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PubMed1996

Infectious RNA transcribed from an engineered full-length cDNA template of the genome of a pestivirus.

Infectious RNA was transcribed for the first time from a full-length cDNA template of the plus-strand RNA genome of a pestivirus. The genome of the C strain, which is a vaccine strain of classical swine fever virus, was sequenced and used to synthesize the template. The cDNA sequence of the C strain was found to be 12,311 nucleotides in length and contained one large open reading frame encoding a polyprotein of 3,898 amino acids. Although there were mostly only small differences between the sequence of the C strain and the published sequences of strains Alfort and Brescia, there was one notable insertion of 13 nucleotides, TTTTCTTTTTTTT, in the 3' noncoding region of the C strain. Furthermore, we showed that the sequences at the 5' and 3' termini of the C strain are highly conserved among pestiviruses. We found that the infectivity of the in vitro transcripts of DNA copies pPRKflc-113 and pPRKflc-133 depended on the correctness of the nucleotide sequence. The in vitro transcripts of pPRKflc-133 were infectious, whereas those of pPRKflc-113 were not. In fact, only 5 amino acids among the complete amino acid sequence determined this difference in infectivity. However, virus FLc-133, which was generated from pPRKflc-133, cannot be differentiated from native C-strain virus. Therefore, we exchanged the region encoding the antigenic N-terminal half of envelope protein E2 in pPRKflc-133 with the equivalent region of strain Brescia. The resulting hybrid virus, FLc-h6, could be differentiated from the C strain and from FLc-133 with monoclonal antibodies directed against envelope proteins Erns and E2 of strain Brescia and the C strain. To be suitable for further vaccine development, viruses generated from pPRKflc-133 should grow at least as well as native C-strain virus. In fact, we found that FLc-133, hybrid virus FLc-h6, and the C strain grew equally well. We concluded that pPRKflc-133 is an excellent tool for developing a classical swine fever marker vaccine and may prove valuable for studying the replication, virulence, cell and host tropism, and pathogenesis of classical swine fever virus.

Animals↗
PubMed1998

High prevalence of infection with hepatitis G virus in patients with hepatic and extrahepatic malignancies.

BACKGROUND/AIMS: The pathogenic role of hepatitis G virus, the recently discovered blood-borne agent, is controversial. Our aim was to ascertain the prevalence of hepatitis G virus infection in hepatic and in extrahepatic malignancies. METHODS: We studied 166 Italian patients (112 male, 54 female, mean age 61.8+/-9.3, mean+/-SD, range 34-85). One hundred and eighteen had cirrhosis, which was complicated by hepatocellular carcinoma in 66 cases. Forty-eight patients had extra-hepatic malignancies. Circulating HGV RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) of both the nonstructural-3 and 5'noncoding regions of the hepatitis G virus genome. Antibodies to the E2 protein of hepatitis G virus were detected by means of an enzyme-linked immunosorbent assay. RESULTS: Ongoing HGV infection was detected in 30/66 (46%) patients with hepatocellular carcinoma, 12/52 (23%) patients with cirrhosis, and 14/48 (29%) patients with extrahepatic malignancies (p<0.05). Evidence of exposure to hepatitis G virus (detection of either HGV RNA or anti-E2 antibodies) was found in 46% of patients with cirrhosis, 66% of patients with hepatocellular carcinoma, and 39% of patients with extrahepatic malignancies. Serum HGV RNA positivity was associated with a hematocrit value < or = 0.35 and with history of exposure to blood products (p<0.005). CONCLUSIONS: Ongoing hepatitis G virus infection is detected at a very high rate in patients with hepatocellular carcinoma, but is also fairly common in extrahepatic malignancies. Hepatitis G virus infection in these patients is likely to originate from exposure to blood products, and to persist because of deficient immune surveillance.

Adult↗
PubMed1987

Interactions of T7 RNA polymerase with T7 late promoters measured by footprinting with methidiumpropyl-EDTA-iron(II).

The interactions of T7 RNA polymerase with T7 late promoters were studied by using quantitative footprinting with methidiumpropyl-EDTA X Fe(II) [MPE-Fe(II)] as the DNA cleaving agent. Class II and class III T7 promoters have a highly conserved 23 base pair sequence from -17 to +6. Among class III promoters the -22 to -18 region is also highly conserved. For a class II promoter, T7 RNA polymerase protects the -17 to -4 region from MPE-Fe(II) cleavage; when GTP is present, protection extends from -17 to +5 (noncoding strand). For a class III promoter, protection extends from -20 to -4 and in the presence of GTP from -20 to +5 (noncoding strand). The protected regions for the coding strands of both promoters were nearly identical with that seen for the noncoding strands. The binding constant for the class III promoter is (4 +/- 1.5) X 10(7) M-1 and in the presence of GTP increases to (10 +/- 1.7) X 10(7) M-1. These binding constants are about 1000 and 200 times greater, respectively, than values reported previously [Ikeda, R. A., & Richardson, C. C. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3614-3618]. The differences in binding constants are probably due to tRNA and high salt used in those earlier experiments. Both tRNA and high salt (greater than 50 mM NaCl and greater than 10 mM MgCl2) inhibit the binding of the polymerase to the promoter. Optimal binding conditions occur at 2-5 mM MgCl2 and 0-10 mM NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2003

Prevalence of GBV-C infection among dental personnel.

Healthcare workers who carry out exposure-prone procedures are theoretically at increased risk of acquiring blood-borne virus infections. GB virus C (GBV-C) is a recently described blood-borne virus that is related distantly to hepatitis C virus. The occupational risk of GBV-C infection to healthcare workers is unknown. This study collected detailed occupational and personal risk data in parallel with a blood specimen, to establish the prevalence and determinants of GBV-C infection among dental healthcare workers. The presence of GBV-C antibodies was detected using commercially available ELISA; GBV-C RNA was detected by nested PCR using primers from the conserved 5' noncoding region. The overall prevalence of GBV-C antibodies among the study population was 11.1% (98/880, 95% confidence interval [CI], 9.1-13.4%) and 4.6% were positive for GBV-C RNA (46/879, 95% CI, 2.5-5.1%), resulting in a cumulative prevalence of 15.7%. These figures are similar to those described in other populations. There was no significant difference in lifetime exposure to GBV-C between dentists (17.7%) and dental nurses/hygienists (14.3%). Significantly more dental nurses/hygienists aged 16-30 years had been exposed to GBV-C compared to dentists of the same age (chi(2) = 13.75; P < 0.001). Conversely, significantly more dentists 46 years or older had evidence of exposure to GBV-C compared to dental nurses/hygienists (chi(2) = 6.79; P = 0.009). The high prevalence of GBV-C infection did not seem to be related to past parenteral exposure, and the data suggest that sexual transmission, rather than occupational transmission, was a more important route for GBV-C infection among this population.

Adolescent↗
PubMed2006

Initiation factor-independent translation mediated by the hepatitis C virus internal ribosome entry site.

The hepatitis C viral mRNA initiates translation using an internal ribosome entry site (IRES) located in the 5' noncoding region of the viral genome. At physiological magnesium ion concentrations, the HCV IRES forms a binary complex with the 40S ribosomal subunit, recruits initiation factor eIF3 and the ternary eIF2/GTP/Met-tRNA(i)Met complex, and joins 60S subunits to assemble translation-competent 80S ribosomes. Here we show that in the presence of 5 mM MgCl2, the HCV IRES can initiate translation by an alternative mechanism that does not require known initiation factors. Specifically, the HCV IRES was shown to initiate translation in a reconstituted system consisting only of purified 40S and 60S subunits, elongation factors, and aminoacylated tRNAs at high magnesium concentration. Analyses of assembled complexes supported a mechanism by which preformed 80S ribosomes can assemble directly on the HCV IRES at high cation concentrations. This mechanism is reminiscent of that employed by the divergent IRES elements in the Dicistroviridae, exemplified by the cricket paralysis virus, which mediates initiation of protein synthesis without initiator tRNA.

Aminoacylation↗
PubMed1994

The 5'-untranslated regions of picornavirus RNAs contain independent functional domains essential for RNA replication and translation.

The role of the 5'-untranslated region (5'UTR) in the replication of enteroviruses has been studied by using a series of poliovirus type 3 (PV3) replicons containing the chloramphenicol acetyltransferase reporter gene in which the 5'UTR was replaced by the 5'UTR of either coxsackievirus B4 or human rhinovirus 14 or composite 5'UTRs derived from sequences of PV3, human rhinovirus 14, coxsackievirus B4, or encephalomyocarditis virus. The results indicate that efficient replication of an enterovirus genome requires a compatible interaction between the 5'-terminal cloverleaf structure and the coding and/or 3'-noncoding regions of the genome. A crucial determinant of this interaction is the stem-loop formed by nucleotides 46 to 81 (stem-loop d). The independence of the cloverleaf structure formed by the 5'-terminal 88 nucleotides and the ribosome landing pad or internal ribosome entry site (IRES) was investigated by constructing a 5'UTR composed of the PV3 cloverleaf and the IRES from encephalomyocarditis virus. Chloramphenicol acetyltransferase gene-containing replicons and viruses containing this recombinant 5'UTR showed levels of replication similar to those of the corresponding genomes containing the complete PV3 5'UTR, indicating that the cloverleaf and the IRES may be regarded as functionally independent and nonoverlapping elements.

Base Sequence↗
PubMed2005

The structure of an enzyme-activating fragment of human telomerase RNA.

The ribonucleoprotein enzyme telomerase ensures the stability and fidelity of linear chromosome ends by elongating the telomeric DNA that is lost during each round of DNA replication. All telomerases contain a catalytic protein component homologous to viral reverse transcriptases (TERT) and an RNA (TR) that provides the template sequence, acts as the scaffold for ribonucleoprotein assembly, and activates the enzyme for catalysis. Vertebrate telomerase RNAs contain three highly conserved structural and functional domains: the template domain, the "CR4-CR5" or "activation" domain essential for activation of the enzymatic activity, and a 3'-terminal "box H/ACA"-homology domain responsible for ribonucleprotein assembly and maturation. Here we report the NMR structure of a functionally essential RNA structural element derived from the human telomerase RNA CR4-CR5 domain. This RNA, referred to as hTR J6, forms a stable hairpin interrupted by a single nucleotide bulge and an asymmetric internal loop. Previous work on telomerase has shown that deletion of the hTR J6 asymmetric internal loop results in an RNA incapable of binding the enzymatic protein component of the RNP and therefore an inactive RNP without telomerase activity. We demonstrate here that the J6 internal loop introduces a twist in the RNA structure that may position the entire domain into the catalytic site of the enzyme.

Catalytic Domain↗
PubMed2001

Attenuated lapinized chinese strain of classical swine fever virus: complete nucleotide sequence and character of 3'-noncoding region.

The complete nucleotide sequence including precise 5'- and 3'-terminal non-coding regions (NCRs) of the attenuated lapinized Chinese strain (HCLV) of Classical Swine Fever Virus (CSFV) was determined from overlapping cDNA clones constructed by separated RT-PCR and rapid amplification of cDNA ends (RACE) methods. The genomic RNA of the HCLV strain consists of 12.310 nucleotides (nts) including 374 nts and 242nts in the 5'- and 3'-NCRs, respectively. It contains one large open reading frame (ORF) encoding a polyprotein of 3,898 amino acids with a calculated molecular weight of 437.6 kDa. There is one notable insertion of 12 continuous nts, CTTTTTTCTTTT in the 3'-NCR of HCLV genomic cDNA when compared with its parental virulent Shimen strain. Sequence alignment of partial 3'-NCR reveals two groups of CSFV vaccine strains carrying similar T-rich insertions at different positions in this region. Computer-predicted secondary structures suggest that T-rich insertion greatly change the structures and thus decrease the promoter functions of 3'-NCRs during the replications of these two groups of CSFV vaccine strains.

3' Untranslated Regions↗
PubMed1995

Two different PCR assays to detect enteroviral RNA in CSF samples from patients with acute aseptic meningitis.

Two polymerase chain reaction (RT-PCR) assays were developed to allow rapid detection of enteroviral RNA in cerebrospinal fluid samples (CSF). Primers homologous to the conserved 5' noncoding region of the enterovirus genome were designed. The RT-PCR product size was approximately 500 bp (479 bp for Poliovirus, 500 bp for Coxsackievirus) and was visualized using ethidium bromide-stained gels. Assay 1 utilized Moloney Murine Leukaemia Virus Reverse Transcriptase (MMLV-RTase) for reverse transcription and Taq polymerase for subsequent PCR. Assay 2 utilized a thermoactive DNA polymerase of Thermus thermophilus (rTth enzyme) for both reverse transcription and DNA amplification. In addition, in Assay 2 reverse transcription and PCR were accomplished within the same reaction tube. Both assays detected between 1 and 0.02 TCID50 of prototype strains of Polio and Coxsackie type B viruses propagated in VERO cell and spiked in a pooled preparation of CSF samples from patients with noninfective neurological disorders. However, Assay 1 was 10-fold more sensitive than Assay 2 when applied to the detection of enteroviral RNA in CSF samples from patients with etiologically well characterized acute aseptic meningitis.

Acute Disease↗
PubMed1993

Cell proteins bind to sites within the 3' noncoding region and the positive-strand leader sequence of measles virus RNA.

The genomic 3' noncoding region (NCR) of nonsegmented negative-strand RNA viruses contains recognition site(s) for the polymerase complex, while the RNA plus-strand leader sequence (LS) is probably involved in RNA encapsidation. It is known that host-encoded factors play a role in transcription and replication of some of this group of viruses. Here we report that cellular proteins interact with the genomic 3' NCR and with the plus-strand LS RNA of an important human pathogen, measles virus (MV), a member of the family Paramyxoviridae. Using gel retardation assay and RNA footprinting analysis, we demonstrated that in Vero cells, host-encoded proteins bind specifically to domains within these two sequences. A polypeptide of about 20 kDa binding to the 3' NCR and two polypeptides of about 22 and 30 kDa interacting with plus-strand LS were detected by RNA-protein UV cross-linking. Different RNA-binding activities were found in cells differing in permissiveness to MV replication. The results suggest a role for host-encoded proteins in MV replication.

Animals↗
PubMed1996

Nucleotide sequence of the 5' noncoding region of hepatitis G virus isolated from Japanese patients: comparison with reported isolates.

The nucleotide sequences of the 5' noncoding region (NCR) of hepatitis G virus (HGV) from sera of Japanese patients were determined. Among these isolates, there was a high degree (> 96.9%) of sequence identity, whereas identity with previously reported GB virus-C or HGV strains was low (> 87.0%). Phylogenetic analyses showed that the HGV strains from Japanese patients clustered in groups distantly separated from previously reported strains. Among the Japanese HGV isolates, the genetic distances corresponded to subtype differences observed within hepatitis C virus (HCV) isolates, whereas the differences between the Japanese isolates and the prototypes corresponded to genetic distances observed between HCV genotypes. The Japanese HGV isolates found in this study should be placed in a new genotype distinct from previously described isolates.

Base Sequence↗
PubMed1991

Presence of poly(A) in a flavivirus: significant differences between the 3' noncoding regions of the genomic RNAs of tick-borne encephalitis virus strains.

A poly(A) tail was identified on the 3' end of the prototype tick-borne encephalitis (TBE) virus strain Neudoerfl. This is in contrast to the general lack of poly(A) in the genomic RNAs of mosquito-borne flaviviruses analyzed so far. Analysis of several closely related strains of TBE virus, however, revealed the existence of two different types of 3' noncoding (NC) regions. One type (represented by strain Neudoerfl) is only 114 nucleotides long and carries a 3'-terminal poly(A) structure. This was also found in several TBE virus strains isolated from different geographic regions over a period of almost 30 years. The other type (represented by strain Hypr) is 461 nucleotides long and not polyadenylated. The sequence homology between the two types of TBE virus 3' NC regions terminates at a specific position 81 nucleotides after the stop codon. The second type of 3' NC region more closely resembles the common flavivirus pattern, including the potential for the formation of a 3'-terminal hairpin structure. However, it lacks primary sequence elements that are conserved among other flavivirus genomes.

Animals↗
PubMed1997

Evaluation of commercially available and in-house reverse transcription-PCR assays for detection of hepatitis G virus or GB virus C.

Serum samples from 96 Spanish hemodialysis patients, as well as serial dilutions of RNA extracted from a reference strain of hepatitis G virus (HGV), were tested for HGV or GB virus C (GBV-C) RNA. Two different reverse transcription (RT)-PCR-based methods of detection were compared for the ability to detect RNA extracted from the samples: an RT-nested PCR assay with primers derived from the 5' noncoding region (5'NC) or nonstructural region 3 (NS3) sequences and a commercially available RT-PCR assay with primers derived from the 5'NC or NS5A sequences. When RT-nested PCR was performed on 10-fold serial dilutions of RNA from the HGV reference strain, the last positive dilution was 10(-7) to 10(-8). With the commercial RT-PCR assay, the last positive dilution was 10(-6) to 10(-7). When equal amounts of RNA extracted from serum samples from 96 hemodialysis patients were tested for HGV or GBV-C RNA, 25 patients (26%) were positive by the RT-nested PCR. However, only 21 (84%) of these 25 positive patients were positive for HGV or GBV-C by the commercial RT-PCR assay. Analysis of the 5'NC and NS3 sequences amplified by RT-nested PCR demonstrated that all but two positive patients had unique HGV or GBV-C sequences. In summary, RT-nested PCR and a commercially available RT-PCR assay for HGV or GBV-C gave concordant results for 96% of the patients tested.

Base Sequence↗
PubMed2006

Development, evaluation, and standardization of a real-time TaqMan reverse transcription-PCR assay for quantification of hepatitis A virus in clinical and shellfish samples.

A standardized real-time reverse transcription-PCR (RT-PCR) assay has been developed for an accurate estimation of the number of genome copies of hepatitis A virus (HAV) in clinical and shellfish samples. Real-time procedures were based on the amplification of a fragment of the highly conserved 5' noncoding region and detection through an internal fluorescent probe, including TaqMan and beacon chemistries, in one- and two-step RT-PCR formats. The best performance in terms of sensitivity and reproducibility was achieved by a one-step TaqMan RT-PCR, with a sensitivity enabling the detection of 0.05 infectious unit and 10 copies of a single-stranded RNA (ssRNA) synthetic transcript. Standard reagents, such as a mengovirus strain and an ssRNA transcript, were employed as controls of nucleic acid extraction and RT-PCR, respectively. The test proved to be highly specific after a broad panel of enteric viruses was tested. Sequence alignment of target regions of the primers and probe proved them to be adequate for the quantification of all HAV genotypes. In addition, a quasispecies analysis of the mutant spectrum indicated that these regions are not prone to variability, thus confirming their robustness.

Animals↗
PubMed1995

Nucleotide sequence and transcription of the left early region of Streptococcus pneumoniae bacteriophage Cp-1 coding for the terminal protein and the DNA polymerase.

Cp-1 is a small virulent bacteriophage infecting Streptococcus pneumoniae. It has a linear, double-stranded genome of about 19 kb that replicates by a protein-priming mechanism. We have determined the nucleotide sequence of the leftmost 4780 bp of the DNA of this bacteriophage; computer analysis revealed that this fragment contains seven open reading frames (ORFs) which could encode polypeptides containing more than 50 amino acids. The ORFs are clustered in two groups separated by noncoding intergenic regions. Two of these ORFs code for the terminal protein and a specific DNA polymerase that participate in the replication of the DNA. The predicted amino acid sequence of the terminal protein shows significant similarity with the terminal protein of Bacillus subtilis phage phi 29, and Cp-1 DNA polymerase is homologous to the subgroup of eukaryotic-type DNA polymerases that use a protein as a primer. Combined Northern blots and primer extension experiments have allowed us to map the 5' initiation sites of left early transcripts. These studies revealed that transcripts elongate from left to right and identified the left early promoters.

Amino Acid Sequence↗
PubMed1995

Interaction of polypyrimidine tract-binding protein with the 5' noncoding region of the hepatitis C virus RNA genome and its functional requirement in internal initiation of translation.

Initiation of translation of the human hepatitis C virus (HCV) RNA genome occurs by internal ribosome entry into the 5' noncoding region (5'NCR) in a cap-independent manner. The internal ribosome entry site of the HCV 5'NCR has been previously defined to encompass almost the entire 5'NCR. Here we report the interaction of polypyrimidine tract-binding protein (PTB) at three distinct regions within the 5'NCR by UV cross-linking assays. All three regions contain a consensus polypyrimidine tract motif. The evidence for the interaction of recombinant PTB at multiple sites within the 5'NCR is based on the use of 5'NCR mutants as competitors and by direct UV cross-linking of the mutant RNAs. Furthermore, the PTB isomers from HeLa nuclear extracts interact with the HCV 5'NCR, as shown by immunoprecipitation of a UV cross-linked complex with anti-PTB serum. Immunodepletion of PTB from translation lysates suggested the functional requirement for PTB during translation initiation of the HCV RNA. Addition of purified PTB to immunodepleted lysates did not restore translation mediated by the HCV 5'NCR, indicating the requirement of PTB-associated factors that were removed during immunodepletion.

Base Sequence↗
PubMed1996

Analysis of cis-acting elements required for replication of barley stripe mosaic virus RNAs.

The replicative abilities of mutant RNA transcripts derived from barley stripe mosaic virus cDNA clones were investigated in barley protoplasts that had been coinoculated with wild-type RNA alpha and -gamma transcripts. The 5' and 3' noncoding regions were required for replication, and lack of a 5' cap structure (GpppG) reduced the replicative ability substantially. All internal deletions within RNA alpha abrogated replication in trans. A 2-base change that produced a truncated alpha a protein lacking the first 16 amino acids also compromised the ability of RNA alpha to be replicated. In contrast, RNA beta transcripts containing deletions involving each ORF and the downstream poly(A) tract were effectively amplified by RNAs alpha and gamma, but collective deletion of all four ORFs drastically reduced accumulation. The intergenic region between beta a and beta b was not absolutely required for replication, but small deletions within this region reduced the abundance of RNA beta by at least 10-fold. Deletions within the first 507 nt of the gamma a ORF abrogated replication. However, transcripts containing deletions within the central and 3' regions of the gamma a ORF, the gamma a--gamma b intergenic region, and the gamma b ORF could be amplified in trans. Two mutants containing extensive deletions encompassing the central region of the gamma a ORF and most of gamma b behaved like defective interfering RNAs because they multiplied to high levels in trans and caused a pronounced reduction in accumulation of the coinoculated wild-type RNAs alpha and gamma.

Base Sequence↗
PubMed2001

Functional formation of domain V of the poliovirus noncoding region: significance of unpaired bases.

Previously we have shown that polioviruses with mutations that disrupt the predicted secondary structure of the 5' noncoding region of domain V are temperature sensitive for growth. Non-temperature-sensitive revertant viruses had mutations that re-formed secondary structure by a direct back mutation of changes in the opposite strand. We mutated unpaired regions and selected revertants of viruses with single base deletions, where no obvious back mutation was available in order to gain information on secondary structure. Results indicated that conservation of length of a three base loop between two double-stranded stems was essential for a functional domain V to form. The requirement for the unpaired "hinge" base at 484 which is implicated in the attenuation of Sabin 2 was also confirmed. Results also underline the necessity for functional folding over local secondary structure stability.

5' Untranslated Regions↗