RNA-dependent RNA Polymerase Protein Research Articles

PARTIAL MOLECULAR CHARACTERIZATION OF A CHINESE ISOLATE OF GRAPEVINE LEAFROLL-ASSOCIATED VIRUS 2 AND PRODUCTION OF ANTISERA TO RECOMBINANT VIRAL PROTEINS

The genes encoding RNA-dependent RNA polymerase (RdRp) and coat protein (CP) of a Chinese isolate of Grapevine leafroll-associated virus 2 (GLRaV-2- CF) were cloned in the vector pET-28a (+) and sequenced. Comparisons of these sequences with those of other GLRaV-2 isolates available from databases revealed homology levels with GLRaV-2-PN and GLRaV- 2-Sem higher than 98% at the nucleotide and amino acid levels. GLRaV-2-CF had lower homology with GLRaV-2-RG, another distinct strain, with similarities of 78% (RdRp) and 79% (CP) at the nucleotide level. The cloned RdRp and CP were successfully expressed in Escherichia coli BL-21 (DE3 plysS) and the resulting recombinant proteins were used to raise antisera in rabbits. Tests using these antisera readily detected the recombinant proteins in Western blots and the native virus proteins in infected grapevine samples.

Complete nucleotide sequence and genome organization of a dsRNA partitivirus infecting Pleurotus ostreatus

The nucleotide sequences of the genomic dsRNA mycovirus infecting Pleurotus ostreatus ( P. ostreatus virus 1; PoV1) were determined and compared to the sequences of the other mycoviruses belonging to partitiviruses and totivirues. PoV1 dsRNA-1 and dsRNA-2 had genomes of 2296 and 2223 nucleotides, respectively. The purified virus preparations contained isometric particles of 28–30 nm in diameter, and also the same two dsRNAs were isolated from purified virus preparations. The sequences of PoV1 dsRNA-1 and dsRNA-2 had GC contents of 48.4 and 51.5%, respectively. dsRNA-1 had 78 and 97 nucleotides of 5′- and 3′-untranslated region (UTR) while dsRNA-2 had 114 and 198 nucleotides of 5′- and 3′-UTR, respectively. Computer analysis of putative open reading frame (ORF) shows that dsRNA-1 and dsRNA-2 contain a single ORF encoding proteins of 82.2 and 71.1 kDa that show high sequence identity with RNA-dependent RNA polymerase and capsid protein of partitiviruses, respectively. When compared to other dsRNA mycoviruses in a phylogenetic analysis they were found to form a distinct virus clade with partitiviruses, and were more distantly related to totiviruses.

VP1, the RNA-dependent RNA polymerase and genome-linked protein of infectious bursal disease virus, interacts with the carboxy-terminal domain of translational eukaryotic initiation factor 4AII

Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is a non-enveloped, double-stranded RNA virus. Viral protein 1 (VP1), the putative RNA-dependent RNA polymerase, occurs in virions both as a free polypeptide and as a genome-linked protein, called VPg. To gain more insight in its function, we initiated a yeast two-hybrid screen. With this approach we identified the carboxy-terminal domain of eukaryotic translation initiation factor 4AII (eIF4AII) as an interactor for VP1. The association between these molecules was confirmed by co-immunoprecipitation analyses. eIF4A plays an essential role in the initiation of translation of both capped and uncapped mRNAs. Its association with IBDV VP1 suggests an involvement of this viral protein in IBDV mRNA translation. An interaction between VP1 and full-length eIF4AII was, however, not observed. In view of the known two-domain structure of eIF4AII it is conceivable that the interaction of VP1 with full-length eIF4AII requires collaborating proteins that open up its structure and expose the VP1-binding site in the carboxy-terminal domain. The biological relevance of the potential VP1-eIF4AII interaction is discussed.

Polymorphisms of NS5B protein relates to early clearance of hepatitis C virus by interferon plus ribavirin: a pilot study.

Although randomized trials have shown enhancement of efficacy for combination therapy with interferon (IFN) alpha-2b and ribavirin compared with IFN monotherapy as first-line treatment for chronic hepatitis C, infection with genotype 1b and high viremia are still associated with significantly low response rates compared with non-1 genotypes and low viremia. We analysed amino acid sequences of the viral RNA-dependent RNA polymerase (RdRP) or nonstructural protein 5B (NS5B), responsible for ribavirin misincorporation into RNA products in patients with genotype 1b-related chronic hepatitis C and high viremia, and examined the relationship between such RdRp polymorphisms, and the initial decline in viral load induced by combination therapy with IFN-alpha and ribavirin. Substitution of glutamic acid to lysine at the 124th position (E124K) and of isoleucine to valine at the 85th position (I85V) were found to be closely associated with a potent decline of viral load and viral clearance at 8 weeks of treatment (five of five patients, coincidence rate 100%). In conclusion, our results suggest that the polymorphisms of E124K and I85V identified in NS5B protein are crucial for early viral clearance in patients with genotype 1b and high viremia by combination therapy with IFN and ribavirin, and that detection of amino acid sequence motifs might enable prediction of clinical efficacy.

Genetic and functional diversification of small RNA pathways in plants.

Multicellular eukaryotes produce small RNA molecules (approximately 21–24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense.

Genome sequence and characterization of a virus (HaRNAV) related to picorna-like viruses that infects the marine toxic bloom-forming alga Heterosigma akashiwo

Heterosigma akashiwo ( Rhaphidophyceae) is a unicellular, flagellated, bloom-forming, toxic alga of ecological and economic importance. Here, we report the results of sequencing and analyzing the genome of an 8.6-kb single-stranded RNA virus (HaRNAV-SOG263) that infects H. akashiwo. Our results show that HaRNAV is related to picorna-like viruses, but does not belong within any currently defined virus family. This is based on the genome organization and sequence comparisons of putative RNA-dependent RNA polymerase (RdRp), helicase, and capsid protein sequences. The genome sequence predicts a single open reading frame (orf) encoding a polyprotein that contains conserved picorna-like protein domains, with putative nonstructural protein domains present in the N-terminus and the structural proteins in the C-terminus of the polyprotein. We have analyzed and compared the virus structural proteins from infectious and noninfectious particles. In this way, we identified structural protein cleavage sites as well as protein processing events that are presumably important for maturation of virus particles. The combination of genome structure and sequence relationships to other viruses suggests that HaRNAV is the first member of a proposed new virus family ( Marnaviridae), related to picorna-like viruses.

High diversity of unknown picorna-like viruses in the sea.

Picorna-like viruses are a loosely defined group of positive-sense single-stranded RNA viruses that are major pathogens of animals, plants and insects. They include viruses that are of enormous economic and public-health concern and are responsible for animal diseases (such as poliomyelitis), plant diseases (such as sharka) and insect diseases (such as sacbrood). Viruses from the six divergent families (the Picornaviridae, Caliciviridae, Comoviridae, Sequiviridae, Dicistroviridae and Potyviridae) that comprise the picorna-like virus superfamily have the following features in common: a genome with a protein attached to the 5' end and no overlapping open reading frames, all the RNAs are translated into a polyprotein before processing, and a conserved RNA-dependent RNA polymerase (RdRp) protein. Analyses of RdRp sequences from these viruses produce phylogenies that are congruent with established picorna-like virus family assignments; hence, this gene is an excellent molecular marker for examining the diversity of picorna-like viruses in nature. Here we report, on the basis of analysis of RdRp sequences amplified from marine virus communities, that a diverse array of picorna-like viruses exists in the ocean. All of the sequences amplified were divergent from known picorna-like viruses, and fell within four monophyletic groups that probably belong to at least two new families. Moreover, we show that an isolate belonging to one of these groups is a lytic pathogen of Heterosigma akashiwo, a toxic-bloom-forming alga responsible for severe economic losses to the finfish aquaculture industry, suggesting that picorna-like viruses are important pathogens of marine phytoplankton.

The cis-acting replication signal at the 3′ end of Flock House virus RNA2 is RNA3-dependent

The nodavirus Flock House virus has a bipartite positive-sense RNA genome consisting of RNAs 1 and 2, which encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The RdRp catalyzes replication of both genome segments and produces from RNA1 a subgenomic RNA (RNA3) that transactivates RNA2 replication. Here, we replaced internal sequences of RNAs 1 and 2 with a common heterologous core and were thereby able to test the RNA termini for compatibility in supporting the replication of chimeric RNAs. The results showed that the 3′ 50 nt of RNA2 contained an RNA3-dependent cis-acting replication signal. Since covalent RNA dimers can direct the synthesis of monomeric replication products, the RdRp can evidently respond to cis-acting replication signals located internally. Accordingly, RNA templates containing the 3′ termini of both RNAs 1 and 2 in tandem generated different replication products depending on the presence or absence of RNA3.

Complete nucleotide sequence of Tulip virus X (TVX-J): the border between species and strains within the genus Potexvirus.

The complete nucleotide sequence of the genomic RNA of Tulip virus X Japanese isolate (TVX-J) has been determined. The sequence is 6056 nucleotides in length, excluding the poly(A) tail at the 3' terminus, and contains five open reading frames (ORFs) coding for proteins of Mr 153, 25, 12, 10, and 22 kDa (ORFs 1 through 5, respectively). The genome organization of TVX-J is similar to that of potexviruses, and the encoded proteins share a high degree of homology to the corresponding proteins of other potexviruses. Phylogenetic analyses based on the RNA-dependent RNA polymerase (RdRp) protein (the methyltransferase, helicase, and polymerase domains) encoded by ORF1 and the capsid protein (CP) encoded by ORF5, revealed a close relationship of TVX-J to Plantago asiatica mosaic virus (PlAMV). Pairwise comparison analyses revealed that the relationship between TVX and PlAMV is intermediate between that of strains and species, though previously they have not been considered related. Due to the relatively distant relationships of their replication apparatus and triple gene blocks, we conclude that TVX and PlAMV should be classified as distinct viruses. In addition, the borderline between species and strains of potexviruses is discussed.

The genome segment B encoding the RNA-dependent RNA polymerase protein VP1 of very virulent infectious bursal disease virus (IBDV) is phylogenetically distinct from that of all other IBDV strains.

Summary. A full-length cDNA clone of the segment B of the very virulent infectious bursal disease virus (IBDV) strain BD 3/99 was constructed and the full-length nucleotide sequence was established. The nucleotide sequence encoding VP1, an RNA-dependent RNA polymerase, of BD 3/99 was aligned with that of 17 other IBDV strains including six very virulent, three classical virulent, five classical attenuated, one antigenic variant and two serotype 2 strains. The VP1 genes of all very virulent strains were 97.5% to 99.8% identical. With the exception of an atypical Australian strain, 002-73, all of the classical virulent or attenuated and antigenic variant strains were also 97.5% to 100% identical. Serotype 2 strains showed only 4–6% divergence from serotype 1 classical virulent or attenuated strains; in contrast, however, the very virulent strains were 10.5% to 12.5% divergent from the classical virulent or attenuated strains as well as serotype 2 strains. Analysis of the deduced amino acid sequence of VP1 revealed 17 common, including 8 unique amino acid substitutions in the very virulent strains. In the phylogenetic tree the very virulent strains formed a distinct cluster and all other strains including classical virulent, attenuated and antigenic variant strains and even serotype 2 strains were grouped together. It to is suggested that the VP1 of very virulent IBDV is phylogenetically distinct from that of all other IBDV strains and probably originated from a hitherto unidentified source.

Characterization and template properties of RNA dimers generated during flock house virus RNA replication.

Flock house virus (FHV) is the best studied member of the Nodaviridae, a family of small, nonenveloped, isometric RNA viruses of insects and fish. Nodavirus genomes comprise two single-stranded positive-sense RNA segments (RNAs 1 and 2) that encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The RdRp replicates both genomic RNAs and also generates a subgenomic RNA (RNA3) that is not encapsidated. Although genomic RNAs replicate through negative-sense intermediates, little is known about these RNAs or the details of the replication mechanism. Negative-sense RNAs 1, 2, and 3, as well as putative dimers of RNAs 2 and 3, have been detected in previous studies. In this study we detected dimers of RNAs 1, 2, and 3 by Northern blot analyses of RNA samples from FHV-infected Drosophila cells, as well as from mammalian and yeast cells supporting FHV RNA replication. Characterization of these RNA species by RT-PCR and sequence determination showed that they contained head-to-tail junctions of FHV RNAs. RNAs containing the complete sequence of RNA2 joined to RNA3 were also detected during replication. To examine the template properties of these dimeric RNAs, we made corresponding cDNAs and transcribed them from a T7 promoter in mammalian cells constitutively expressing T7 RNA polymerase, together with RNA1 to provide the RdRp. Although heterologous terminal extensions inhibit FHV RNA replication, monomeric RNA2 was resolved and replicated from complete or partial homodimer templates and from an RNA2-RNA3 heterodimer.

Simultaneous quantitation of two orchid viruses by the TaqMan ® real-time RT-PCR

Simultaneous quantitation of two orchid viruses, cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV), were carried out using the TaqMan ® real-time RT-PCR, a novel detection technique that combines RT-PCR with the power of fluorescent detection. Four TaqMan ® probes were synthesized, targeting at the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of both viruses. The reporter dye FAM (6-carboxyfluorescein) was used to label the 5′ terminus of probes specific to CymMV, while TET (tetrachloro-6-carboxyfluorescein) was used for the ORSV probes. TAMRA (6-carboxy-tetramethyl-rhodamine), which was attached at the 3′ terminus of each probe, was used as the universal quencher. With increasing amounts of standard RNA templates, the respective threshold cycle ( C T) values were determined and a linear relationship was established between these C T values and the logarithm of initial template amounts. The amounts of starting templates in mixed-infected Oncidium flowers and leaves were estimated from the standard curves. As little as 10 4 copies or 5 fg each of CymMV and ORSV could be detected simultaneously with either the RdRp or CP gene as the target. This system offers a sensitive, high throughput and rapid method for plant virus detection.

Molecular Beacons: A New Approach to Plant Virus Detection

ABSTRACT Molecular beacons are single-stranded nucleic acid molecules with a stem-loop conformation. The stem portion consists of complementary sequences at the 5' and 3' terminals of the molecule, while the loop portion consists of probe sequences that are complementary to the target sequences of choice. A fluorescent moiety is attached to one end, while a quenching moiety is attached to the opposite end. Reverse transcription-polymerase chain reactions are carried out with primers that amplify specific genome sequences of interest, yielding targets complementary to their respective molecular beacons for subsequent detection. Here, we have designed four molecular beacons specific to the RNA-dependent RNA polymerase and coat protein genes of two orchid viruses, namely Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). This technology is successfully applied to detect as little as 0.5 ng of viral RNA of both orchid viruses simultaneously in 100 mg of coinfected Oncidium orchid leaves. This rapid and specific technique is applicable to the orchid industry, which routinely carries out virus indexing and screening for virus-resistant cultivars. We belief that use of this molecular beacon approach can be extended to the detection of multiple plant viruses in various crops.

Complete nucleotide sequences of garlic viruses A and C, members of the newly ratified genus Allexivirus.

Complete genomic sequences of garlic viruses A (Gar V-A) and C (Gar V-C), members of an unassigned virus group recently identified in garlic plants, were determined. Their respective genomes consist of 8 660 and 8 405 nucleotides. The genomic structure and organization of these viruses are similar to shallot virus X (ShVX) which is the type species of the newly ratified genus Allexivirus. Phylogenetic analysis based on the amino acid sequences of the putative proteins including RNA-dependent RNA polymerase (RdRP), DNA helicase, or viral coat protein showed that the GarV-type viruses should be included in the genus Allexivirus. Furthermore, the amino acid sequence in the RdRP hypervariable region is highly divergent among the viruses in the genus Allexivirus, suggesting that they evolved independently from a hypothetical ancestor virus(es).

Metal ion catalysis of RNA cleavage by the influenza virus endonuclease.

The influenza virus RNA-dependent RNA polymerase protein complex contains an associated RNA endonuclease activity, which cleaves host mRNA precursors in the cell nucleus at defined positions 9-15 nucleotides downstream of the cap structure. This reaction provides capped oligoribonucleotides, which function as primers for the initiation of viral mRNA synthesis. The endonuclease reaction is dependent on the presence of divalent metal ions. We have used a number of divalent and trivalent metal ions alone and in combination to probe the mechanism of RNA cleavage by the influenza virus endonuclease. Virus-specific cleavage was observed with various metal ions, and maximum cleavage activity was obtained with 100 microM Mn2+ or 100 microM Co2+. This activity was about 2-fold higher than that observed with Mg2+ at the optimal concentration of 1 mM. Activity dependence on metal ion concentration was cooperative with Hill coefficients close to or larger than 2. Synergistic activation of cleavage activity was observed with combinations of different metal ions at varying concentrations. These results support a two-metal ion mechanism of RNA cleavage for the influenza virus cap-dependent endonuclease. The findings are also consistent with a structural model of the polymerase, in which the specific endonuclease active site is spatially separated from the nucleotidyl transferase active site of the polymerase module.

Diagnostic PCR between dream and reality — The clinicians' view

Simultaneous quantitation of two orchid viruses, cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV), were carried out using the TaqMan® real-time RT-PCR, a novel detection technique that combines RT-PCR with the power of fluorescent detection. Four TaqMan® probes were synthesized, targeting at the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of both viruses. The reporter dye FAM (6-carboxyfluorescein) was used to label the 5′ terminus of probes specific to CymMV, while TET (tetrachloro-6-carboxyfluorescein) was used for the ORSV probes. TAMRA (6-carboxy-tetramethyl-rhodamine), which was attached at the 3′ terminus of each probe, was used as the universal quencher. With increasing amounts of standard RNA templates, the respective threshold cycle (CT) values were determined and a linear relationship was established between these CT values and the logarithm of initial template amounts. The amounts of starting templates in mixed-infected Oncidium flowers and leaves were estimated from the standard curves. As little as 104 copies or 5 fg each of CymMV and ORSV could be detected simultaneously with either the RdRp or CP gene as the target. This system offers a sensitive, high throughput and rapid method for plant virus detection.

Non-structural plum pox potyvirus proteins detected by immunogold labelling

Plum pox potyvirus (PPV) induces in infected Nicotiana clevelandii cells characteristic crystalline inclusions known as nuclear inclusions (NI) when located in the nucleus and as dense material (Dm) when located in the cytoplasm. Crystalline inclusions contain protease (NIa) and RNA-dependent RNA polymerase (NIb) proteins. It is now well established for all potyviruses that cylindrical inclusions contain CI helicase ATPase protein (Martin et al., 1992). The intracellular location of other non-structural PPV proteins remains unknown. Using Escherichia coli expression vectors, specific antibodies were obtained against P1, P3, 6K2 and NIb PPV proteins for which antibodies were not yet available. As expected, NIb antiserum labelled crystalline inclusions. P1, P3 and 6K2 proteins were present in both types of crystalline inclusions found in the nucleus and in the cytoplasm of PPV-infected leaves of N. clevelandii, suggesting that nuclear inclusions and dense material were composed of the same proteins. This composition is discussed.

Expression of the three influenza virus polymerase proteins in a single cell allows growth complementation of viral mutants.

Transformed cell lines derived from murine C127 cells were constructed that express the influenza virus RNA-dependent RNA polymerase proteins (PA, PB1, and PB2). Cell lines that express only one or all three of the proteins were tested for their ability to complement temperature-sensitive viral mutants incubated at the nonpermissive temperature. Two cell lines were isolated that express all three polymerase genes and complement the growth of PB2 temperature-sensitive mutants at the nonpermissive temperature. One of these lines also complemented PA temperature-sensitive mutants. The viral titers obtained in these two cell lines were 12-fold to 1000-fold higher than the viral titers obtained upon growth of the corresponding temperature-sensitive mutant in C127 cells at the nonpermissive temperature.

Poliovirus RNA-dependent RNA polymerase and host cell protein synthesize product RNA twice the size of poliovirion RNA in vitro.

The poliovirus RNA-dependent RNA polymerase required an oligouridylate primer or a HeLa cell protein (host factor) to initiate RNA synthesis on poliovirion RNA in vitro. The polymerase synthesized template-sized product RNA in the oligouridylate-primed reaction. In the host factor-dependent reaction, the largest product RNA synthesized by the polymerase was twice the size of the template RNA. About half of the product RNA recovered from this reaction was shown to exist in the form of a snapback sequence. Time-course reactions and pulse-chase experiments showed that the product RNA was only slightly larger than the template RNA at early reaction times and that with time it increased in size to form the dimer-sized product RNA. Inhibition of the elongation reaction by adding only [alpha-32P]UTP and ATP resulted in the formation of template-sized product RNA. The dimer-sized product RNA was unaffected by phenol extraction or proteinase K treatment but was converted to template-sized molecules by S1 nuclease. Dimer-sized poliovirus RNA that was sensitive to S1 nuclease was also isolated from poliovirus-infected cells. The results from this study indicate that the labeled negative-strand product RNA synthesized in vitro was covalently linked to the positive-strand template RNA. Thus, in vitro, the primer-dependent poliovirus RNA polymerase may initiate RNA synthesis in the presence of the host factor by using the 3' end of the template RNA as a primer.

RNA polymerase activity and protein synthesis in brome mosaic virus-infected protoplasts

RNA-dependent RNA polymerase activity and protein synthesis in brome mosaic virus (BMV)-infected and uninfected protoplasts were investigated during the course of viral replication. In protoplast homogenates, the membrane-bound RNA polymerase activity resistant to actinomycin D was enhanced by BMV infection up to 30-fold that found in mock-inoculated protoplasts. The activity was first detected 7 to 8 hr postinfection. It reached a maximum at around 30 hr postinfection, then decreased gradually while virus and the viral antigen in the protoplasts, as measured by infectivity and fluorescent antibody staining, respectively, continued to increase. The bound enzyme activity was not enhanced by inoculation with BMV RNA 3 and/or RNA 4. Four proteins with molecular weights of 120, 110, 36, and 19.5 × 103 were observed in BMV-infected protoplasts from three plant species (a systemic host, a local lesion host, and a nonhost of BMV).