Viral RNA In Infected Cells Research Articles

Inhibition of foot-and-mouth disease virus replication by small interfering RNA.

Foot-and-mouth disease, caused by foot-and-mouth disease virus (FMDV), is one of the most dangerous diseases of cloven-hoofed animals and is a constant threat to the dairy and beef industries in the Middle East and other regions of the world, despite intensive vaccination programmes. In this work, the ability of specific small interfering (si)RNAs to inhibit virus replication in BHK-21 cells was examined. By using bioinformatic computer programs, all FMDV sequences in public-domain databases were analysed. The analysis revealed three regions of at least 22 bp with 100 % identity in all FMDV entries. From these sequences, three specific siRNA molecules were prepared and used to test the ability of siRNAs to inhibit virus replication. By using real-time quantitative PCR to measure the amount of viral RNA in infected cells, it was shown that virus replication was inhibited in cells that were transfected with siRNAs. When viral titres were examined, 100 % inhibition of growth could be demonstrated in cells transfected with a mixture of all three anti-FMDV siRNAs, compared with control cells transfected with anti-LacZ siRNA.

In vitro RNA replication directed by replicase complexes isolated from the subgenomic replicon cells of hepatitis C virus.

Replication of hepatitis C virus (HCV) RNA in virus-infected cells is believed to be catalyzed by viral replicase complexes (RCs), which may consist of various virally encoded nonstructural proteins and host factors. In this study, we characterized the RC activity of a crude membrane fraction isolated from HCV subgenomic replicon cells. The RC preparation was able to use endogenous replicon RNA as a template to synthesize both single-stranded (ss) and double-stranded (ds) RNA products. Divalent cations (Mg2+ and Mn2+) showed different effects on RNA synthesis. Mg2+ ions stimulated the synthesis of ss RNA but had little effect on the synthesis of ds RNA. In contrast, Mn2+ ions enhanced primarily the synthesis of ds RNA. Interestingly, ss RNA could be synthesized under certain conditions in the absence of ds RNA, and vice versa, suggesting that the ss and ds RNA were derived either from different forms of replicative intermediates or from different RCs. Pulse-chase analysis showed that radioactivity incorporated into the ss RNA was chased into the ds RNA and other larger RNA species. This observation indicated that the newly synthesized ss RNA could serve as a template for a further round of RNA synthesis. Finally, 3' deoxyribonucleoside triphosphates were able to inhibit RNA synthesis in this cell-free system, presumably through chain termination, with 3' dGTP having the highest potency. Establishment of the replicase assay will facilitate the identification and evaluation of potential inhibitors that would act against the entire RC of HCV.

Method for Detection of Extraneous Active Bovine Viral Diarrhoea Virus and Classical Swine Fever Virus in Animal Viral Vaccines by RT-PCR, Which Amplify Negative-strand Viral RNA in Infected Cells

An oligonucleotide sense primer, Pst324α, was designed and used for synthesizing cDNA from negative-strand viral RNA in infected cells and used for rapid detection of active extraneous bovine viral diarrhoea virus (BVDV) and classical swine fever virus (CSFV) in animal viral vaccines by culturing a sample in cells followed by reverse transcriptase–polymerase chain reaction (RT-PCR). Active and inactivated viruses of BVDV No. 12–43 strain and CSFV GPE−strain were inoculated to bovine testicle and swine testicle cells for incubation. After the complete extraction of RNA from these cells, cDNA was synthesized using Pst324α, and PCR was carried out using primers 324 and 326 (novel RT-PCR). Amplification of novel RT-PCR products was observed in cells infected with active viruses but not in cells inoculated with inactivated viruses, inoculums and cultured media after incubation. This novel RT-PCR was able to amplify viral sequences from cells infected with only a small number of infectious particles (less than 10 TCID50) at three days postinoculation and was as sensitive as the general RT-PCR using a random primer and the interference and immunofluorescent antibody (FA) methods. The results of experiments on detection of BVDV RNA from vaccines contaminated with active and inactivated BVDV showed that the sensitivity of the novel RT-PCR was almost the same as the sensitivities of the interference and FA methods. These results suggest that the novel RT-PCR is easier and more rapid than the interference method for detection of active BVDV and that the novel RT-PCR is a reliable means for detection of active extraneous BVDV for quality control of animal vaccines.

Inhibition of PKR activation by the proline-rich RNA binding domain of the herpes simplex virus type 1 Us11 protein.

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The gamma 34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the gamma 34.5 gene specifies a regulatory subunit for protein phosphatase 1 alpha, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. gamma 34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of gamma 34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation.

Association of Human Immunodeficiency Virus Type 1 Vif with RNA and Its Role in Reverse Transcription

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication, although the functional target of Vif remains elusive. HIV-1 vif mutant virions derived from nonpermissive H9 cells displayed no significant differences in the amount, ratio, or integrity of their protein composition relative to an isogenic wild-type virion. The amounts of the virion-associated viral genomic RNA and tRNA(3)(Lys) were additionally present at normal levels in vif mutant virions. We demonstrate that Vif associates with RNA in vitro as well as with viral genomic RNA in virus-infected cells. A functionally conserved lentivirus Vif motif was found in the double-stranded RNA binding domain of Xenopus laevis, Xlrbpa. The natural intravirion reverse transcriptase products were markedly reduced in vif mutant virions. Moreover, purified vif mutant genomic RNA-primer tRNA complexes displayed severe defects in the initiation of reverse transcription with recombinant reverse transcriptase. These data point to a novel role for Vif in the regulation of efficient reverse transcription through modulation of the virion nucleic acid components.

Effects of dexamethasone on rhinovirus infection in cultured human tracheal epithelial cells.

To examine the effects of glucocorticoid on rhinovirus (RV) infection, primary cultures of human tracheal epithelial cells were infected with either RV2 or RV14. Viral infection was confirmed by demonstrating that viral RNA in infected cells and viral titers of supernatants and lysates from infected cells increased with time. RV14 infection upregulated the expression of mRNA and protein of intercellular adhesion molecule-1 (ICAM-1), the major RV receptor, on epithelial cells, and it increased the production of interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor-alpha in supernatants. Dexamethasone reduced the viral titers of supernatants and cell lysates, viral RNA of infected cells, and susceptibility of RV14 infection in association with inhibition of cytokine production and ICAM-1 induction. In contrast to RV14 infection, dexamethasone did not alter RV2 infection, a minor group of RVs. These results suggest that dexamethasone may inhibit RV14 infection by reducing the surface expression of ICAM-1 in cultured human tracheal epithelial cells. Glucocorticoid may modulate airway inflammation via reducing the production of proinflammatory cytokines and ICAM-1 induced by rhinovirus infection.

Colocalization and membrane association of murine hepatitis virus gene 1 products and De novo-synthesized viral RNA in infected cells.

Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (pol) gene, is first translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases. Genetic complementation analyses suggest that the majority of the gene 1 products are required for viral RNA synthesis. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofluorescent-staining studies with four polyclonal antisera to localize various MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation experiments showed that these antisera detected proteins representing the two papain-like proteases and the 3C-like protease encoded by open reading frame (ORF) 1a, the putative polymerase (p100) and a p35 encoded by ORF 1b, and their precursors. De novo-synthesized viral RNA was labeled with bromouridine triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antisera colocalized with newly synthesized viral RNA in the cytoplasm, particularly in the perinuclear region of infected cells. Several cysteine and serine protease inhibitors, i.e., E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, suggesting that the processing of the MHV gene 1 polyprotein is tightly associated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA colocalized with the Golgi apparatus in HeLa cells. However, in murine 17CL-1 cells, the viral proteins and viral RNA did not colocalize with the Golgi apparatus but, instead, partially colocalized with the endoplasmic reticulum. Our results provide clear physical evidence that several MHV gene 1 products, including the proteases and the polymerase, are associated with the viral RNA replication-transcription machinery, which may localize to different membrane structures in different cell lines.

The human immunodeficiency virus type 1 Tat antagonist, Ro 5-3335, predominantly inhibits transcription initiation from the viral promoter.

Tat, the transcriptional transactivator protein of the human immunodeficiency virus type 1 (HIV-1), is required for viral replication in vitro. The Tat antagonist, Ro 5-3335, and its analog, Ro 24-7429, have been shown to inhibit replication of HIV-1 and to reduce steady-state viral RNA in infected cells (M.-C. Hsu et al., Science 254:1799-1802, 1991, and M.-C. Hsu et al., Proc. Natl. Acad. Sci. USA 90:6395-6399, 1993). Analysis of HIV-1 long terminal repeat-driven reporter gene transcription in a recombinant adenovirus by nuclear run-on assay indicated that the drug predominantly inhibits Tat-dependent initiation and also exerts a measurable effect on elongation. This result may imply a common mechanism for Tat-mediated transcription initiation and elongation.

Structural Genes, Not the LTRs, Are the Primary Determinants of Reticuloendotheliosis Virus A-Induced Runting and Bursal Atrophy

Reticuloendotheliosis virus strain A (REV-A) and chicken syncytial virus (CSV), two replication competent avian retroviruses, differ in the extent to which they induce a runting syndrome that includes anemia, lymphoid organ atrophy, and reduced body size. We have isolated an infectious clone of CSV, the less pathogenic of the two viruses, and compared it to REV-A. Partial DNA sequence analysis suggests that it differs from REV-A by no more than 1 to 2% at the nucleotide level. Analysis of viral interference indicates that these two viruses use the same cell receptor for infection of both fibroblasts and hematopoietic cells, DNA sequence of the CSV and REV-A long terminal repeats (LTRs) reveals that these structures differ principally by two small insertions (5 and 19 bp) present in the U3 region of REV-A. The larger of these may encode enhancer sequences that have been reported to influence transcription rates in vitro . Measurement of steady-state levels of viral RNA in infected cells, however, as well as circulating virus in infected chicks indicates that the different pathogenic responses elicited by these two viruses are not due to large differences in viral transcription or replication. Chimeric viruses were constructed in which the LTRs from one virus were used to express the structural genes of the second virus. Infection of 1-day-old chicks by parental virus as well as the reciprocal chimeric constructs demonstrated that the ability to induce both runting and bursal atrophy segregated with the structural genes of REV-A. Infection of birds with additional chimeric viruses in which the env genes of REV-A and CSV were exchanged indicated that the pathogenic response resulting from REV-A infection was due to at least two regions of the viral genome encoding structural genes.

Biochemical and genetic evidence for a pseudoknot structure at the 3' terminus of the poliovirus RNA genome and its role in viral RNA amplification.

The sequences in the plus-stranded poliovirus RNA genome that dictate the specific amplification of viral RNA in infected cells remain unknown. We have analyzed the structure of the 3' noncoding region of the viral genome by thermodynamic-based structure calculation and by chemical and enzymatic probing of in vitro-synthesized RNAs and provide evidence for the existence of an RNA pseudoknot structure in this region. To explore the functional significance of this structure, revertants of a mutant bearing a lesion in the proposed pseudoknot and exhibiting a temperature-sensitive defect in viral RNA synthesis were isolated and mapped. The results of this genetic analysis established a correlation between the structure of the 3' terminus of the viral RNA and its function in vivo in RNA amplification. Furthermore, phylogenetic analysis indicated that a similar structure could be formed in coxsackievirus B1, a related enterovirus, which further supports a role for the pseudoknot structure in viral RNA amplification in infected cells.

Translational control by messenger RNA competition for eukaryotic initiation factor 2.

Translation of globin mRNA in a micrococcal nuclease-treated reticulocyte lysate was studied in the presence of increasing amounts of Mengovirus RNA, under conditions in which the number of translation initiation events remains constant as judged by the transfer of label from N-formyl[35S]methionyl-tRNAf into protein. The translation of globin mRNA is progressively inhibited by low concentrations of Mengovirus RNA, free of detectable traces of double-stranded RNA, concomitant with the increasing synthesis of Mengovirus RNA-directed products. On a molar basis, Mengovirus RNA apparently competes about 35 times more effectively than globin mRNA for a critical component in translation. The competition is relieved by the addition of highly purified eukaryotic initiation factor 2 (eIF-2). Addition of eIF-2 does not stimulate overall protein synthesis, but shifts it in favor of globin synthesis. No stimulation of globin mRNA translation by eIF-2 is seen when Mengovirus RNA is absent. These experiments show that Mengovirus RNA competes, directly or indirectly, with globin mRNA for eIF-2. In direct binding experiments using isolated mRNA and eIF-2, Mengovirus RNA is shown to compete with globin mRNA for eIF-2 and to exhibit a 30-fold higher affinity for this factor. The binding of Mengovirus RNA to eIF-2 is much more resistant to increasing salt concentrations than is the binding of globin mRNA, again reflecting its high affinity. These results reveal a direct correlation between the ability of these mRNA species to compete in translation and their ability to bind to initiation factor eIF-2. They suggest that the affinity of a given mRNA species for eIF-2 is essential in determining its translation, relative to that of other mRNA species. Messenger RNA competition for eIF-2 may contribute significantly to the selective translation of viral RNA in infected cells.

Presence of double stranded regions of viral RNA in infected cells.

Ribonuclease treatment of rhinovirus-infected human embryo lung cells after cell disruption reveals that double stranded RNA is present in the preparation before nucleic acids are extracted with phenol. This shows that the hydrogen bonding between complementary molecules of viral RNA which occurs in infected cells is not a result of the extraction of RNA with phenol.

Evidence for splicing of avian sarcoma virus 5′-terminal genomic sequences onto viral-specific RNA in infected cells

The 5'-terminal nucleotide sequences of the avian sarcoma virus (ASV) genome are transcribed by the reverse transcriptase in vitro into a DNA transcript that represents the entire distance ( approximately 100 nucleotides) between the tRNA(Trp) primer molecule and the 5' terminus. We have used these DNA(100) transcripts in hybridization reactions with ASV-specific RNA from infected avian cells and find nucleotide sequences complementary to these transcripts on all of the various size classes of viral mRNA identified. Similar hybridization results were obtained with a specific DNA transcript complementary to viral genomic nucleotide sequences between the tRNA(Trp) primer molecule and up to, but not including, the terminal redundant sequences (DNA(70)), indicating that the observed hybridization of DNA(100) to all size classes of viral RNA in infected cells did not reflect hybridization of DNA(100) to the terminal redundant sequences at the 3' end of the viral genome. Escherichia coli RNase H hydrolysis of RNA.DNA hybrids consisting of genomic 35S RNA obtained from virus and DNA(100) transcripts indicated that viral genomic sequences complementary to these DNA transcripts were not present at sites distal to the ends of the RNA genome and therefore not adjacent to the corresponding gene sequences representing the various species of viral mRNA from infected cells. These studies suggest that the 5'-terminal genomic nucleotide sequences, or a portion thereof, are somehow added or "spliced" onto each ASV-specific mRNA species in infected cells either during or after transcription of proviral DNA for some as yet undetermined purpose.

Infectious Viral DNA in Rous Sarcoma Virus-transformed Nonproducer and Producer Animal Cells

Nonproducer and producer RSV-transformed cells and producer nontransforming virus-infected cells harbor viral DNA specifying the respective avian tumor virus. In nonproducer Rous sarcoma cells, the residing viral DNA is linear, double-stranded and covalently linked to the chromosomal DNA. Both double-stranded and single-stranded forms of RSV DNA transfect chicken cells. The progeny virus is indistinguishable from the DNA parent with respect to the morphological, biological and antigenic properties. Unlike the DNA extracted from nonproducer RSV-transformed mammalian cells, that extracted from producer RSV-transformed chicken cells gives rise, in transfection experiments, to both sarcoma virus and its nontransforming derivative. The DNA from nontransforming virus-infected chicken cells generates only nontransforming viruses. The frequency of nontransforming virus recovery is different from that of sarcoma virus recovery, the latter being a nonlinear function of the amount of transfecting DNA, while the former may suggest a linear relationship. On the other hand, the end-point dilution of transfecting DNA for sarcoma virus recovery is approximately the same as that for nontransforming virus recovery. The following is assumed: Sarcoma viruses and nontransforming derivatives are recovered from two different species of viral DNA which carry or lack, respectively, the transforming genetic material. The size of double-stranded viral DNA is substantially smaller than 20 times 10(6) daltons. In transfection experiments, the sarcoma virus DNA is first integrated into the host cell genome before being expressed, while the nontransforming viral DNA apparently bypasses the integration step. The latter DNA generates the progeny virus when taken up, carried, and transcribed in a permissive cell.

Replicative forms of Sendai virus RNA.

The formation of replicative forms of Sendai virus RNA in virus-infected cells was studied. Several RNA species with sedimentation coefficients 7 S to 48S were revealed in the course of infection, and a certain sequence in their appearance was observed. Replicative forms of Sendai virus RNA were found as multistranded replicative intermediate (RI) with sedimentation coefficients ranging from 20S to 30S, and as double-stranded replicative form (RF) with a sedimentation coefficient of about 22–23S. Parental RNA was found in the intranuclear structures and in the cytoplasm. Participation of parental RNA in the formation of double-stranded RF was evidenced.