Showing papers on "RNA-dependent RNA polymerase published in 2000"

The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen.

Abstract: The ribonucleoside analog ribavirin (1-beta-D-ribofuranosyl-1,2, 4-triazole-3-carboxamide) shows antiviral activity against a variety of RNA viruses and is used in combination with interferon-alpha to treat hepatitis C virus infection. Here we show in vitro use of ribavirin triphosphate by a model viral RNA polymerase, poliovirus 3Dpol. Ribavirin incorporation is mutagenic, as it templates incorporation of cytidine and uridine with equal efficiency. Ribavirin reduces infectious poliovirus production to as little as 0. 00001% in cell culture. The antiviral activity of ribavirin correlates directly with its mutagenic activity. These data indicate that ribavirin forces the virus into 'error catastrophe'. Thus, mutagenic ribonucleosides may represent an important class of anti-RNA virus agents.

Hepatitis C Virus-Encoded Enzymatic Activities and Conserved RNA Elements in the 3′ Nontranslated Region Are Essential for Virus Replication In Vivo

Abstract: Hepatitis C virus (HCV) infection is a widespread major human health concern. Significant obstacles in the study of this virus include the absence of a reliable tissue culture system and a small-animal model. Recently, we constructed full-length HCV cDNA clones and successfully initiated HCV infection in two chimpanzees by intrahepatic injection of in vitro-transcribed RNA (A. A. Kolykhalov et al., Science 277:570-574, 1997). In order to validate potential targets for development of anti-HCV therapeutics, we constructed six mutant derivatives of this prototype infectious clone. Four clones contained point mutations ablating the activity of the NS2-3 protease, the NS3-4A serine protease, the NS3 NTPase/helicase, and the NS5B polymerase. Two additional clones contained deletions encompassing all or part of the highly conserved 98-base sequence at the 3' terminus of the HCV genome RNA. The RNA transcript from each of the six clones was injected intrahepatically into a chimpanzee. No signs of HCV infection were detected in the 8 months following the injection. Inoculation of the same animal with nonmutant RNA transcripts resulted in productive HCV infection, as evidenced by viremia, elevated serum alanine aminotransferase, and HCV-specific seroconversion. These data suggest that these four HCV-encoded enzymatic activities and the conserved 3' terminal RNA element are essential for productive replication in vivo.

Functional modulation of Escherichia coli RNA polymerase

Abstract: ▪ Abstract The promoter recognition specificity of Escherichia coli RNA polymerase is modulated by replacement of the σ subunit in the first step and by interaction with transcription factors in the second step. The overall differentiated state of ∼2000 molecules of the RNA polymerase in a single cell can be estimated after measurement of both the intracellular concentrations and the RNA polymerase-binding affinities for all seven species of the σ subunit and 100–150 transcription factors. The anticipated impact from this line of systematic approach is that the prediction of the expression hierarchy of ∼4000 genes on the E. coli genome can be estimated.

In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies.

Abstract: To study the efficiency of RNA-based vaccines, RNA coding for the model antigen beta-galactosidase (beta-gal) was transcribed in vitro from a lacZ gene flanked by stabilizing Xenopus laevis beta-globin 5' and 3' sequences and was protected from RNase degradation by condensation with the polycationic peptide protamine. The liposome-encapsulated condensed RNA-peptide complex, the condensed RNA-peptide complex without liposome or naked, unprotected RNA, was injected into BALB/c (H-2(d)) mice. All preparations led to protein expression in the local tissue, activation of L(d)-restricted specific cytotoxic T lymphocytes (CTL) and production of IgG antibodies reactive against beta-gal. RNA-triggered CTL were as efficient in the lysis of lacZ-transfected target cells as CTL triggered by a lacZ-DNA eukaryotic expression vector. Immunization with RNA transcribed from a cDNA library from the beta-gal-expressing cell line P13.1 again led to beta-gal-specific CTL and IgG induction. Thus, both naked and protected RNA can be used to elicit a specific immune response in vivo, whereby the protected RNA is stable in vitro for a longer period of time. RNA vaccines can be produced in high amounts and have the same major advantages as DNA vaccines but lack the potentially harmful effect of DNA integration into the genome.

A Structural Model of Transcription Elongation

Abstract: The path of the nucleic acids through a transcription elongation complex was tracked by mapping cross-links between bacterial RNA polymerase (RNAP) and transcript RNA or template DNA onto the x-ray crystal structure. In the resulting model, the downstream duplex DNA is nestled in a trough formed by the β′ subunit and enclosed on top by the β subunit. In the RNAP channel, the RNA/DNA hybrid extends from the enzyme active site, along a region of the β subunit harboring rifampicin resistance mutations, to the β′ subunit “rudder.” The single-stranded RNA is then extruded through another channel formed by the β-subunit flap domain. The model provides insight into the functional properties of the transcription complex.

Current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (PRRS) virus: comparison of the North American and European isolates.

Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the recently recognized Arteriviridae family within the genus Arterivirus, order Nidovirales, which also includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). Mature viral particles are composed of an envelope 50–72 nm in diameter, with an isometric core about 20–30 nm enclosing a linear positive-stranded RNA genome of approximately 15 kb. The virions are assembled by the budding of preformed nucleocapsids into the lumen of the smooth endoplasmic reticulum and/or Golgi apparatus. The mature virions are then released by exocytosis. The viral genome contains eight open reading frames (ORFs) which are transcribed in cells as a nested set of subgenomic mRNAs. The ORF1a and ORF1b situated at the 5′end of the genome represent nearly 75% of the viral genome and code for proteins with apparent replicase and polymerase activities. The major structural proteins consist of a 25 kDa envelope glycoprotein (GP5), an 18–19 kDa unglycosylated membrane protein (M), and a 15 kDa nucleocapsid (N) protein, encoded by ORFs 5, 6 and 7, respectively. The N protein is the more abundant protein of the virion and is highly antigenic, which therefore makes it a suitable candidate for the detection of virus-specific antibodies and diagnosis of the disease. Four to five domains of antigenic importance have been identified for the N protein, a common conformational antigenic site for European and North American strains being localized in the central region of the protein. In cells and virions, both M and GP5 occur in heterodimeric complexes linked by disulfide bonds. The expression products of ORFs 2 and 4 are also incorporated into virus particles as additional minor membrane-associated glycoproteins designated as GP2 and GP4, with Mr of 29 and 31 kDa, respectively. The structural nature of the ORF3 product, a highly glycosylated protein with an apparent Mr of 42 kDa, is still being debated, in view of the apparently conflicting data on its presence in virus particles. Nonetheless, the GP3 of North American and European strains has been shown to be antigenic, providing protection for piglets against PRRSV infection in the absence of a noticeable neutralizing humoral response. Pigs exposed to the native form of GP5 by means of DNA immunization develop specific neutralizing and protecting antibodies. The GP5 is involved in antigenic variability, apoptosis, and possibly antibody-dependent enhancement phenomena. The GP4 also possesses antigenic determinants that trigger the immune system to produce neutralizing antibodies. Each of the PRRSV structural proteins carries common and type-specific antigenic determinants that permit the ability to differentiate between European and North American strains. The potential use of the PRRSV structural proteins in subunit recombinant-type vaccines is also discussed.

Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals.

Abstract: Transcript elongation by RNA polymerase is discontinuous and interrupted by pauses that play key regulatory roles. We show here that two different classes of pause signals punctuate elongation. Class I pauses, discovered in enteric bacteria, depend on interaction of a nascent RNA structure with RNA polymerase to displace the 3' OH away from the catalytic center. Class II pauses, which may predominate in eukaryotes, cause RNA polymerase to slide backwards along DNA and RNA and to occlude the active site with nascent RNA. These pauses differ in their responses to antisense oligonucleotides, pyrophosphate, GreA, and general elongation factors NusA and NusG. In contrast, substitutions in RNA polymerase that increase or decrease the rate of RNA synthesis affect both pause classes similarly. We propose that both pause classes, as well as arrest and termination, arise from a common intermediate that itself binds NTP substrate weakly.

Single-molecule study of transcriptional pausing and arrest by E. coli RNA polymerase.

Abstract: Using an optical-trap/flow-control video microscopy technique, we followed transcription by single molecules of Escherichia coli RNA polymerase in real time over long template distances. These studies reveal that RNA polymerase molecules possess different intrinsic transcription rates and different propensities to pause and stop. The data also show that reversible pausing is a kinetic intermediate between normal elongation and the arrested state. The conformational metastability of RNA polymerase revealed by this single-molecule study of transcription has direct implications for the mechanisms of gene regulation in both bacteria and eukaryotes.

Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg

Abstract: The first step in the replication of the plus-stranded poliovirus RNA is the synthesis of a complementary minus strand. This process is initiated by the covalent attachment of UMP to the terminal protein VPg, yielding VPgpU and VPgpUpU. We have previously shown that these products can be made in vitro in a reaction that requires only synthetic VPg, UTP, poly(A), purified poliovirus RNA polymerase 3Dpol, and Mg2+ (A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280–284, 1998). Since such a poly(A)-dependent process cannot confer sufficient specificity to poliovirus RNA replication, we have developed a new assay to search for a viral RNA template in conjunction with viral or cellular factors that could provide this function. We have now discovered a small RNA hairpin in the coding region of protein 2C as the site in PV1(M) RNA that is used as the primary template for the in vitro uridylylation of VPg. This hairpin has recently been described in poliovirus RNA as being an essential structure for the initiation of minus strand RNA synthesis (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590–4600, 2000). The uridylylation reaction either with transcripts of cre(2C) RNA or with full-length PV1(M) RNA as the template is strongly stimulated by the addition of purified viral protein 3CDpro. Deletion of the cre(2C) RNA sequences from minigenomes eliminates their ability to serve as template in the reaction. A similar signal in the coding region of VP1 in HRV14 RNA (K. L. McKnight and S. M. Lemon, RNA 4:1569–1584, 1998) and the poliovirus cre(2C) can be functionally exchanged in the assay. The mechanism by which the VPgpUpU precursor, made specifically on the cre(2C) template, might be transferred to the site where it serves as primer for poliovirus RNA synthesis, remains to be determined.

De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus

Abstract: Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn(2+) than in the presence of Mg(2+). When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a "copy-back" mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3' end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (>/=50 microM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.

Identification of a cis-Acting Replication Element within the Poliovirus Coding Region

Abstract: Poliovirus, the archetypal picornavirus, is arguably one of the best characterized of all viruses. The availability of infectious molecular clones (33) has enabled the application of reverse genetics to understand the function of the nonstructural proteins and the noncoding regions (NCR) of the virus genome (reviewed in reference 41). The 7.4-kb single-stranded positive (messenger)-sense RNA genome encodes a single polyprotein and contains the necessary signals for virus replication in the cell cytoplasm. Posttranslational cleavage of the polyprotein by virus-encoded proteases (2Apro and 3Cpro) yields the four capsid proteins (VP1 to VP4), the RNA-dependent RNA polymerase (3Dpol), and the accessory proteins required for replication. The input RNA acts as a template for the synthesis of a negative strand which, in turn, is used as a template for synthesis of genome sense RNA. The virus-encoded protein VPg (3A) is implicated as a protein primer for both positive- and negative-sense strand initiation, as are RNA sequences occupying the 5′ NCR of the virus genome. A cloverleaf (CL) structure of 88 nucleotides (nt) at the 5′ end of the genome interacts with the virus 3CproDpol and either the VPg-containing precursor 3AB or the cellular poly-C binding protein type 2 (PCBP2) (2, 3, 11, 28). This 5′ NCR ribonucleoprotein complex is required for replication and may also be involved in the suppression of virus translation (10). RNA sequences and structures within the 3′ NCR presumed to be involved in replication are much less well defined. Chimeric polioviruses in which the 3′ NCR is replaced by the analogous region of other picornaviruses generally retain viability (35), even though there is little sequence or structural homology between substituted 3′ NCR. These results are reflected in the cross-competition for cellular proteins seen between the poliovirus, coxsackievirus, and rhinovirus 3′ NCR in vitro (21). In contrast to the gross changes to 3′ NCR structure, defined mutations affecting formation of a pseudoknot (20, 25), or—in the rhinovirus type 14 3′ NCR—within a conserved sequence at the base of the structure or the terminal loop (22, 35), adversely affect replication. Characterization of revertants selected from such mutants have identified interacting RNA structures (25) or proteins (22). These results suggest that, although there is a degree of flexibility in the 3′ NCR sequences and structures that can be accommodated, this region of the genome plays an important function in the replication cycle of the virus. However, this conclusion is at odds with the observation that poliovirus or rhinovirus genomes from which the 3′ NCR is deleted retain the ability to replicate, albeit rather poorly (22, 39, 40). Therefore, the 3′ NCR cannot be an obligatory component for the initiation of negative-strand synthesis, which in vitro studies show can be primed by 3Dpol and VPg alone (29), but presumably influences the efficiency of the process. The encapsidation of newly synthesized genomes completes the formation of infectious virions. The specificity of this process (5, 14, 32) can be readily monitored using subgenomic replicons, in which the capsid-encoding P1 region of the genome is replaced with a reporter gene such as that coding for chloramphenicol acetyl transferase (CAT) (30). We have used such replicons to demonstrate that RNA sequences involved in packaging are not located within the P1, 5′ NCR, 3′ NCR, or VPg-encoding regions (5, 30). A characteristic of 5′ NCR sequences involved in translation and replication and of sequences in the 3′ NCR and an expected feature of encapsidation signals is that they all form secondary structures (1, 35–37). In an attempt to identify further functional stem-loop structures, we searched the polyprotein-coding region of the poliovirus genome for such elements. A candidate structure was identified computationally within the 2C-encoding region and disrupted by site-directed mutagenesis. Our results suggest that the 61-nt stem-loop is a location-independent cis-acting replication element (CRE) required in the positive sense for function. This is the first report of a CRE within the polyprotein-encoding region of poliovirus. The recent identification of an analogous element in human rhinovirus type 14 (HRV14) (18, 19), although distinctly different in sequence, structure, and location, and the presence of similar elements in other positive-sense RNA viruses may reflect a replication mechanism evolutionarily conserved by this widely divergent group of viruses.

Strategy for Systematic Assembly of Large RNA and DNA Genomes: Transmissible Gastroenteritis Virus Model

Abstract: A systematic method was developed to assemble functional full-length genomes of large RNA and DNA viruses. Coronaviruses contain the largest single-stranded positive-polarity RNA genome in nature. The approximately 30-kb genome, coupled with regions of genomic instability, has hindered the development of a full-length infectious cDNA construct. We have assembled a full-length infectious construct of transmissible gastroenteritis virus (TGEV), an important pathogen in swine. Using a novel approach, six adjoining cDNA subclones that span the entire TGEV genome were isolated. Each clone was engineered with unique flanking interconnecting junctions which determine a precise systematic assembly with only the adjacent cDNA subclones, resulting in an intact TGEV cDNA construct of approximately 28.5 kb in length. Transcripts derived from the full-length TGEV construct were infectious, and progeny virions were serially passaged in permissive host cells. Viral antigen production and subgenomic mRNA synthesis were evident during infection and throughout passage. Plaque-purified virus derived from the infectious construct replicated efficiently and displayed similar plaque morphology in permissive host cells. Host range phenotypes of the molecularly cloned and wild-type viruses were similar in cells of swine and feline origin. The recombinant viruses were sequenced across the unique interconnecting junctions, conclusively demonstrating the marker mutations and restriction sites that were engineered into the component clones. Full-length infectious constructs of TGEV will permit the precise genetic modification of the coronavirus genome. The method that we have designed to generate an infectious cDNA construct of TGEV could theoretically be used to precisely reconstruct microbial or eukaryotic genomes approaching several million base pairs in length.

PRRSV, the virus.

Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-strand RNA virus that belongs to the Arteriviridae family. PRRSV grows in primary alveolar macrophages and in monkey kidney cell lines. The genomic RNA is approximately 15 kb. The genome encodes the RNA replicase (ORF1a and ORF1b), the glycoproteins GP2 to GP5, the integral membrane protein M, and the nucleocapsid protein N (ORFs 2 to 7). A comparison of nucleotide sequences of different strains indicates that European and North American strains represent two distinct antigenic types. Various PRRSV-specific monoclonal antibodies and recombinant structural proteins have been produced. Well-defined PRRSV mutants can be generated with the recently developed infectious cDNA clone of PRRSV.

De Novo Initiation of RNA Synthesis by Hepatitis C Virus Nonstructural Protein 5B Polymerase

Abstract: RNA-dependent RNA polymerase (RdRp) encoded by positive-strand RNA viruses is critical to the replication of viral RNA genome. Like other positive-strand RNA viruses, replication of hepatitis C virus (HCV) RNA is mediated through a negative-strand intermediate, which is generated through copying the positive-strand genomic RNA. Although it has been demonstrated that HCV NS5B alone can direct RNA replication through a copy-back primer at the 3' end, de novo initiation of RNA synthesis is likely to be the mode of RNA replication in infected cells. In this study, we demonstrate that a recombinant HCV NS5B protein has the ability to initiate de novo RNA synthesis in vitro. The NS5B used HCV 3' X-tail RNA (98 nucleotides) as the template to synthesize an RNA product of monomer size, which can be labeled by ?gamma-(32)Pnucleoside triphosphate. The de novo initiation activity was further confirmed by using small synthetic RNAs ending with dideoxynucleotides at the 3' termini. In addition, HCV NS5B preferred GTP as the initiation nucleotide. The optimal conditions for the de novo initiation activity have been determined. Identification and characterization of the de novo priming or initiation activity by HCV NS5B provides an opportunity to screen for inhibitors that specifically target the initiation step.

Structures of two RNA domains essential for hepatitis C virus internal ribosome entry site function.

Abstract: Translation of the hepatitis C virus (HCV) polyprotein is initiated at an internal ribosome entry site (IRES) element in the 5' untranslated region of HCV RNA. The HCV IRES element interacts directly with the 40S subunit, and biochemical experiments have implicated RNA elements near the AUG start codon as required for IRES-40S subunit complex formation. The data we present here show that two RNA stem loops, domains IIId and IIIe, are involved in IRES-40S subunit interaction. The structures of the two RNA domains were solved by NMR spectroscopy and reveal structural features that may explain their role in IRES function.

RNA polymerase III-based expression of therapeutic RNAs

Abstract: A transcribed non-naturally occuring RNA molecule comprising a desired RNA molecule, wherein the 3' region of the RNA is able to base-pair with at least 8 bases at the 5' terminus of the same RNA molecule.

Genetic analysis of the compatibility between polymerase proteins from human and avian strains of influenza A viruses.

Abstract: In order to determine how efficiently the polymerase proteins derived from human and avian influenza A viruses can interact with each other in the context of a mammalian cell, a genetic system that allows the in vivo reconstitution of active ribonucleoproteins was used. The ability to achieve replication of a viral-like reporter RNA in COS-1 cells was examined with heterospecific mixtures of the core proteins (PB1, PB2, PA and NP) from two strains of human viruses (A/Puerto Rico/8/34 and A/Victoria/3/75), two strains of avian viruses (A/Mallard/NY/6750/78 and A/FPV/-Rostock/34), and a strain of avian origin (A/Hong Kong/156/97) that was isolated from the first human case of H5N1 influenza in Hong Kong in 1997. In accordance with published observations on reassortant viruses, PB2 amino acid 627 was identified as a major determinant of the replication efficiency of heterospecific complexes in COS-1 cells. Moreover, the results showed that replication of the viral-like reporter RNA was more efficient when PB2 and NP were both derived from the same avian or human virus or when PB1 was derived from an avian virus, whatever the origin of the other proteins. Furthermore, the PB1 and PB2 proteins from the A/Hong- Kong/156/97 virus exhibited intermediate properties with respect to the corresponding proteins from avian or human influenza viruses, suggesting that some molecular characteristics of PB1 and PB2 proteins might at least partially account for the ability of the A/Hong Kong/156/97 virus to replicate in humans.

Poliovirus Requires a Precise 5′ End for Efficient Positive-Strand RNA Synthesis

Abstract: Poliovirus infectious RNA can be synthesized in vitro using phage DNA-dependent RNA-polymerases. These synthetic transcripts contain several extra nucleotides at the 5' end, which are deleted during replication to generate authentic viral genomes. We removed those 5'-end extra nucleotides utilizing a hammerhead ribozyme to produce transcripts with accurate 5' ends. These transcripts replicate substantially more rapidly in cell culture, demonstrating no lag before replication; they also replicate more efficiently in Xenopus laevis oocytes and in in vitro translation-replication cell extracts. In both systems, an exact 5' end is necessary for synthesis of positive-strand RNA but not negative-strand RNA.

The end of the (DNA) line

Abstract: Telomerases contain an essential RNA subunit (TER), as well as an essential protein reverse transcriptase subunit (TERT). The RNA subunit includes a short template region that is copied into telomeric DNA, but otherwise it is large and divergent. However, phylogenetic studies have revealed a conserved core secondary structure for TER. Much of the divergence can be accounted for by the acquisition of different types of RNA domains that function in RNA stabilization. Some of the nontemplate portions of TER, which include regions in the conserved core, are important for aspects of telomerase enzymatic activity independent of their role in telomerase assembly. Mutational studies indicate that telomerase enzyme function results from a collaboration of both protein and RNA functional groups contributed by TERT and TER.