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The nucleotide sequence (2617 nucleotides) of virus-like double-stranded (ds) RNA 3a in a diseased isolate, Log1/3-8d2(Ld), of the ascomycete fungusOphiostoma novo-ulmihas been determined. One strand of the dsRNA contains an open reading frame (ORF) with the potential to encode a protein of 718 amino acids, and the complementary strand contains two smaller ORFs with the potential to encode proteins...
Infection of plants or protoplasts with turnip crinkle virus (TCV) results in the synthesis of the genomic RNA and two subgenomic (sg) RNAs of 1.7 kb and 1.45 kb, respectively. Both of the sgRNA promoters were characterized previously and their secondary structures predicted by computer analysis [J. Wang and A. E. Simon (1997).Virology232, 174–186]. Secondary structure-sensitive chemical and enzymatic...
The nucleotide sequences of three mitochondrial virus double-stranded (ds) RNAs, RNA-4 (2599 nucleotides), RNA-5 (2474 nucleotides), and RNA-6 (2343 nucleotides), in a diseased isolate Log1/3–8d2(Ld) of the Dutch elm disease fungusOphiostoma novo-ulmihave been determined. All these RNAs are A-U-rich (71–73% A + U residues). Using the fungal mitochondrial genetic code in which UGA codes for tryptophan,...
RNA-dependent RNA polymerase (RdRp) activity has been detected in mitochondria from an isolate of Ophiostoma novo-ulmi infected with O. novo-ulmi mitovirus 6 (OnuMV6). The reaction products corresponded to the double-stranded and single-stranded forms of OnuMV6 RNA. Western blot analysis using antibodies raised against a conserved RdRp region has detected a protein of ca. 80 kDa in OnuMV6-infected...
RNA initiation by Qβ replicase directed by the short-sequence CCA at the 3′-end of all RNAs amplified by this enzyme has been studied. Most CCA repeats in an RNA consisting of 12 CCAs serve as independent sites of de novo RNA initiation, with initiation occurring opposite the 3′-C residue of each CCA. Qβ replicase is thus capable of internal initiation remote from the 3′-end, although predominant...
We recently developed a model for flavivirus infection in mice and hamsters using the Modoc virus (MODV), a flavivirus with no known vector (P. Leyssen, A. Van Lommel, C. Drosten, H. Schmitz, E. De Clercq, and J. Neyts, 2001, Virology 279, 27–37). We now present the coding and noncoding sequence of MODV. The Modoc virus genome was determined to be 10,600 nucleotides in length with a single open reading...
The insect nodavirus Flock house virus (FHV) has a small genome divided between two segments of positive-sense RNA, RNA1 and RNA2. RNA1 encodes the RNA-dependent RNA polymerase (RdRp) catalytic subunit and templates the synthesis of a subgenomic RNA (RNA3) that encodes two small nonstructural proteins. Replication of RNA2, which encodes a precursor to the viral capsid proteins, suppresses RNA3 synthesis...
Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the positive-strand RNA virus family Arteriviridae. Although considerable research has focused on this important pathogen, little is known about the function of most PRRSV proteins. To examine characteristics of putative nonstructural proteins (nsp) encoded in ORF1b, which have been identified by nucleotide similarity to domains...
The GB virus-B (GBV-B) nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp) with greater than 50% sequence similarity to the hepatitis C virus (HCV) NS5B. Recombinant GBV-B NS5B was reported to possess RdRp activity (W. Zhong et al., 2000, J. Viral Hepat. 7, 335-342). In this study, the GBV-B RdRp was examined more thoroughly for different RNA synthesis activities, including...
The plus-strand RNA genome of tomato bushy stunt virus (TBSV) contains a 351-nucleotide (nt)-long 3'-untranslated region. We investigated the role of the 3'-proximal 130 nt of this sequence in viral RNA accumulation within the context of a TBSV defective interfering (DI) RNA. Sequence comparisons between different tombusviruses revealed that the 3' portion of the 130-nt sequence is highly conserved...
A mycovirus, named oyster mushroom spherical virus (OMSV), was isolated from cultivated oyster mushrooms with a severe epidemic of oyster mushroom Die-back disease. OMSV was a 27-nm spherical virus encapsidating a single-stranded RNA (ssRNA) of 5.784 kb with a coat protein of approximately 28.5 kDa. The nucleotide sequence of the virus revealed that its genomic RNA was positive strand, containing...
To continue the molecular characterization of RNA-dependent RNA polymerases of dsRNA bacteriophages (Cystoviridae), we purified and biochemically characterized the wild-type (wt) and a temperature-sensitive (ts) point mutant of the polymerase subunit (Pol) from bacteriophage φ12. Interestingly, initiation by both wt and the ts φ12 Pol was notably more sensitive to increased temperatures than the elongation...
We hypothesized that mutations in the HCV NS5B polymerase, which occur during infection, may affect RNA-dependent RNA polymerase (RdRp) activity. NS5B proteins corresponding to a genotype 1a infectious clone and mutants identified in chimpanzees following inoculation with the clone were expressed and purified and their in vitro RdRp activity was compared to a NS5B genotype 1b control. A Gln-65-to-His...
The L RNA of three Lassa virus strains originating from Nigeria, Ghana/Ivory Coast, and Sierra Leone was sequenced and the data subjected to structure predictions and phylogenetic analyses. The L gene products had 2218–2221 residues, diverged by 18% at the amino acid level, and contained several conserved regions. Only one region of 504 residues (positions 1043–1546) could be assigned a function,...
The RNA-dependent RNA polymerase of the Sendai virus (SeV) consists of the large protein (L) and the phosphoprotein (P). P plays a crucial role in the enzyme by positioning L (which carries the polymerase activity) onto the matrix for transcription and replication formed by the RNA and the nucleoprotein, the N-RNA. P has a modular structure with distinct functional domains: an N-terminal domain involved...
The genomic RNA of Turnip yellow mosaic virus (TYMV) has an 82-nucleotide-long tRNA-like structure at its 3'-end that can be valylated and then form a stable complex with translation elongation factor eEF1A.GTP. Transcription of this RNA by TYMV RNA-dependent RNA polymerase (RdRp) to yield minus strands has previously been shown to initiate within the 3'-CCA sequence. We have now demonstrated that...
Infectious bronchitis virus (IBV) produces six subgenomic (sg) mRNAs, each containing a 64 nucleotide (nt) leader sequence, derived from the 5' end of the genome by a discontinuous process. Several putative functional domains such as a papain-like proteinase (PL pro ), main protease (M pro ), RNA-dependent RNA polymerase (RdRp), and RNA helicase encoded by the replicase...
The RNA-dependent RNA polymerase (RdRp) of SARS coronavirus (SARS-CoV) is essential for viral replication and a potential target for anti-SARS drugs. We report here the cloning, expression, and purification of the N-terminal GST-fused SARS-CoV RdRp and its polymerase catalytic domain in Escherichia coli. During purification, the full-length GST-RdRp was found to cleave into three main fragments: an...
Several host translation elongation factors have been suggested to play essential roles in the replication and translation of viral RNAs in plants, animals and bacteria. Here, we show the interaction between eukaryotic translation elongation factor 1A (eEF1A) and Tobacco mosaic virus (TMV) RNA-dependent RNA polymerase (RdRp) in vivo by immunoprecipitation. The tobacco eEF1A interacted not only with...
The influenza virus RNA-dependent RNA polymerase interacts with the serine-5 phosphorylated carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II). It was proposed that this interaction allows the viral RNA polymerase to gain access to host mRNA-derived capped RNA fragments required as primers for the initiation of viral mRNA synthesis. Here, we show, using a chromatin immunoprecipitation...
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