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PubMed2003

Rescue of hepatitis A virus from cDNA-transfected but not virion RNA-transfected mouse Ltk- cells.

Hepatitis A virus (HAV) has stringent replication requirements and a restricted host-range. Mouse Ltk- cells do not support growth of HAV upon infection or transfection of virion RNA. However, low levels of HAV were rescued from Ltk- cells transiently transfected with its infectious cDNA. Ltk- stable transfectants that expressed HAV antigens and produced infectious HAV were selected and termed Ltk-pJH15 cells. After a few serial passages, HAV became undetectable in the Ltk-pJH15 cells. Multiple rounds of single cell cloning of HAV antigen positive Ltk-pJH15 cells resulted in the isolation of clone E8 that produced higher levels of HAV for at least 5 passages. HAV produced in E8 cells was similar to the parental virus as shown by infectivity assays. Luciferase assays using a bi-cistronic construct containing the HAV 5' noncoding region showed similar levels of HAV IRES-dependent translation in Ltk- and Ltk-pJH15 cells, which suggested that HAV IRES-dependent translation was not a limiting factor for HAV growth in these cells. The availability of the Ltk-pHJ15 cells will allow the identification of cellular factors required for HAV growth, which could lead to the development of a mouse model to study pathogenesis of HAV.

Animals↗
PubMed2000

Molecular epidemiology of rabies virus isolates from Israel and other middle- and Near-Eastern countries.

A total of 226 isolates of rabies virus from different areas of Israel, including three human isolates and one sample from South Lebanon were identified between 1993 and 1998 by direct immunofluorescence using monoclonal antibodies to the viral nucleoprotein (N). An epidemiological survey based on nucleotide sequence analysis of 328 bp from the C terminus of the N coding region and the noncoding region between the nucleoprotein and the phosphoprotein (NS gene) was performed. Phylogenetic analysis of the isolates from Israel showed that they were related geographically, but not according to host species. Five variants, related groups distributed among four geographical regions, were identified. In each region, rabies virus was isolated from more than one animal species. A comparison of the sequence analysis of rabies virus samples from the rest of world revealed a 2-nucleotide change that distinguished the Middle East variants from the rest.

Adult↗
PubMed1995

Interactions between the IBV nucleocapsid protein and RNA sequences specific for the 3' end of the genome.

The infectious bronchitis virus (IBV) nucleocapsid protein was expressed as a fusion protein in bacteria. The coding sequence differed from the native protein only in the addition of six histidine residues at the amino terminus which were used for enrichment with a nickel affinity column. In gel shift assays, the mobility of labelled G RNA was decreased with increasing concentrations of the fusion protein. Competitive gel shift assays with labelled G RNA indicated that the protein interacted with relatively high avidities to several unlabelled RNAs representing sequences at the 3' noncoding end of the IBV genome. Cache Valley virus (a bunyavirus) mRNA transcribed from the smaller segment cDNA also inhibited the interaction with IBV G RNA to the same extent as homologous unlabelled G RNA. In contrast, interactions of the fusion proteins with a region from 99 to 249 bases from the 3' terminus of the IBV genome and bovine liver RNA were relatively weak. The binding of IBV nucleocapsid protein with RNA probably requires specific sequences and/or structures that are present at a number of sites on the genome, and may represent a common mechanism used by similar viral proteins whose functions depend on binding to RNA.

Animals↗
PubMed1986

Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene.

The 3' end of the 20-kb genome of the Purdue strain of porcine transmissible gastroenteritis coronavirus (TGEV) was copied into cDNA after priming with oligo(dT) and the double-stranded product was cloned into the PstI site of the pUC9 vector. One clone of 2.0-kb contained part of the poly(A) tail and was sequenced in its entirety using the chemical method of Maxam and Gilbert. Another clone of 0.7 kb also contained part of the poly(A) tail and was sequenced in part to confirm the primary structure of the most 3' end of the genome. Two potential, nonoverlapping genes were identified within the 3'-terminal 1663-base sequence from an examination of open reading frames. The first gene encodes a 382-amino acid protein of 43,426 mol wt, that is the apparent nucleocapsid protein on the basis of size, chemical properties, and amino acid sequence homology with other coronavirus nucleocapsid proteins. It is flanked on its 5' side by at least part of the matrix protein gene. The second encodes a hypothetical 78-amino acid protein of 9101 mol wt that is hydrophobic at both ends. A 3'-proximal noncoding sequence of 276 bases was also determined and a conserved stretch of 9 nucleotides near the poly(A) tail was found to be common among TGEV, the mouse hepatitis coronavirus, and the avian infectious bronchitis coronavirus.

Amino Acid Sequence↗
PubMed1992

Analysis of the gB promoter of herpes simplex virus type 1: high-level expression requires both an 89-base-pair promoter fragment and a nontranslated leader sequence.

To investigate the cis-acting sequences involved in regulation of a herpes simplex virus gamma 1 gene, deletion analyses of the glycoprotein B (gB) gene promoter were performed. In transfection assays with gB-chloramphenicol acetyltransferase plasmids, high-level constitutive expression from the gB promoter was found with an 89-bp sequence (-69 to +20). Additional sequences in the 5'-transcribed noncoding leader region (+20 to +136) were required for full stimulation by herpes simplex virus infection. Plasmids with progressive deletions of the gB leader sequence demonstrated that chloramphenicol acetyltransferase expression in infected cells was proportional to the length of the leader region retained. In recombinant viruses containing a gB-gC gene fusion, a similar 83-bp (-60 to +23) region of the gB gene was found to promote accurately initiated gC mRNA from the viral genome with the same kinetics as the wild-type gB gene. Although the kinetics of expression remained the same, RNA abundance was greater with a 298-bp (-260 to +38) promoter than with the 83-bp promoter.

Animals↗
PubMed1980

The gene for polypeptide IX of adenovirus type 2 and its unspliced messenger RNA.

A 606 base pair segment of adenovirus type 2 DNA which includes the gene for polypeptide IX and its flanking regions has been sequenced. Comparison with the sequence of a cDNA copy of the 9S mRNA for polypeptide IX and direct analysis of its capped oligonucleotide show that the gene and its mRNA are colinear. The 9S mRNA has a length of 485 nucleotides, excluding the poly(A) tract and the m7G of the cap, and contains one single open translational reading frame which is sufficiently long to encode polypeptide IX. This reading frame contains three AUG codons, the first of which is used for initiation of protein synthesis. The mRNA has a 5' noncoding region of 24 nucleotides, excluding the m5G of the cap, and encodes a polypeptide which is 139 amino acids long and unusually rich in serine and alanine. The sequence TATATAA is found 25 nucleotides upstream from the cap site. The polyadenylation site has been identified but cannot be located unambiguously because of the presence of two A residues in the DNA sequence at this position. The 3' noncoding region is 41--43 nucleotides long and contains the hexanucleotide sequence AAUAAA. The mRNA for polypeptide IVa2 is encoded adjacent to that for polypeptide IX and is transcribed in the opposite direction. Sequence analysis revealed an overlap of 11--16 nucleotides, depending on the precise location of the polyadenylation sites, between 3' ends of these mRNAs.

Adenoviruses, Human↗
PubMed1998

Yellow fever 5' noncoding region as a potential element to improve hepatitis C virus production through modification of translational control.

The lengthy 5' noncoding region (5' NCR) of hepatitis C virus (HCV) RNA forms a highly ordered secondary structure, very conserved among different strains. It includes an internal ribosome entry site (IRES) element, responsible for the cap-independent translation initiation of HCV RNA. Similarly to the IRES of hepatitis A virus (HAV), another human hepatitis virus, HCV IRES, activity in internal initiation of translation is weak. Furthermore, both viruses exhibit a poor growth phenotype that may result at least partially from an inhibitory control of translation. To enhance HCV translation, as a preliminary step in designing constructs for improvement in viral production, we sought to evaluate a chimeric construct containing the yellow fever virus (YFV) 5' NCR fused to the initiation codon of the HCV coding sequence. YF viral RNA, as the majority of eukaryotic messenger RNAs, is translated by a ribosome scanning mechanism in a cap-dependent manner. The efficiency of translation initiation of the parental HCV construct was compared in vitro in rabbit reticulocyte lysates with that of the chimeric construct containing YFV 5' NCR. Surprisingly, the related distanced YFV 5' NCR was fivefold more active than was the wild-type HCV IRES in directing that function. Furthermore, chimeric transcripts were shown to be effective in vivo after transfection of eukaryotic cells. Taken together, these results raise the following question: why has the HCV genus evolved to the acquisition of an IRES element within its 5' NCR among the Flaviviridae family?

5' Untranslated Regions↗
PubMed1987

Continuing coevolution of virus and defective interfering particles and of viral genome sequences during undiluted passages: virus mutants exhibiting nearly complete resistance to formerly dominant defective interfering particles.

We quantitatively analyzed the interference interactions between defective interfering (DI) particles and mutants of cloned vesicular stomatitis virus passaged undiluted hundreds of times in BHK-21 cells. DI particles which predominated at different times in these serial passages always interfered most strongly (and very efficiently) with virus isolated a number of passages before the isolation of the DI particles. Virus isolated at the same passage level as the predominant DI particles usually exhibited severalfold resistance to these DI particles. Virus mutants (Sdi- mutants) isolated during subsequent passages always showed increasing resistance to these DI particles, followed by decreasing resistance as new DI particles arose to predominate and exert their own selective pressures on the virus mutant population. It appears that such coevolution of virus and DI particle populations proceeds indefinitely through multiple cycles of selection of virus mutants resistant to a certain DI particle (or DI particle class), followed by mutants resistant to a newly predominant DI particle, etc. At the peak of resistance, virus mutants were isolated which were essentially completely resistant to a particular DI particle; i.e., they were several hundred thousand-fold resistant, and they formed plaques of normal size and numbers in the presence of extremely high multiplicities of the DI particle. However, they were sensitive to interference by other DI particles. Recurring population interactions of this kind can promote rapid virus evolution. Complete sequencing of the N (nucleocapsid) and NS (polymerase associated) genes of numerous Sdi- mutants collected at passage intervals showed very few changes in the NS protein, but the N gene gradually accumulated a series of stable nucleotide and amino acid substitutions, some of which correlated with extensive changes in the Sdi- phenotype. Likewise, the 5' termini (and their complementary plus-strand 3' termini) continued to accumulate extensive base substitutions which were strikingly confined to the first 47 nucleotides. We also observed addition and deletion mutations in noncoding regions of the viral genome at a level suggesting that they probably occur at a high frequency throughout the genome, but usually with lethal or debilitating consequences when they occur in coding regions.

Amino Acid Sequence↗
PubMed1995

Vaccine-associated cases of poliomyelitis over a 30 year period in East Germany.

A report is presented about studies on poliovirus type 3 isolates from vaccine-associated cases or contacts of cases of paralytic poliomyelitis, observed over a period of 30 years in East Germany (former GDR). In the viral isolates, some mutations were found in comparison to the Sabin vaccine type 3 strain, distributed over the whole genome. The significance of these mutations has been discussed, especially the mutation at position 472 in the 5' noncoding region found in all the isolates investigated. In five isolates, intertypic recombination between Sabin type 3 and Sabin type 1 vaccine strain occurred. Primary and secondary structures were analysed for the recombination sites.

Child↗
PubMed1989

Delineation of regulatory domains of early (beta) and late (gamma 2) genes by construction of chimeric genes expressed in herpes simplex virus 1 genomes.

The expression of the gamma 2 class of viral genes in cells infected with herpes simplex virus 1 requires viral DNA synthesis and functional viral products made earlier in infection. To identify the sequences required for gamma 2 gene expression, we constructed recombinant viruses in which regions of the thymidine kinase gene (tk), a beta gene normally expressed early in infection, were replaced by specific domains of a gamma 2 gene. The phenotypic attributes examined were (i) sensitivity or resistance of expression in cells exposed to sufficient phosphonoacetate to block viral DNA synthesis, properties of gamma 2 and beta genes, respectively; (ii) expression early in infection, a property of beta genes; and (iii) expression late in infection, a property of gamma 2 genes. We report that replacement of nucleotides -200 to +51 of tk with nucleotides -77 to +104 of the gamma 2 gene conferred upon tk all of the tested attributes of gamma 2 genes. The tk sequence in the 5' transcribed noncoding domain downstream of nucleotide +51 played no apparent role in the expression of the chimeric genes. Similarly, tk sequence downstream of -16 and gamma 2 sequence upstream of -12, when juxtaposed in correct orientations, yielded a chimeric gene that was poorly expressed. In contrast, the chimeric gene consisting of tk sequence upstream of -16 fused to gamma 2 sequence downstream of -12 had the attributes of both beta and gamma 2 genes in that it was expressed both early and late in infection and was partially resistant to phosphonoacetate. The capacity for expression late in infection encoded in the gamma 2 5' transcribed noncoding domain was observed in cells infected with a recombinant virus in which gamma 2 nucleotides +17 to +104 were inserted into the 5' transcribed noncoding domain of the tk gene. We conclude that whereas in the beta genes exemplified by the tk gene the regulatory domains are mainly upstream from nucleotide +51, the sequence(s) that confers gamma 2 regulation is downstream from the TATAA box.

Animals↗
PubMed1991

Splice site skipping in polyomavirus late pre-mRNA processing.

Polyomavirus late nuclear primary transcripts contain tandem repeats of the late strand of the viral genome, as a result of inefficient transcription termination and polyadenylation. Pre-mRNA processing involves the splicing of short noncoding late leader exons to each other (removing genome-length introns) and the splicing of the last leader to a coding body exon (such as for the major virion structural protein, VP1). As a result, cytoplasmic mRNAs contain 1 to 12 tandem leader exons at their 5' ends that are followed by a single coding exon. To understand more about how polyomavirus exons are spliced together, we studied a double-genome construct consisting of two tandem but nonidentical polyomavirus late transcription units. The alternating leader exons are distinguishable from one another but retain identical flanking RNA-processing signals, as for the alternating VP1 exons. We transfected this construct and derivatives of it into mouse cells and determined which leader exons are spliced to which others and which VP1 exons are utilized. Results showed that leader exons are almost never skipped during splicing and are spliced sequentially to one another. On the other hand, VP1 exons were often skipped, with the VP1 exon closest to the polyadenylation site splicing to the nearest upstream leader exon. Splice site replacement experiments showed that VP1 exon skipping is not due to a relative weakness of its 3' splice site or to any sequence upstream of the VP1 3' splice site. Exon skipping is also not the result of sequences within the VP1 exon. Rather, VP1 3' splice site skipping can be eliminated by replacing the inefficient late polyadenylation signal with an efficient one, or by inserting a 5' splice site between the VP1 3' splice site and the late polyadenylation site. Thus, sequences that compose the distal border of the VP1 exon can influence usage of the upstream 3' splice site.

3T3 Cells↗
PubMed1992

Reversion of Q beta RNA phage mutants by homologous RNA recombination.

Q beta phage RNAs with inactivating insertion (8-base) or deletion (17-base) mutations within their replicase genes were prepared from modified Q beta cDNAs and transfected into Escherichia coli spheroplasts containing Q beta replicase provided in trans by a resident plasmid. Replicase-defective (Rep-) Q beta phage produced by these spheroplasts were detected as normal-sized plaques on lawns of cells containing plasmid-derived Q beta replicase, but were unable to form plaques on cells lacking this plasmid. When individual Rep- phage were isolated and grown to high titer in cells containing plasmid-derived Q beta replicase, revertant (Rep+) Q beta phage were obtained at a frequency of ca. 10(-8). To investigate the mechanism of this reversion, a point mutation was placed into the plasmid-derived Q beta replicase gene by site-directed mutagenesis. Q beta mutants amplified on cells containing the resultant plasmid also yielded Rep+ revertants. Genomic RNA was isolated from several of the latter phage revertants and sequenced. Results showed that the original mutation (insertion or deletion) was no longer present in the phage revertants but that the marker mutation placed into the plasmid was now present in the genomic RNAs, indicating that recombination was one mechanism involved in the reversion of the Q beta mutants. Further experiments demonstrated that the 3' noncoding region of the plasmid-derived replicase gene was necessary for the reversion-recombination of the deletion mutant, whereas this region was not required for reversion or recombination of the insertion mutant. Results are discussed in terms of a template-switching model of RNA recombination involving Q beta replicase, the mutant phage genome, and plasmid-derived replicase mRNA.

Amino Acid Sequence↗
PubMed1996

Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes.

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.

Base Sequence↗
PubMed1999

Complete nucleotide sequence of an Indian strain of Japanese encephalitis virus: sequence comparison with other strains and phylogenetic analysis.

The RNA genome of an Indian strain of Japanese encephalitis virus (JEV), GP78, was reverse transcribed and the cDNA fragments were cloned in bacterial plasmids. Nucleotide sequencing of the cDNA clones covering the entire genome of the virus established that the GP78 genome was 10,976 nucleotides long. An open reading frame of 10,296 bases, capable of coding for a 3,432 amino acid polyprotein, was flanked by 95- and 585-base long 5'- and 3'-non-coding regions, respectively. When compared with the nucleotide sequence of the JaOArS982 strain, the JEV GP78 genome had a number of nucleotide substitutions that were scattered throughout the genome except for the 5'-noncoding region, the sequence of which was fully conserved. Comparison of the complete genome sequences of different JEV isolates showed a 1.3-4.1% nucleotide sequence divergence among them, which resulted in 0.6-1.8% amino acid sequence divergence. Analysis based on the complete genome sequences of different JEV isolates showed that the GP78 isolate from India was phylogenetically closer to the Chinese SA14 isolate.

Adult↗
PubMed1989

Sequences of VP9 genes from short and supershort rotavirus strains.

Segment 10 genes from a short (RV-5, serotype G2) and a supershort (B37, a new G serotype) strain were cloned and their sequences compared to the (corresponding) segment 11 sequences of Wa, SA11, and UK rotaviruses. The determined nucleotide sequences were 817 (RV-5) and 947 (B37) bases in length and showed extensively conserved 5' noncoding and protein coding regions. The major open reading frame codes for a protein of 200 (RV-5) or 198 (B37) amino acids, and the newly proposed second open reading frame can code for a protein of 92 amino acids. Compared to long strain gene segments, the base sequences of the short and supershort strains were found to contain extended, AT-rich 3' noncoding regions which were not significantly homologous to each other, to other parts of the VP9 gene, or to other rotavirus genes that have been sequenced. The function(s) of these 3' regions is not apparent.

Amino Acid Sequence↗
PubMed1993

Comparative sequence analysis of four complete primary structures of plum pox virus strains.

The complete nucleotide sequence of plum pox virus (PPV) strain SK 68 was determined from a series of overlapping cDNA clones. The exact 5' terminus was determined by direct RNA sequencing. The RNA sequence was 9786 nucleotides in length, excluding a 3' terminal poly(A) sequence. The large open reading frame starts at nucleotide position 147 and is terminated at position 9568. Comparison of cistrons from other plum pox virus strains with those predicted for the SK 68 strain indicated the same genomic organizations. Comparison of sequences leads to the following conclusions: (1) The genetic organization of all four PPV strains is identical, containing one large polyprotein gene and two noncoding regions at the 5' and 3' ends; (2) pairwise comparison of the genomic sequence of PPV SK 68 with other PPV strains shows 11% alteration. Sequence differences among strains are spread in a uniform manner upon the genome, except for the P1, HC-pro, and two noncoding regions, which are more conserved (with a 4% and 6.6% change). The stability of the noncoding regions is probably linked to their role in replication. The sequence variation has little effect on the amino acid sequence of the corresponding polypeptides, as changes occur preferentially in the third position of the reading frame triplets, except in the case of the 5' end of the coat protein gene (2.7% average difference in amino acid level, while in the case of coat protein it is 7.7%).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Conservation of the 3' terminal nucleotide sequences of Ebola and Marburg virus.

The 3' RNA base sequences of several Marburg (MBG) and Ebola (EBO) virus isolates have been determined. A comparison of these 3' terminal noncoding sequences with those of other negative strand RNA viruses suggests a unique phylogenic niche for Marburg and Ebola viruses. The translation initiation site and 35 N-terminal amino acids of the 3' proximal coding gene of a Zaire strain of Ebola virus was predicted. In addition, putative leader RNA sequences preceding the first gene are discussed in terms of possible regulatory functions.

Base Sequence↗