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The ds-RNA induced by TYMV infection in the nuclei of infected Chinese cabbage leaf cells became labelled with [32P]phosphate most rapidly before production of virus could be detected. In contrast, the ds-RNA associated with the chloroplasts became labelled most rapidly during the period of maximum virus production.
Turnip yellow mosaic virus (TYMV) RNA has two extensively overlapping open reading frames (ORFs) encoding noncapsid proteins. The longest of these, ORF-206, is essential for RNA replication. We have investigated the expression and role of ORF-69 (encoding a protein with a MW of 69K) using specific antiserum and mutant genomic RNAs designed to interrupt ORF-69 expression. TYC69 antiserum immunoprecipitated a protein with an apparent MW of 75K (p69) from in vitro translations of TYMV RNA. Mutant RNAs with stop codons inserted at nucleotides 139, 178, and 178/224 in ORF-69 expressed ORF-69 at very low levels in vitro. These mutants replicated to wild-type levels in turnip or Chinese cabbage protoplasts, but were not recovered from any leaves of inoculated plants. These results suggest that ORF-69 products are dispensible for replication, but are required for viral spread. Revertant and pseudorevertant viruses with uninterrupted ORF-69 were recovered from plants showing delayed onset of symptoms after inoculation with two of the above mutants, indicating the importance of ORF-69 expression in establishing a systemic infection in plants. ORF-69 expression could be detected using Western blots in extracts from young, symptomatic leaves of infected plants. A single band with an apparent MW of 75-80K was detected in leaves infected with cDNA-derived viral RNAs, while a doublet was detected after infection with type strain viral RNA, suggesting the presence of two ORF-69 alleles or differential post-translational modification.
Dengue virus type 2 (DEN-2), a member of the Flaviviridae family, has a positive-strand RNA genome, 10,723 nucleotides (nt) in length and encoding a single polyprotein precursor consisting of 3391 amino acids (aa). In order to construct a full-length cDNA clone, the viral genome was cloned into 5' (nt 1-2203 under the control of the T7 promoter (pT7)) and 3' (nt 2203-10,723) constructs. A full-length DEN-2 cDNA under pT7 control was assembled in vitro after excising the two cDNA inserts from the 5' and 3' constructs, and joining them with T4 DNA ligase. The RNA produced by in vitro transcription of the cDNA using T7 RNA polymerase was infectious, as shown by transfection of permissive BHK-21 and Vero cells, and propagation of the virus particles released into the culture media. The virus particles stably maintained the conservative mutation introduced into the 5' construct, and the cells infected with the infectious RNA-derived virus synthesized virus-specific DEN-2 antigens, as shown by immunofluorescence and immunoprecipitations. The full-length infectious clone for DEN-2 should be useful for the study of molecular mechanisms involved in viral RNA replication and virus assembly.
Replication complexes (RC) of the encephalomyocarditis (EMC) virus were shown previously to contain components that exhibit marked preference for nucleoside diphosphates over nucleoside triphosphates (NTP) as substrates for viral RNA synthesis [Koonin and Agol (1983), Virology 129, 309-318]. These NDP-preferring components have now been found to posses the following properties. When RC preparations were fractionated by sucrose density gradient centrifugation, the fractions containing NDP-preferring components exhibited a considerably higher nucleotide kinase activity as compared to either the fractions containing NTP-preferring components or corresponding fractions from mock-infected cells. When NDP-preferring RC were incubated with ADP and three other NTP, very low concentrations of endogenously generated ATP ensured a greater rate of RNA synthesis than did much higher concentrations of exogenous ATP. When an equimolar mixture of differently labelled UDP and UTP was used as a substrate for NDP-preferring RC, the label from UDP predominated in the newly synthesized RNA, even though the UDP-derived UTP constituted a minor portion of the total UTP pool. When labelled UDP was diluted with unlabelled uridine nucleotides, unlabelled UTP proved to be far less efficient than unlabelled UDP in diminishing the specific radioactivity of UMP incorporated into RNA by NDP-preferring RC. These data are interpreted in the sense that the NTP generated by the built-in nucleotide kinase system are not freed into the external milieu but rather form a separate pool preferentially used for synthesis of viral RNA by NDP-preferring RC. It is suggested that this functional compartmentation of NTP may be significant for the replication of viral RNA in vivo.
The 3C protease from foot-and-mouth disease virus (FMDV 3C(pro)) is critical for viral pathogenesis, having vital roles in both the processing of the polyprotein precursor and RNA replication. Although recent structural and functional studies have revealed new insights into the mechanism and function of the enzyme, key questions remain that must be addressed before the potential of FMDV 3C(pro) as an antiviral drug target can be realised.
In addition to the four RNAs known to be encapsidated by Prunus necrotic ringspot virus (PNRSV) and Apple mosaic virus (ApMV), an additional small RNA (RNA 5) was present in purified preparations of several isolates of both viruses. RNA 5 was always produced following infection of a susceptible host by an artificial mixture of RNAs 1, 2, 3, and 4 indicating that it was a product of viral replication. RNA 5 does not activate the infectivity of mixtures that contain the three genomic RNAs (RNA 1 + RNA 2 + RNA 3) nor does it appear to modify symptom expression. Results from hybridization studies suggested that RNA 5 had partial sequence homology with RNAs 1, 2, 3, and 4. Cloning and sequencing the RNA 5 of isolate CH 57/1-M of PNRSV, and the 3' termini of the RNA 1, RNA 2 and RNA 3 of this isolate indicated that it was a copy of the 3' untranslated terminal region (3'-UTR) of the genomic RNA 3.
BACKGROUND & AIMS: Modulation of the host innate immune response is an attractive means of inhibiting hepatitis C virus (HCV) replication. Having previously determined that expression of the interferon-sensitive gene (ISG)15 protease USP18 is increased in the liver biopsy specimens of patients who do not respond to interferon (IFN)-alfa therapy, we hypothesized that USP18 might hinder the ability of IFN to inhibit HCV replication. METHODS: The role of USP18 in IFN antiviral activity was examined using an in vitro model of HCV replication that reproduces the full viral life cycle. USP18 was silenced specifically using small inhibitory RNAs (siRNAs), and the dose response of HCV replication and infectious virus production to IFN-alfa was measured. RESULTS: The siRNA knockdown of USP18 in human cells consistently potentiated the ability of IFN to inhibit HCV-RNA replication and infectious virus particle production by a factor of 1-2 log(10). USP18 knockdown also resulted in a number of cellular changes consistent with increased sensitivity to IFN. Decreasing USP18 expression led to increased cellular protein ISGylation in response to exogenous IFN-alfa, prolonged tyrosine phosphorylation of signal transducer and activation of transcription (STAT1), and a general enhancement of IFN-stimulated gene expression. CONCLUSIONS: These data suggest that USP18 modulates the anti-HCV type I IFN response, and is a possible therapeutic target for the treatment of HCV infection.
Tubulin acts as a positive transcription factor for in vitro RNA synthesis by two different negative-strand viruses: Sendai virus, a paramyxovirus; vesicular stomatitis virus (VSV), a rhabdovirus. A monoclonal antibody directed against beta-tubulin completely inhibited not only mRNA synthesis and RNA replication catalyzed in vitro by extracts of cells infected with either virus but also mRNA synthesis by detergent-disrupted purified virions. The synthesis of both a leader-like RNA and the NP mRNA directed by detergent-disrupted purified Sendai virions was shown to be totally dependent on the addition of purified tubulin. The addition of purified tubulin, although not required, also stimulated mRNA synthesis directed by detergent-disrupted VSV virions 2- to 7-fold. Finally, there appears to be an association between tubulin and the L protein of VSV, since both monoclonal and polyclonal anti-tubulin antisera specifically immunoprecipitated not only tubulin but also the L protein of two different VSV serotypes from the soluble protein fraction of infected cells.
In order to study mechanisms of induction of IFN-alpha by Newcastle disease virus (NDV), we used two replicon systems which are based respectively on DNA and RNA of the Semliki forest virus (SFV) and transfected these into baby hamster kidney cells (BHK) which do not produce interferon-alpha. Co-incubation of BHK cells which were transfected with the two vector systems, with human PBMCs, showed that production of IFN-alpha takes place by two different ways. When using the DNA-based SFV vector, only transfectants expressing cell surface HN molecules of NDV (and not the mock-transfected cells) elicited such a response via interaction of these HN molecules with viral receptors on PBMCs. In contrast, BHK cells transfected with RNA which had been in vitro transcribed from the RNA-based SFV vector without foreign gene as insert (mock-transfected) elicited a comparable IFN-alpha response. Transfer by transfection of poly(I:C), an analogue of double stranded RNA (dsRNA), into the BHK cells induced also by itself the production of IFN-alpha. Therefore induction of "danger signals" (as double-strand RNA replicative intermediates) might be responsible for this discrepancy observed in IFN-alpha induction in PBMCs between the two studied SFV vector systems based on transfection of DNA and on RNA. These observations highlight two ways of IFN-alpha induction which additively may explain the high interferonogenic capacity of NDV as virus: i) via cell-surface expressed HN after transfection of the DNA-based SFV replicon and ii) via transfection of self-amplifying RNA.
A protein similar to that previously demonstrated on poliovirus RNA and replicative intermediate RNA (VPg) was found on all sizes of nascent viral RNA molecules and on the polyuridylic acid isolated from negative-strand RNA. 32P-labeled nascent chains were released from their template RNA and fractionated by exclusion chromatography on agarose. Fingerprint analysis using two-dimensional polyacrylamide gels of RNase T1 oligonucleotides derived from nascent chains of different lengths showed that a size fractionation of nascent chains was achieved. VPg was recovered from nascent chains varying in length from 7,500 nucleotides (full-sized RNA) to about 500 nucleotides. No other type of 5' terminus could be demonstrated on nascent RNA, and the yield of VPg was consistent with one molecule of the protein on each nascent chain. These results are consistent with the concept that the protein is added to the 5' end of the growing RNA chains at a very early stage, possibly as a primer of RNA synthesis. Analysis of the polyuridylic acid tract isolated from the replicative intermediate and double-stranded RNAs indicated that a protein of the same size as that found on the nascent chains and virion RNA is also linked to the negative-strand RNAs. It is likely that a similar mechanism is responsible for initiation of synthesis of both plus- and minus-strand RNAs.
The antiviral effect of interferon inductors, such as poly-I--poly-C, phage f2 RNA replicative form and low molecular inductor GSN and their influence on cellular DNA synthesis were studied in the cultures of lymphoblastoid (inplanting lines Raji Namalva) and somatic human cells. The Semliki forest virus used as the test organism multiplicated well in cells Raji accumulating up to 9 lg BOU/ml. The two-strand RNA was less active in the lymphoid cells than in the somatic ones. GSN was 10 times more active and less toxic in cells Raji as compared to the fibroblasts. The lymphoblastoid interferon had higher antiviral activity as compared to the fibroblast interferon in the system of Raji--Semliki forest virus than in the system of the human embryon fibroblast--Venezuela Horse Encephalytic Virus. Romantadin actively inhibited (100 times) production of the alfavirus in both the somatic and lymphoblastoid cells.
The structural lesion in the temperature-sensitive mutant E1 of the New Jersey serotype of vesicular stomatitis virus has been assigned to the NS protein. Although the packaged wild-type and mutant NS proteins were similarly phosphorylated, the mutant NS protein migrated faster than the wild-type NS protein in polyacrylamide slab gels electrophoresed in the presence of sodium dodecyl sulfate. The resolution appears to be the result of conformational rather than size differences since the two proteins comigrated in polyacrylamide gels which contained 4 M urea in addition to sodium dodecyl sulfate. Peptide maps, obtained by limited proteolysis of 32P-labeled wild-type and mutant NS proteins with Staphylococcus aureus V8 protease and papain, revealed striking differences which suggested that the mutant alteration could involve an aspartic or glutamic acid residue. Since NS proteins obtained from naturally occurring revertants of E1 were indistinguishable from the wild-type protein in all of these analyses, the structural alteration in the mutant NS protein correlates with the functional lesion. Because E1 is defective in the RNA replication pathway at the restrictive temperature, a replicative role is proposed for the NS protein.
Japanese encephalitis virus (JEV) core protein was detected in both the nucleoli and cytoplasm of mammalian and insect cell lines infected with JEV or transfected with the expression plasmid of the core protein. Mutation analysis revealed that Gly(42) and Pro(43) in the core protein are essential for the nuclear and nucleolar localization. A mutant M4243 virus in which both Gly(42) and Pro(43) were replaced by Ala was recovered by plasmid-based reverse genetics. In C6/36 mosquito cells, the M4243 virus exhibited RNA replication and protein synthesis comparable to wild-type JEV, whereas propagation in Vero cells was impaired. The mutant core protein was detected in the cytoplasm but not in the nucleus of either C6/36 or Vero cell lines infected with the M4243 virus. The impaired propagation of M4243 in mammalian cells was recovered by the expression of wild-type core protein in trans but not by that of the mutant core protein. Although M4243 mutant virus exhibited a high level of neurovirulence comparable to wild-type JEV in spite of the approximately 100-fold-lower viral propagation after intracerebral inoculation to 3-week-old mice of strain Jcl:ICR, no virus was recovered from the brain after intraperitoneal inoculation of the mutant. These results indicate that nuclear localization of JEV core protein plays crucial roles not only in the replication in mammalian cells in vitro but also in the pathogenesis of encephalitis induced by JEV in vivo.
Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cells has been shown to possess an RNA-dependent RNA polymerase (RdRp) activity. Our initial attempt to produce the full-length BVDV NS5B with a C-terminal hexahistidine tag in Escherichia coli failed due to the expression of insoluble products. Prompted by a recent report that removal of the C-terminal hydrophobic domain significantly improved the solubility of hepatitis C virus (HCV) NS5B, we constructed a similar deletion of 24 amino acids at the C terminus of BVDV NS5B. The resulting fusion protein, NS5BDeltaCT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primer-independent (de novo) mechanisms. Furthermore, BVDV RdRp was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template. In addition to the previously described polymerase motifs A, B, C, and D, alignments with other flavivirus sequences revealed two additional motifs, one N-terminal to motif A and one C-terminal to motif D. Extensive alanine substitutions showed that while most mutations had similar effects on both elongative and de novo RNA syntheses, some had selective effects. Finally, deletions of up to 90 amino acids from the N terminus did not significantly affect RdRp activities, whereas deletions of more than 24 amino acids at the C terminus resulted in either insoluble products or soluble proteins (DeltaCT179 and DeltaCT218) that lacked RdRp activities.
Coronaviruses are positive-strand, RNA-dependent RNA polymerase-utilizing viruses that require a polymerase template switch, characterized as discontinuous transcription, to place a 5'-terminal genomic leader onto subgenomic mRNAs (sgmRNAs). The usually precise switch is thought to occur during the synthesis of negative-strand templates for sgmRNA production and to be directed by heptameric core donor sequences within the genome that match an acceptor core (UCUAAAC in the case of bovine coronavirus) near the 3' end of the 5'-terminal genomic leader. Here it is shown that a 22-nucleotide (nt) donor sequence engineered into a packageable bovine coronavirus defective interfering (DI) RNA and made to match a sequence within the 65-nt virus genomic leader caused a template switch yielding an sgmRNA with only a 33-nt minileader. By changing the donor sequence, acceptor sites between genomic nt 33 and 97 (identical between the DI RNA and the viral genome) could be used to generate sgmRNAs detectable by Northern analysis (approximately 2 to 32 molecules per cell) by 24 h postinfection. Whether the switch was intramolecular only was not determined since a potentially distinguishing acceptor region in the DI RNA rapidly conformed to that in the helper virus genome through a previously described template switch known as leader switching. These results show that crossover acceptor sites for discontinuous transcription (i) need not include the UCUAAAC core and (ii) rest within a surprisingly wide 5'-proximal "hotspot." Overlap of this hotspot with that for leader switching and with elements required for RNA replication suggests that it is part of a larger 5'-proximal multifunctional structure.
Polysomes from reovirus-infected L cells were analyzed. Although all 10 transcription products of the viral genome were represented in polysomes and presumably can be translated, fewer than 10 reovirus-specific polypeptides were detected in infected cells. The 5'-terminal sequence of all species of reovirus ribonucleic acid (RNA) from polysomes was ppGpYp, as previously found for the genome double-stranded RNA and the in vitro single-stranded products of the virion-associated RNA polymerase. RNA isolated from both heavy (>30 ribosomes) and light (5 to 8 ribosomes) polysomes includes similar amounts of large, medium, and small classes of reovirus single-stranded RNA, suggesting that linkage of the genome segments observed in virions may occur at the single-strand stage of RNA replication.
The confinement of membrane proteins by lipid-lipid interactions into specialized detergent-insoluble membrane (DIM) microdomains has been proposed as a general mechanism to recruit selectively lipid-modified proteins and specific transmembrane proteins. Poliovirus capsid VP4 protein and its precursors are myristoylated at the NH(2)-terminal Gly residue. To determine whether poliovirus uses DIMs during its replicative cycle, we isolated DIMs from poliovirus-infected HeLa cells and identified the presence of capsid proteins and their precursors, proteinases 2A and 3C, and other viral proteins involved in poliovirus RNA replication such as protein 2C and the polymerase 3D. The morphology of these DIMs was similar to that of the previously described rosette-like vesicles associated with replication complexes isolated from poliovirus-infected cells. To examine the possible role of the myristoyl moiety in the targeting of poliovirus structural proteins to DIMs, we generated a chimeric protein consisting of the nine amino-terminal amino acids from VP4 fused to the amino terminus of the green fluorescent protein (GFP). The selected VP4 sequence was sufficient to confer N-myristoylation and targeting to DIMs to the GFP chimera. Mutations within this sequence known to affect both myristoylation and poliovirus assembly abrogated the targeting of the GFP chimera. These results indicate that the myristoylated amino-terminal nonapeptide from poliovirus VP4 protein constitutes a signal for incorporation into DIMs.
Although combination therapy with interferon and ribavirin has improved the treatment for chronic hepatitis C virus (HCV) infection, the detailed anti-HCV effect of ribavirin in clinical concentrations remains uncertain. To detect the anti-HCV effect of ribavirin in lower concentrations, a sensitive and accurate assay system was developed using the reporter replicon system with an HCV genotype 2a subgenomic replicon (clone JFH-1) that exhibits robust replication in various cell lines. This reporter replicon was generated by introducing the luciferase reporter gene (instead of the neomycin resistance gene) into the subgenomic JFH-1 replicon. To assess the replication of this reporter replicon, luciferase activity was measured serially up to day 3 after transient transfection of Huh7 cells. The luciferase activity increased exponentially over the time course of the experiment. After adjustment for transfection efficiency and transfected cell viability, the impacts of interferon and ribavirin were determined. The administration of interferon and ribavirin resulted in dose-dependent suppression of replicon RNA replications. The 50% inhibitory concentration of interferon and ribavirin was 1.80 IU/ml and 3.70 microg/ml, respectively. In clinical concentrations, replications were reduced to 0.09% and 53.74% by interferon (100 IU/ml) and ribavirin (3 microg/ml), respectively. Combination use of ribavirin and interferon enhanced the anti-HCV effect of interferon by 1.46- to 1.62-fold. In conclusion, we developed an accurate and sensitive replicon system, and the antivirus effect of interferon and ribavirin was easily detected within their clinical concentrations by this replicon system. This system will provide a powerful tool for screening new antiviral compounds against HCV.