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[Characteristics of messenger RNA for interferon and of interferon-inducing RNA isolated from Newcastle disease virus infected cells].

Two kinds of RNA capable of producing interferon in a heterologous cell system occur in the cells producing interferon in response to induction by Newcastle disease virus. One of them has messenger activity for interferon and is presented by a single-stranded structure with sedimentation constant of 10-22 S. This RNA appears to belong to cellular messenger RNA. The other is capable of inducing interferon production by heterogenous cells, resistant to treatment with RN-ase and has sedimentation constant greater than 35 S. In our opinion, this RNA is the primary replicative RNA of the virus-inducer.

Culture Techniques↗
PubMed2001

Generation and genetic characterization of avian reovirus temperature-sensitive mutants.

There currently is little known about the genetic and biological functions of avian reovirus (ARV), an atypical member of the family Reoviridae and the prototype of all nonenveloped viruses that induce syncytia formation. In this study, we created ARV temperature-sensitive (ts) mutants by chemical mutagenesis of ARV strain 138. We developed a novel efficiency of lysis (EOL) screening technique and used it and the classical efficiency of plating (EOP) assay to identify 17 ARV ts mutants. Pairwise mixed infections of these mutants and evaluation of recombinant progeny ts status led to their organization into seven recombination groups. This indicates that these new groups of mutants represent the majority of the ARV genome. To phenotypically characterize the ts mutants, progeny double-stranded RNA (dsRNA) produced at permissive and nonpermissive temperature was measured. Some mutants were capable of dsRNA synthesis at the restrictive temperature (RNA(+)), which indicates the effects of their ts lesions occur after RNA replication. Most mutants were RNA(-), which suggests their mutations affect stages in viral replication that precede progeny genome synthesis.

Animals↗
PubMed2004

Hepatitis C virus subgenomic replicons in the human embryonic kidney 293 cell line.

Hepatitis C virus (HCV) infects liver cells and its replication in other cells is incompletely defined. Human hepatoma Huh-7 cells harboring subgenomic HCV replicons were used in somatic cell fusion experiments with human embryonic kidney 293 cells as a means of examining the permissiveness of 293 cells for HCV subgenomic RNA replication. 293 cells were generally not permissive for replication of Huh-7 cell-adapted replicons. However, upon coculturing of the two cell lines, we selected rare replicon-containing cells, termed 293Rep cells, that resembled parental 293 cells. Direct metabolic labeling of cells with (33)P in the presence of actinomycin D and Northern blotting to detect the negative strand of the replicon demonstrated functional RNA replicons in 293Rep cells. Furthermore, Western blots revealed that 293Rep cells expressed the HCV nonstructural proteins as well as markers of the naïve 293 cells but not Huh-7 cells. Propidium iodide staining and fluorescence-activated cell sorting analysis of 293Rep cells revealed that clone 293Rep17 closely resembled naïve 293 cells. Transfection of total RNA from 293Rep17 into naïve 293 cells produced replicon-containing 293 cell lines with characteristics distinct from those of Huh-7-derived replicon cell lines. Relative to Huh-7 replicons, the 293 cell replicons were less sensitive to inhibition by alpha interferon and substantially more sensitive to inhibition by poly(I)-poly(C) double-stranded RNA. This study established HCV subgenomic replicons in nonhepatic 293 cells and demonstrated their utility in expanding the study of cellular HCV RNA replication.

Cell Fusion↗
PubMed1984

Recombination and rescue between temperature-sensitive mutants of poliovirus type 1.

Four different temperature-sensitive (ts) mutants derived from the Mahoney strain of poliovirus type 1, were crossed in an infectious center recombination test. Evidence for recombination was obtained in three crosses, with a different segregation of an unselected marker, resistance to guanidine, in each case. Evidence for genetic complementation between ts mutants was not found, except with one set of RNA- mutants, ts 221 and ts 035. The marked virus yield enhancement which was observed in cells mixedly infected by these two mutants resulted from a nonreciprocal rescue of ts 035 by ts 221. The effects of ts 221 input multiplicity and of guanidine inhibition of viral RNA replication on the rescue were analyzed. The results showed that yield enhancement of ts 035 in mixed infection could be correlated to the low level RNA replication of ts 221 at the nonpermissive temperature.

Animals↗
PubMed2000

Template/primer requirements and single nucleotide incorporation by hepatitis C virus nonstructural protein 5B polymerase.

Nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3' terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3' terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di- or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3' terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique beta-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.

Genome, Viral↗
PubMed2004

Mutational Analysis of Hepatitis C Virus NS5B in the Subgenomic Replicon Cell Culture.

The hepatitis C virus (HCV) NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme of HCV RNA replication. We previously identified five novel residues of NS5B in a JK-1 isolate indispensable for RdRP activity in vitro (Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S. (2001) Hepatology 33, 728-737). We addressed the role of these residues in HCV RNA replication using a HCV replicon system derived from an M1LE isolate (Kishine, H., Sugiyama, K., Hijikata, M., Kato, N., Takahashi, H., Noshi, T., Nio, Y., Hosaka, M., Miyanari, Y., and Shimotohno, K. (2002) Biochem. Biophys. Res. Commun. 293, 993-999). The five residues of NS5B in M1LE were found to be critical for HCV replication in vivo and also indispensable for RdRP activity in vitro along with purified bacterial recombinant proteins. We also found a chimeric replicon of JK-1 and M1LE in which only the NS5B sequence derived from JK-1 could not replicate in Huh-7 cells. The residues responsible for the phenomenon were mapped by several chimeric and substituted forms of NS5B M1LE and/or JK-1 isolates in the HCV RNA replicon. Two residues, amino acids 220 and 288, were critical, and two residues, amino acids 213 and 231, were important for efficient HCV replication. Mutant JK-1 NS5B harboring all four residues of M1LE was replication-competent in the chimeric replicon and was as efficient as the original M1LE replicon. By comparing the replication competence in vivo and RdRP activity in vitro with various chimeric and mutated versions of NS5B, the HCV replication ability was found to correlate well with the RdRP activity. However, heat- and dilution-sensitive NS5Bs exhibiting weaker RdRP activity in vitro were found to be replication-incompetent, suggesting that HCV replication requires RdRP activity higher than a certain critical threshold.

Amino Acid Sequence↗
PubMed2006

Inhibition of HIV-1 replication by RNA interference of p53 expression.

p53 expression and activation have been associated to faster human immunodeficiency virus (HIV) disease progression, most probably by inducing CD4+ T cell death but also through its cooperative effect in the control of viral gene transcription by viral regulatory proteins. Here, we show that RNA interference of p53 in HIV-1 reporter (HeLa P4-R5 MAGI) and lymphoid (SupT1) cell lines blocked HIV-1 and Tat-induced transcription from the HIV-1 promoter and HIV-1 replication in acutely infected cells, suggesting a cooperative role of p53 in HIV-1 transcription. Contrary to SupT1 cells, which encode several mutations on the p53 DNA binding domain, death of HIV-1-induced syncytia was reduced in cocultures of HeLa P4-R5 MAGI with persistently infected HIV-1 cells. To our knowledge, this is the first demonstration of the effect of the loss of function of p53 in HIV-1 replication, which is independent on its classical DNA binding activity. Our results suggest two independent roles for p53 in HIV-1 infection: cooperation in HIV long-terminal repeat transcription and virus-induced cell death.

Cell Death↗
PubMed1997

In vivo DNA expression of functional brome mosaic virus RNA replicons in Saccharomyces cerevisiae.

To facilitate manipulation of brome mosaic virus (BMV) RNA replicons in Saccharomyces cerevisiae and for yeast genetic analysis of BMV RNA replication, gene expression, and host interactions, we constructed DNA plasmids from which BMV RNA3 and RNA3 derivatives can be transcribed in vivo from the galactose-inducible yeast GAL1 promoter and terminated by a self-cleaving ribozyme at or near their natural 3' ends. In galactose-induced yeast harboring such plasmids, expression of BMV RNA replication proteins 1a and 2a led to synthesis of negative-strand RNA3, amplification of positive-strand RNA3 to levels over 45-fold higher than those of DNA-derived RNA3 transcripts, and synthesis of the RNA3-encoded subgenomic mRNA for coat protein. Although the GAL1 promoter initiated transcription from multiple sites, 1a and 2a selectively amplified RNA3 with the authentic viral 5' end. As expected, reporter genes substituted for the 3'-proximal coat protein gene could not be translated directly from DNA-derived RNA3 transcripts, so their expression depended on 1a- and 2a-directed subgenomic mRNA synthesis. In yeast in which DNA transcription of B3CAT, an RNA3 derivative with the chloramphenicol acetyltransferase (CAT) gene replacing the coat gene, was induced, CAT activity remained near background levels in the absence of 1a and 2a but increased over 500,000-fold when 1a and 2a were expressed. Similarly, a plasmid encoding B3URA3, an RNA3 derivative with the yeast URA3 gene replacing the coat gene, conferred uracil-independent growth to ura3- yeast only after 1a and 2a expression and galactose induction. Once its 1a- and 2a-dependent replication was initiated, B3URA3 was maintained in dividing yeast as a free RNA replicon, even after repression of the GAL1 promoter or the loss of the B3URA3 cDNA plasmid. These findings should be useful for many experimental purposes.

Base Sequence↗
PubMed1999

The major attenuating mutations of the respiratory syncytial virus vaccine candidate cpts530/1009 specify temperature-sensitive defects in transcription and replication and a non-temperature-sensitive alteration in mRNA termination.

The live-attenuated respiratory syncytial virus vaccine candidate cpts530/1009 was previously shown to contain two separate amino acid changes in the L protein, mutations 530 and 1009 (Phe-521-->Leu and Met-1169-->Val, respectively, according to the amino acid sequence of the L protein). Each mutation independently specifies temperature-sensitive (ts) and attenuation phenotypes. In this study, we examined the effects of these mutations on transcription and RNA replication, using complete infectious recombinant virus as well as a plasmid-based minireplicon system, the latter under conditions in which effects on replication and transcription are uncoupled. In comparison with recombinant wild-type virus, the 530 and 1009 viruses were partially restricted at 37 degrees C for RNA replication, mRNA synthesis, and virus growth. The 1009 virus was partially restricted for RNA synthesis and virus growth even at 32 degrees C, which suggested that the 1009 mutation has a non-ts component in addition to the ts component. Interestingly, the synthesis of polycistronic readthrough mRNAs was elevated 1.6- to 3.8-fold for the 1009 virus, and this defect was non-ts. Studies with the minigenome system showed that the 530 and 1009 mutations each directly affect both replication and transcription, that the effect on replication was marginally greater than on transcription for the 530 mutation, and that the increase in readthrough mRNA associated with the 1009 mutation also was observed with the minigenome system.

Mutation↗
PubMed2000

Functional analysis of the genomic and antigenomic promoters of human respiratory syncytial virus.

The promoters involved in transcription and RNA replication by respiratory syncytial virus (RSV) were examined by using a plasmid-based minireplicon system. The 3' ends of the genome and antigenome, which, respectively, contain the 44-nucleotide (nt) leader (Le) and 155-nt trailer-complement (TrC) regions, should each contain a promoter for RNA replication. The 3' genome end also should have the promoter for transcription. Substitution for the Le with various lengths of TrC demonstrated that the 3'-terminal 36 nt of TrC are sufficient for extensive (but not maximal) replication and that when juxtaposed with a transcription gene-start (GS) signal, this sequence was also able to direct transcription. It was also shown that the region of Le immediately preceding the GS signal of the first gene could be deleted with either no effect or with a slight decrease in transcription initiation. Thus, the TrC is competent to direct transcription even though it does not do so in nature, and the partial sequence identity it shares with the 3' end of the genome likely represents the important elements of a conserved promoter active in both replication and transcription. Increasing the length of the introduced TrC sequence incrementally to 147 nt resulted in a fourfold increase in replication and a nearly complete inhibition of transcription. These two effects were unrelated, implying that transcription and replication are not interconvertible processes mediated by a common polymerase, but rather are independent processes. The increase in replication was specific to the TrC sequence, implying the presence of a nonessential, replication-enhancing cis-acting element. In contrast, the inhibitory effect on transcription was due solely to the altered spacing between the 3' end of the genome and GS signal, which implies that the transcriptase recognizes the first GS signal as a promoter element. Neither the enhancement of replication nor the inhibition of transcription was due to increased base-pairing potential between the 3' and 5' ends. The relative strengths of the Le and TrC promoters for directing RNA synthesis were compared and found to be very similar. Thus, these findings highlighted a high degree of functional similarity between the RSV antigenomic and genomic promoters, but provided a further distinction between promoter requirements for transcription and replication.

5' Untranslated Regions↗
PubMed2002

DNA-directed expression of an animal virus RNA for replication-dependent colony formation in Saccharomyces cerevisiae.

To date, the insect nodavirus flock house virus (FHV) is the only virus of a higher eukaryote that has been shown to undergo a full replicative cycle and produce infectious progeny in the yeast Saccharomyces cerevisiae. The genome of FHV is composed of two positive-sense RNA segments: RNA1, encoding the RNA replicase, and RNA2, encoding the capsid protein precursor. When yeast cells expressing FHV RNA replicase were transfected with a chimeric RNA composed of a selectable gene flanked by the termini of RNA2, the chimeric RNA was replicated and transmitted to daughter cells indefinitely. In the work reported here, we developed a system in which a selectable chimeric RNA replicon was transcribed from an inducible RNA polymerase II (polII) promoter in vivo in yeast. To render marker gene expression absolutely dependent on RNA replication, the primary polII transcript was made negative in sense and contained an intron that blocked the translation of cryptic transcripts from the opposite DNA strand. The RNA products of DNA-templated transcription, processing, and RNA replication were characterized by Northern blot hybridization and primer extension analysis. Marker gene expression and colony growth under selective conditions depended strictly on FHV RNA replication, with background colonies arising at a frequency of fewer than 1 in 10(8) plated cells. The utility of the system was demonstrated by introducing a second chimeric replicon and showing that at least two different selectable markers could be simultaneously expressed by means of RNA replication. This is the first example of FHV RNA1-dependent selectable marker expression initiated in vivo and will greatly facilitate the identification and characterization of the requirements and inhibitors of RNA replication.

Animals↗
PubMed2003

Replication of the hepatitis C virus in cell culture.

Studies of hepatitis C virus (HCV) replication in cell culture have been greatly facilitated by the development of genetically engineered viral genomes that are capable of self-amplifying to high levels in a human hepatoma cell line. Since the original description of this 'replicon' model in 1999, important improvements have been made. Most notably, cell culture adaptive mutations were identified in various non-structural proteins that enhance RNA replication by several orders of magnitude. More recently, the permissiveness of the host cell was determined as an additional important factor contributing to efficient RNA replication. These discoveries allowed the development of transient replication assays, selectable full length genomes and a variety of novel replicons that will be useful for basic studies and facilitate the development of antiviral drugs. Ultimately, the replicon system may help to decipher the molecular basis of interferon-alpha (IFN-alpha) resistance.

Adaptation, Biological↗
PubMed2001

Characterization of cell lines carrying self-replicating hepatitis C virus RNAs.

Subgenomic selectable RNAs of the hepatitis C virus (HCV) have recently been shown to self-replicate to high levels in the human hepatoma cell line Huh-7 (V. Lohmann, F. Körner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartenschlager, Science 285:110-113, 1999). Taking advantage of this cell culture system that allows analyses of the interplay between HCV replication and the host cell, in this study we characterized two replicon-harboring cell lines that have been cultivated for more than 1 year. During this time, we observed no signs of cytopathogenicity such as reduction of growth rates or ultrastructural changes. High levels of HCV RNAs were preserved in cells passaged under continuous selection. When selective pressure was omitted replicon levels dropped, but depending on culture conditions the RNAs persisted for more than 10 months. A tight coupling of the amounts of HCV RNA and proteins to host cell growth was observed. Highest levels were found in exponentially growing cells, followed by a sharp decline in resting cells, suggesting that cellular factors required for RNA replication and/or translation vary in abundance and become limiting in resting cells. Studies of polyprotein processing revealed rapid cleavages at the NS3/4A and NS5A/B sites resulting in a rather stable NS4AB5A precursor that was processed slowly into individual products. Half-lives (t(1/2)s) of mature proteins ranged from 10 to 16 h, with the exception of the hyperphosphorylated form of NS5A, which was less stable (t(1/2), approximately 7 h). Results of immunoelectron microscopy revealed an association of the majority of viral proteins with membranes of the endoplasmic reticulum, suggesting that this is the site of RNA replication. In summary, replicon-bearing cells are a good model for viral persistence, and they allow the study of various aspects of the HCV life cycle.

Hepacivirus↗
PubMed2003

An internally located RNA hairpin enhances replication of Tomato bushy stunt virus RNAs.

Defective interfering (DI) RNAs of Tomato bushy stunt virus (TBSV), a plus-sense RNA virus, comprise four conserved noncontiguous regions (I through IV) derived from the viral genome. Region III, a 70-nucleotide-long sequence corresponding to a genomic segment located 378 nucleotides upstream of the 3' terminus of the genome, has been found to enhance DI RNA accumulation by approximately 10-fold in an orientation-independent manner (D. Ray and K. A. White, Virology 256:162-171, 1999). In this study, a more detailed structure-function analysis of region III was conducted. RNA secondary-structure analyses indicated that region III contains stem-loop structures in both plus and minus strands. Through deletion analyses of a DI RNA, a primary determinant of region III activity was mapped to the 5'-proximal 35-nucleotide segment. Compensatory-type mutational analyses showed that a stem-loop structure in the minus strand of this subregion was required for enhanced DI RNA replication. The same stem-loop structure was also found to function in a position-independent manner in a DI RNA (albeit at reduced levels) and to be important for efficient accumulation within the context of the TBSV genome. Taken together, these observations suggest that the 5'-proximal segment of region III is a modular RNA replication element that functions primarily through the formation of an RNA hairpin structure in the minus strand.

Base Sequence↗
PubMed2005

Mapping of the RNA promoter of Newcastle disease virus.

The RNA promoters of the genome and antigenome of Newcastle disease virus (NDV) were studied by mutational analysis of their 3' terminal ends. Similarly to other paramyxoviruses, NDV RNA replication follows the rule of six, and the genomic and antigenomic promoters require two discontinuous regions: conserved region I (first 18 nucleotides) and conserved region II (nucleotides 73-90). Proper spacing between those regions and the phase of six in region II is critical for efficient RNA promoter activity. As expected, the gene start signal at the 3' end of the NDV genome was required for mRNA transcription, but not for RNA replication. Surprisingly, mutation of the polyadenylation signal in the 5' end did not affect gene expression transcription. Although the conserved region I of NDV (avulavirus) promoter appears to be more similar to that of Sendai virus (SeV) (respirovirus), conserved region II is analogous to that of rubulaviruses.

Cell Line↗
PubMed2003

Molecular analysis of the cucumber leaf spot virus genome.

Full-length clones of the genome of the Aureusvirus, Cucumber leaf spot virus (CLSV), have been constructed and infectious T7 polymerase derived synthetic transcripts have been produced. Mutational analysis of the genome indicates a role for p84 in viral RNA replication, the CP in systemic movement, p27 in viral cell-to-cell movement and p17 in symptom induction. A CLSV mutant lacking ORFs for the CP, p27 and p17 (CLSV YX) was capable of replication and systemic movement in transgenic Nicotiana benthamiana plants expressing the Red clover necrotic mosaic virus (RCNMV) movement protein (MP) suggesting that p25 and p84 are sufficient for viral RNA replication and that the RCNMV MP can permit CLSV cell-to-cell as well as systemic movement. Moreover, CLSV YX induced severe necrosis in both inoculated and uninoculated leaves of transgenic plants suggesting that CLSV p25 and/or p84 are important determinants of the necrotic phenotype. Another mutant similar to CLSV YX but expressing only limited amino-terminal portions of CP, p27 and p17 failed to produce necrosis or to move systemically in RCNMV MP transgenic N. benthamiana plants. These results suggest that these short translated regions or cis-acting sequences present in the CLSV CP, p27 and/or p17 ORFs suppress the necrosis induced by p25/p84 and also suppress systemic movement mediated by the RCNMV MP.

Amino Acid Sequence↗
PubMed1999

Cytopathogenic and noncytopathogenic RNA replicons of classical swine fever virus.

To determine the minimal requirements for autonomous RNA replication of classical swine fever virus (CSFV), genomes having in-frame deletions within the genes for structural and flanking nonstructural proteins were constructed, based on an infectious cDNA clone of CSFV Alfort/187. RNA was transcribed in vitro from the respective plasmids and transfected into SK-6 swine kidney cells. The replication competence of the RNA was determined by immunostaining transfected cells for CSFV NS3 protein and by analysis of cell extracts for viral RNA, as well as protein synthesis at different times after transfection. The genes encoding N(pro), C, E(rns), E1, E2, p7, and NS2 proved to be dispensable for RNA replication, but the efficiency of replication varied strongly between individual constructs. RNA replicons containing the complete NS2-NS3 gene persisted in transfected cells and continued to replicate without causing any obvious morphological or functional damage to the cells, whereas genomes lacking the NS2 gene replicated more efficiently and induced a cytopathic effect. These findings suggest that NS2, although it is not essential for pestivirus RNA replication, has a regulatory function therein. Both cytopathogenic and noncytopathogenic replicons were packaged into virus particles provided in trans by a cotransfected full-length helper virus genome.

Animals↗