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PubMed1991

Mutation and replacement of the 16-kDa protein gene in RNA-1 of tobacco rattle virus.

The function of the 16-kDa protein encoded by tobacco rattle virus (TRV) RNA-1 was investigated by a mutational analysis of the 16-kDa protein gene. Transcripts of TRV RNA-1 produced from a full-length cDNA clone of TRV RNA-1 (SYM strain) remained infectious when the 16-kDa protein gene was disrupted by premature termination codons and a deletion which removed 73% of the coding region. A deletion which included the intergenic region between the 29-kDa protein gene and the 16-kDa protein gene, the entire 16-kDa protein coding region, and 57% of the 3' noncoding region was not infectious. Transcripts in which the 16-kDa protein coding region was replaced by the tobacco mosaic virus (TMV) (L strain) coat protein gene were also infectious and expressed TMV coat protein in infected tissue. Inclusion of the TMV origin of assembly sequence in the chimaeric constructs resulted in the accumulation of TMV-like virus particles in infected tissue.

Blotting, Northern↗
PubMed2000

Mov34 protein from mouse brain interacts with the 3' noncoding region of Japanese encephalitis virus.

The plus-sense RNA genome of Japanese encephalitis virus (JEV) contains noncoding regions (NCRs) of 95 and 585 bases at its 5' and 3' ends, respectively. The last 83 nucleotides of the 3'-NCR are predicted to form stable stem-loop (SL) structures. The shape of this 3'-SL structure is highly conserved among divergent flaviviruses even though only small stretches of nucleotide sequence contained within these structures are conserved. These SL structures have been predicted to function as cis-acting signals for RNA replication and as such may bind to viral and cellular proteins that may be involved in viral replication. We have studied the interaction of the JEV 3'-NCR RNA with host proteins using gel retardation assays. We show that the JEV 3'-SL structure RNA forms three complexes with proteins from the S100 cytoplasmic extract prepared from the neonatal mouse brain. These complexes could be obtained in the presence of 200 mM KCl, indicating that the RNA-protein interaction may be physiologically relevant. UV-induced cross-linking and Northwestern blotting analyses detected three proteins with apparent molecular masses of 32, 35, and 50 kDa that bound to the JEV 3'-SL structure RNA. Screening of the neonatal mouse brain cDNA library with the JEV 3'-SL structure RNA identified a 36-kDa Mov34 protein interacting with it. Competition experiments using the RNA extracted from JEV virions established that the 36-kDa Mov34 protein indeed bound to the JEV genome. Murine Mov34 belongs to a family of proteins whose members have been shown to be involved in RNA transcription and translation. It is, therefore, likely that the murine Mov34 interaction with JEV 3'-NCR has a role in RNA replication.

3' Untranslated Regions↗
PubMed2000

[Sequence analysis of the 5' and 3' terminal regions of dengue type 2 virus 04 strain].

OBJECTIVE: To analyze the sequences of 5' and 3' terminal region of dengue type 2 virus 04 strain. METHODS: Total RNA was isolated from dengue type 2 virus 04 strain (D2-04) infected C6/36 cells. With this RNA as template, the cDNA of both 5' and 3' termini of D2-04 were amplified using RACE method respectively. The cDNAs were separately inserted into pGEM-T vector and the recombinant plasmids containing the 5' terminus 535 bp and 3' terminus 503 bp were obtained. The nucleotide sequences of the inserted cDNA fragment were determined. The nucleotide sequences of the 5' and 3' noncoding regions of D2-04 were compared with other dengue type 2 viruses such as JAM, NGC, S1, 16681 and PD-53. RESULTS: The results showed that they shared 98.96%, 98.96%, 93.75%, 98.95%, 97.92% and 97.72%, 97.80%, 90.65%, 94.26%, 94.22% homology with D2-04 strain respectively. CONCLUSIONS: Except SI, the homology between D2-04 and other type 2 viruses was higher than 94%.

Base Sequence↗
PubMed2005

A map of the diversity of RNA3 recombinants appearing in plants infected with Cucumber mosaic virus and Tomato aspermy virus.

In order to better understand the role of recombination in creating the diversity of viral genomes that is acted on by selection, we have studied in detail the population of recombinant RNA3 molecules occurring in tobacco plants coinfected with wild-type strains of cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) under conditions of minimal selection pressure. Recombinant RNA3s were observed in 9.6% of the samples. Precise homologous recombination predominated since it was observed at 28 different sites, primarily in six hot spots. Imprecise homologous recombination was observed at two sites, particularly within a GU repeat in the 5' noncoding region. Seven of the eight aberrant homologous recombination sites observed were clustered in the 3' noncoding region. These results have implications on the role of recombination in host adaptation and virus evolution. They also provide essential baseline information for understanding the potential epidemiological impact of recombination in transgenic plants expressing viral sequences.

Base Sequence↗
PubMed1991

Nucleotide sequence and mutation rate of the H strain of hepatitis C virus.

Patient H is an American patient who was infected with hepatitis C virus (HCV) in 1977. The patient became chronically infected and has remained so for the past 13 years. In this study, we compared the nucleotide and predicted amino acid sequences of the HCV genome obtained from plasma collected in 1977 with that collected in 1990. We find that the two HCV isolates differ at 123 of the 4923 (2.50%) nucleotides sequenced. We estimate that the mutation rate of the H strain of HCV is approximately 1.92 x 10(-3) base substitutions per genome site per year. The nucleotide changes were exclusively base substitutions and were unevenly distributed throughout the genome. A relatively high rate of change was observed in the region of the HCV genome that corresponds to the non-structural protein 1 gene region of flaviviruses, where 44 of 960 (4.6%) nucleotides were different. Within this region there was a 39-nucleotide domain in which 28.2% of the nucleotides differed between the two isolates. In contrast, relatively few nucleotide substitutions were observed in the 5' noncoding region, where only 2 of 276 (0.7%) nucleotides were different. Our results suggest that the mutation rate of the HCV genome is similar to that of other RNA viruses and that genes appear to be evolving at different rates within the virus genome.

Amino Acid Sequence↗
PubMed1982

Nucleotide sequence of the 5' noncoding region and part of the gag gene of Rous sarcoma virus.

Several functions of the retrovirus genome involve structural features in the vicinity of its 5' terminus. In an effort to further elucidate the relationship between structure and function in retrovirus RNA, we have determined the sequence of the first 1,010 nucleotides at the 5' end of the genome of Rous sarcoma virus by using the Maxam-Gilbert method to sequence suitable domains in cloned Rous sarcoma virus DNA. The results (i) locate the initiation codon for the gag gene of Rous sarcoma virus 372 nucleotides from the 5' end of viral RNA; (ii) demonstrate that this codon is preceded by three methionine codons that are apparently not used in translation; (iii) sustain previous conclusions that the principal site to which ribosomes bind on the Rous sarcoma virus genome in vitro does not contain the initiation codon for gag; (iv) permit deduction of the amino acid sequence of a viral structural protein, p19; (v) confirm the amino-terminal sequence of Pr76gag; and (vi) substantiate the identification of a splice donor site described in the accompanying manuscript (Hackett et al., J. Virol., 41:527-534, 1982).

Avian Sarcoma Viruses↗
PubMed1988

Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5' noncoding region.

Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, we have determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. We found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element maps between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. We also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation.

Base Sequence↗
PubMed2004

Development and evaluation of an efficient 3'-noncoding region based SARS coronavirus (SARS-CoV) RT-PCR assay for detection of SARS-CoV infections.

The severe acute respiratory syndrome (SARS) epidemic originating from China in 2002 was caused by a previously uncharacterized coronavirus that could be identified by specific RT-PCR amplification. Efforts to control future SARS outbreaks depend on the accurate and early identification of SARS-CoV infected patients. A real-time fluorogenic RT-PCR assay based on the 3'-noncoding region (3'-NCR) of SARS-CoV genome was developed as a quantitative SARS diagnostic tool. The ideal amplification efficiency of a sensitive SARS-CoV RT-PCR assay should yield an E value (PCR product concentration increase per amplification cycle) equal to 2.0. It was demonstrated that the 3'-NCR SARS-CoV based RT-PCR reactions could be formulated to reach excellent E values of 1.81, or 91% amplification efficacy. The SARS-CoV cDNA preparations derived from viral RNA extract and the cloned recombinant plasmid both exhibit the identical amplification characteristics, i.e. amplification efficacy using the same PCR formulation developed in this study. The viral genomic copy (or genomic equivalences, GE) per infectious unit (GE/pfu) of SARS-CoV used in this study was also established to be approximate 1200-1600:1. The assay's detection sensitivity could reach 0.005 pfu or 6-8 GE per assay. It was preliminarily demonstrated that the assay could efficiently detect SARS-CoV from clinical specimens of SARS probable and suspected patients identified in Taiwan. The 3'-NCR based SARS-CoV assay demonstrated 100% diagnostic specificity testing samples of patients with acute respiratory disease from a non-SARS epidemic region.

3' Untranslated Regions↗
PubMed1998

A 68-nucleotide sequence within the 3' noncoding region of simian hemorrhagic fever virus negative-strand RNA binds to four MA104 cell proteins.

The 3' noncoding region (NCR) of the negative-strand RNA [3'(-)NCR RNA] of the arterivirus simian hemorrhagic fever virus (SHFV) is 209 nucleotides (nt) in length. Since this 3' region, designated 3'(-)209, is the site of initiation of full-length positive-strand RNA and is the template for the synthesis of the 5' leader sequence, which is found on both full-length and subgenomic mRNAs, it is likely to contain cis-acting signals for RNA synthesis and to interact with cellular and viral proteins to form replication complexes. Gel mobility shift assays showed that cellular proteins in MA104 S100 cytoplasmic extracts formed two complexes with the SHFV 3'(-)209 RNA, and results from competition gel mobility shift assays demonstrated that these interactions were specific. Four proteins with molecular masses of 103, 86, 55, and 36 kDa were detected in UV-induced cross-linking assays, and three of these proteins (103, 55, and 36 kDa) were also detected by Northwestern blotting assays. Identical gel mobility shift and UV-induced cross-linking patterns were obtained with uninfected and SHFV-infected extracts, indicating that the four proteins detected are cellular, not viral, proteins. The binding sites for the four cellular proteins were mapped to the region between nt 117 and 184 (68-nt sequence) from the 3' end of the SHFV negative-strand RNA. This 68-nt sequence was predicted to form two stem-loops, SL4 and SL5. The 3'(-)NCR RNA of another arterivirus, lactate dehydrogenase-elevating virus C (LDV-C), competed with the SHFV 3'(-)209 RNA in competition gel mobility shift assays. UV-induced cross-linking assays showed that four MA104 cellular proteins with the same molecular masses as those that bind to the SHFV 3'(-)209 RNA also bind to the LDV-C 3'(-)NCR RNA and equine arteritis virus 3'(-)NCR RNA. However, each of these viral RNAs also bound to an additional MA104 protein. The binding sites for the MA104 cellular proteins were shown to be located in similar positions in the LDV-C 3'(-)NCR and SHFV 3'(-)209 RNAs. These data suggest that the binding sites for a set of the cellular proteins are conserved in all arterivirus RNAs and that these cell proteins may be utilized as components of viral replication complexes.

Animals↗
PubMed1997

Sequence analysis of hepatitis C virus variants producing discrepant results with two different genotyping assays.

Methods for identifying the genotype of hepatitis C virus (HCV) in clinical specimens are frequently based upon the direct characterisation of viral RNA sequences by polymerase chain reaction (PCR) amplification, or by serologically based methods, in which the infecting genotype is inferred from the pattern of antibody reactivity to type-specific peptides or recombinant proteins used as antigens in an Enzyme Linked Immunosorbent Assay (ELISA). Although genotyping by direct, PCR-based methods show generally highly concordant results with the genotype inferred from serological typing assays (> 95% agreement), there exist a small number of samples that produce discrepant results. To investigate the underlying reasons for the discrepancies, we obtained eleven samples from haemophiliacs and four samples from patients with chronic hepatitis C that produced discordant results between a PCR based assay (InnoLipa I and II) and a serotyping assay (Murex HC02). Nucleotide sequences in the 5'noncoding region (5'NCR), core, and NS4 region were used to identify the genotype of the circulating virus and to identify amino acid changes in NS4 that might alter antigenicity. In 14 samples, sequence analysis of all three regions was concordant with the results of the InnoLipa assay. There were few if any amino acid substitutions in NS4 that might have accounted for the discrepant serotyping results, which were found predominantly in samples from individuals with a history of multiple exposure to HCV. It remains unclear whether the detection of antibody in such discrepant samples corresponds to previous expression of a different genotype than detected by PCR, or whether the virus population in plasma is more restricted in genotype diversity than the population in the liver or at other sites of viral replication.

Amino Acid Sequence↗
PubMed2006

Real-time quantitative assay of HCV RNA using the duplex scorpion primer.

A novel real-time quantitative method for detecting HCV in serum was established in which the duplex scorpion primer was used to provide a unimolecular probing mechanism for hybridizing the highly conserved 5' noncoding region (5' NCR) of the HCV genome specifically. Through methodological evaluation, we found this new method had a wide linearity, high sensitivity, repeatability and specificity. Compared to the commercial TaqMan method, this method was found to be more sensitive and less costly, and the final results were obtained more quickly. Therefore, it could be applied to diagnose and monitor HCV infection in clinical practice.

DNA Primers↗
PubMed2000

L* protein of Theiler's murine encephalomyelitis virus is required for virus growth in a murine macrophage-like cell line.

We sought to confirm the importance of L* protein for growth of Theiler's murine encephalomyelitis virus (TMEV) in a macrophage-like cell line, J774-1. The protein is out of frame with the polyprotein and synthesized in DA but not GDVII subgroup strains of TMEV. A recombinant virus, DANCL*/GD, which substitutes the DA 5' noncoding and L* coding regions for the corresponding regions of GDVII and synthesizes L* protein, grew with little restriction in J774-1 cells. In contrast, another recombinant virus, DANCL*-1/GD, which has an ACG rather than an AUG as the starting codon of L* protein at nucleotide 1079, resulting in no synthesis of L* protein, did not grow well. No significant difference between the rates of adsorption to J774-1 cells of these viruses was observed. RNase protection assay demonstrated that DANCL*/GD viral RNA significantly increased, whereas only a minimal increase was observed for DANCL*-1/GD. The present study suggests that L* protein is required for virus growth in macrophages.

Amino Acid Sequence↗
PubMed1985

Gene expression from both intronless and intron-containing Rous sarcoma virus clones is specifically inhibited by anti-sense RNA.

To distinguish the inhibitory effect of anti-sense RNA on translation from the effect on splicing, a plasmid (pLC32) was constructed from a cDNA clone of the Rous sarcoma virus (RSV) envelope gene (env) mRNA. Transcription of this plasmid results in the synthesis of RNA identical to the RSV env gene mRNA which does not require splicing to be expressed. Plasmids derived from pLC32 were also constructed in which the env gene coding sequence and 5' noncoding leader sequences were inserted in the opposite orientation relative to the RSV long terminal repeats (LTRs). pLC32 DNA transfected by the calcium phosphate coprecipitation technique efficiently rescued infectious virus from quail cells infected with an RSV mutant deleted in the env gene [R(-)Q cells], indicating that the intron sequences are dispensable in env gene expression. When the inverted constructs were cotransfected with pLC32, significantly less infectious virus was produced. The extent of the inhibition depended upon the concentration ratio of the two plasmids. The maximum inhibition (80%) occurred when the ratio of inverted constructs to pLC32 was 12:1. The inhibition is specific for the inverted orientation since cotransfection of pLC32 with several other plasmids containing viral LTRs and defective src and env genes at similar concentrations did not inhibit the production of infectious virus. In addition, the inverted constructs did not interfere with the expression of an LTR-driven chloramphenicol acetyltransferase gene. When cotransfected with a wild-type Prague A RSV DNA plasmid (pJD100), the inverted constructs also greatly inhibited expression and replication of virus in R(-)Q quail cells. These data suggest that the specific inhibition is caused by hybridization of complementary RNA transcribed from the inverted constructs to the env mRNA, thereby blocking its expression. The fact that expression of both intron-containing and intronless clones are inhibited to the same extent suggest that inhibition by anti-sense RNA from the env exon regions does not act at the level of RNA splicing.

Animals↗
PubMed2003

Complete nucleotide sequence of spring beauty latent virus, a bromovirus infectious to Arabidopsis thaliana.

Spring beauty latent virus (SBLV), a bromovirus, systemically and efficiently infected Arabidopsis thaliana, whereas the well-studied bromoviruses brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) did not infect and poorly infected A. thaliana, respectively. We constructed biologically active cDNA clones of SBLV genomic RNAs and determined their complete nucleotide sequences. Interestingly, SBLV RNA3 contains both the box B motif in the intercistronic region, as does BMV, and the subgenomic promoter-like sequence in the 5' noncoding region, as does CCMV. Sequence comparisons of SBLV, BMV, CCMV, and broad bean mottle virus demonstrated that SBLV is closely related to BMV and CCMV.

Arabidopsis↗
PubMed1990

A region of the 5' noncoding region of foot-and-mouth disease virus RNA directs efficient internal initiation of protein synthesis within cells: involvement with the role of L protease in translational control.

Plasmids encoding bicistronic mRNAs have been constructed and used to identify a region from the 5' noncoding region of foot-and-mouth disease virus (FMDV) which directs efficient internal initiation of protein synthesis within cells. The loss of about 30 nucleotides (nt) from the 5' terminus or about 50 nt from the 3' terminus of the 435-nt region completely abolished the activity of this region. The expression of the FMDV L protease severely inhibited the expression of other genes unless they were preceded by this element. The regulation of protein synthesis mediated by FMDV is discussed.

Animals↗
PubMed1996

Comparisons of the genomic cis-elements and coding regions in RNA beta components of the hordeiviruses barley stripe mosaic virus, lychnis ringspot virus, and poa semilatent virus.

Nucleotide sequences of the genomic RNA beta components of hordeiviruses poa semilatent virus (PSLV) and lychnis ringspot virus (LRSV) were determined. PSLV and LRSV closely resemble barley stripe mosaic virus (BSMV), type hordeivirus, in the gene arrangement of their RNAs beta, comprising 5'-proximal beta a (coat protein) gene and downstream triple gene block (TGB) coding for the beta b, beta c, and beta d putative transport proteins. The beta a, beta b, beta c, and beta d proteins of the three hordeiviruses showed significant sequence similarity, with the respective proteins of PSLV and BSMV being closer to each other than to their counterparts of LSRV. Comparisons of the TGB-encoded proteins of hordeiviruses, potexviruses, carlaviruses, and furoviruses indicate that the first and second TGB genes belong to the monophyletic groups, whereas the third gene may have multiple ancestry. LRSV, PSLV, and BSMV showed remarkable variation in the 3'-untranslated regions of their genomic RNAs. Among the three hordeiviruses, LRSV has the shortest 3'-noncoding region that lacks tentative pseudoknot-forming elements conserved upstream of the 3'-tRNA-like structure in the BSMV and PSLV genomes. On the other hand, LRSV RNA beta, like that of BSMV, contained the internal poly(A) sequence that is absent from PSLV RNA.

Adaptation, Physiological↗
PubMed1994

Structure of human rhinovirus 3C protease reveals a trypsin-like polypeptide fold, RNA-binding site, and means for cleaving precursor polyprotein.

The structure of human rhinovirus-14 3C protease (3Cpro) has been determined at 2.3 A resolution and refined to an R factor of 0.22. This cysteine protease folds into two topologically equivalent six-stranded beta barrels and in this sense is similar to trypsin-like serine proteases. However, there are differences in the lengths and positioning of individual beta strands as well as in loops connecting elements of secondary structure. The catalytic residues Cys-146, His-40, and Glu-71 are positioned as in serine proteases, but the oxyanion hole is moved 1-1.2 A away. Residues that bind to the 5' noncoding region of rhinovirus genomic RNA are located on the opposite side of the molecule from the active site. Interactions between individual 3Cpro molecules in the crystal lattice suggest a model for intermolecular proteolytic cleavage of the 3CD polyprotein.

3C Viral Proteases↗
PubMed1988

An internal 5'-noncoding region required for translation of poliovirus RNA in vitro.

A truncated poliovirus RNA that contains the entire 5'-noncoding region as well as some capsid protein-coding sequences was produced from cloned cDNA inserted into an SP6 transcription vector and subsequently was translated in a mixed rabbit reticulocyte-HeLa cell lysate. Deletions or modifications of regions of the 5'-noncoding sequences had significant effects upon the efficiency of translation. The presence of a 60-nucleotide sequence located at positions 567 to 627 appeared to be essential for active ribosome binding and translation of this uncapped RNA.

Cell-Free System↗