV radiation. In the present work, the UV sensitivity of U3, U4, and U5 snRNA synthesis is shown to be also high. The synthesis of U1, U2, U3, U4, and U5 snRNAs progressively decreased during the first 2 h after UV irradiation (this was not observed in polyadenylated RNA) and had not returned to normal rates 6 h after UV exposure. In contrast, the restoration of 5.8S rRNA synthesis began immediately after UV irradiation and was essentially complete 6 h later. A small fraction of U1 and U5 (and possibly U2 and U3) snRNA synthesis remained unaffected by high UV doses, when cell radiolabeling began 10 min after UV irradiation. The present data suggest that a factor other than the level of pyrimidine dimers in DNA (possibly, steps in the post-irradiation DNA repair process) plays an important role in the mechanism of UV-induced inhibition of U1-U5 snRNA synthesis.     

RNA interference in mammalian cells by chemically-modified RNA

RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.     

Reaction of colloidal silica with membranes of intact mammalian cells

Colloidal silical particles were produced at a size that permitted reaction with human erythrocytes and rat macrophages without affecting cell integrity. Binding of colloid was shown by increased electrophoretic mobility of red cells and also resulted in changes in the surface topography of red cells as seen with scanning electron microscopy. The degree to which colloid binds to red cells was determined by microprobe analysis of single intact cells. Furthermore, the capacity of red cells to bind silica was increased if sialic acid residues were removed enzymatically from the cell surface.     

Attachment and growth of mammalian cells on microcarriers with different ion exchange capacities

In the design of microcarriers for animal cell growth, the exchange capacity has been considered a critical factor. However, charge densities of microcarriers under culture conditions are not the same as the exchange capacities. Furthermore, the charge density requirement for optimum attachment is not necessarily the same as that required for optimum growth. We demonstrate that charge is not the sole factor affecting the attachment and growth of animal cells on microcarriers. We also show that supplemental serum in the growth medium has a negative effect on cell attachment to microcarriers.     

Covalent attachment of RNA to nascent DNA in mammalian cells

We have used the technique of phosphate transfer analysis to test for the presence of phosphodiester bonds linking ribonucleotides (on the 5′ side) to deoxyribonucleotides (on the 3′ side) in DNA newly synthesized within lysates or purified nuclei of mammalian cells. We have found that such covalent junctions between RNA and DNA are present at a frequency of one junction per newly synthesized DNA strand. The junctions are located close to the ends of the nascent DNA strands. The stretches of RNA at the junction are very short compared to the stretches of DNA. These properties are consistent with the conclusion by Reichard, Eliasson, and Söderman (1974) that short stretches of RNA are present on the 5′ ends of nascent DNA strands produced during replication of polyoma virus.     

Early nucleolar preribosomal RNA - protein in mammalian cells.

Early steps of ribosomal maturation have been studied by analysis of nucleolar extracts using different extraction procedures. Early 45-S nucleolar RNA is found to be associated with slowly sedimenting elements, distinct from previously described 80-S and 55-S nucleolar preribosomes. This early 45-S RNA has been shown to be of preribosomal type according to the following criteria. (a) When hybridized with nucleolar DNA, competition with rRNA can be observed; (b) its biosynthesis is sensitive to low doses of actinomycin D; (c) it is methylated at an early stage; (d) it contains no linked poly(A) segments. This early 45-S RNA seems to be specifically linked with proteins. The protein content of these early RNA-protein complexes is significantly lower than that for 80-S preribosomes. 45-S RNA sensitivity to nucleolytic activities that can take place in the course of preribosome extraction has been found to be higher in these RNA-protein complexes than in 80-S preribosomes. Identical results were obtained when different mammalian cell species were studied (rat, hamster, or HeLa cells).     

Stability of nuclear RNA in mammalian cells

The kinetics of entry of [³H]adenosine into ATP, cellular RNA, and nuclear RNA of mouse L cells were determined and analyzed. A molar accumulation curve for RNA was estimated from the specific radioactivities of RNA and ATP; this curve was resolved graphically into stable and unstable components. The stability of the unstable component (mostly heterogeneous nuclear RNA) was estimated by applying first-order decay analysis. Heterogeneous, nuclear RNA decays with an apparently uniform half-life of 23 minutes, considerably greater than some previous estimates. It is synthesized at an instantaneous rate of 5.4 × 10^?2 pg/cell per minute and reaches a steady-state level of 1.8 pg/cell in the nucleus, or 7% of the total cellular RNA. Only about 2% of the heterogeneous RNA synthesized in L cells enters polysomes as messenger RNA. The implications of these values are discussed with reference to similarly determined values for sea urchin embryos.