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PubMed1985

Constitutive and conditional suppression of exogenous and endogenous genes by anti-sense RNA.

Plasmid DNA directing transcription of the noncoding (anti-sense) DNA strand can specifically inhibit the expression of several test genes as well as normal, endogenous genes. The anti-sense plasmid constructions can be introduced into eukaryotic cells by transfection or microinjection and function in both transient and stable transformation assays. Anti-sense transcripts complementary to as little as 52 bases of 5' untranslated target gene mRNA specifically suppress gene activity as well as, or more efficiently than, anti-sense transcripts directed against the protein coding domain alone. Conditional anti-sense inhibition is accomplished with the use of hormone-inducible promoter sequences. Suppression of endogenous actin gene activity by anti-sense RNA is detected as a decrease in growth rate and as a reduction in the number of actin microfilament cables. These observations suggest that anti-sense RNA may be generally useful for suppressing the expression of specific genes in vivo and may be a potential molecular alternative to classical genetic analysis.

Acetyltransferases↗
PubMed1988

Replication of chimeric tobacco mosaic viruses which carry heterologous combinations of replicase genes and 3' noncoding regions.

Three tobacco mosaic virus (TMV)-L (tomato strain)-derived chimeras, designated OL1, LG11, or LK31, were constructed by replacing the 3' noncoding region with the corresponding sequence of TMV-OM (common strain), cucumber green mottle mosaic virus (CGMMV), or TMV-Cc (cowpea strain), respectively. The genomic RNAs of TMV-L, -OM, and CGMMV carry histidine-accepting tRNA-like structures at their 3' termini, while the genome of TMV-Cc accepts valine. The three chimeric viruses were able to multiply in both tobacco protoplasts and plants. Multiplication of OL1 in protoplasts was similar to that of the parental strain, L, but in the cases of LG11 and LK31 multiplication was decreased. Sequence analyses of progeny RNAs revealed that viruses with chimeric sequences propagated. These data suggested that TMV-L replicase recognizes the 3' terminal structures of TMV-OM, CGMMV, and TMV-Cc and can initiate minus-strand RNA synthesis. The relationship between the virus-coded component(s) of TMV replicase and the 3' terminal region may not be so stringent.

Base Sequence↗
PubMed2001

Transcription and RNA replication of tacaribe virus genome and antigenome analogs require N and L proteins: Z protein is an inhibitor of these processes.

Tacaribe virus (TV), the prototype of the New World group of arenaviruses, comprises a single phylogenetic lineage together with four South American pathogenic producers of hemorrhagic disease. The TV genome consists of two single-stranded RNA segments called S and L. A reconstituted transcription-replication system based on plasmid-supplied TV-like RNAs and TV proteins was established. Plasmid expression was driven by T7 RNA polymerase supplied by a recombinant vaccinia virus. Plasmids were constructed to produce TV S segment analogs containing the negative-sense copy of chloramphenicol acetyltransferase (CAT) flanked at the 5' and 3' termini by sequences corresponding to those of the 5' and 3' noncoding regions of the S genome (minigenome) or the S antigenome (miniantigenome). In cells expressing N and L proteins, input minigenome or miniantigenome produced, respectively, encapsidated miniantigenome or minigenome which in turn produced progeny minigenome or progeny miniantigenome. Both minigenome and miniantigenome in the presence of N and L mediated transcription, which was analyzed as CAT expression. Coexpression of the small RING finger Z (p11) protein was highly inhibitory to both transcription and replication mediated by the minigenome or the miniantigenome. The effect depended on synthesis of Z protein rather than on plasmid or the RNA and was not ascribed to decreased amounts of plasmid-supplied template or proteins (N or L). N and L proteins were sufficient to support full-cycle RNA replication of a plasmid-supplied S genome analog in which CAT replaced the N gene. Replication of this RNA was also inhibited by Z expression.

Arenaviruses, New World↗
PubMed1999

Primary structure of open reading frame 2 and 3 of the hepatitis E virus isolated from Morocco.

The nucleotide sequence from position 5,014 to 7,186 of the hepatitis E virus (HEV) genome was determined using a set of 10 polymerase chain reaction (PCR) fragments amplified directly from a pool of fecal specimens obtained from patients with well-documented epidemic HEV infection in Morocco. This sequence contains the 3'-terminal region of open reading frame 1 (ORF1), full length ORF2 and ORF3, and a portion of the 3'-noncoding region. The HEV Morocco nucleotide sequence was compared with the corresponding sequences of 13 HEV strains. A region of ORF2 that overlaps with ORF3 was found to be the most conserved region of ORF2, whereas a protein segment encoded by this region was found to be the most variable. Theoretical RNA secondary structure analysis predicted that this region may be folded into a strong secondary structure that may constrain nucleotide sequence variability. In addition, the nucleotide sequence comparison revealed that the HEV Morocco sequence is most homologous to the sequences of the HEV Asian strains compared with the HEV Mexico, swine, and US strains. Phylogenetic analysis performed on the entire ORF2 and ORF3 sequences and on a small fragment of ORF2 allowed classification of the HEV Morocco strain together with a few other known African strains as a separate subtype within the Asian-African genotype.

Base Sequence↗
PubMed1989

A cellular protein that binds to the 5'-noncoding region of poliovirus RNA: implications for internal translation initiation.

Initiation of translation on poliovirus mRNA occurs by internal binding of ribosomes to a region within the 5'-noncoding portion of the mRNA. The mechanistic details and trans-acting factors involved in this event are not understood fully. We used a mobility-shift electrophoresis assay to identify a specific RNA-protein complex, which can form between an RNA fragment that contains nucleotides 559-624 of the poliovirus 5' UTR (untranslated region) and a component or components of a HeLa cell extract. Complex formation was reduced greatly in a reticulocyte lysate or a wheat-germ extract. A 52-kD polypeptide (p52) has been identified as part of the protein-RNA complex by use of an UV cross-linking assay. This polypeptide apparently is not a known translation initiation or elongation factor. The possible involvement of p52 in translation initiation of poliovirus protein synthesis is discussed.

Base Sequence↗
PubMed1998

Utilization of a mammalian cell-based RNA binding assay to characterize the RNA binding properties of picornavirus 3C proteinases.

Using an assay capable of detecting sequence-specific RNA/protein interactions in mammalian cells, we demonstrate that the poliovirus and rhinovirus 3C proteinases are able to bind structured target RNA sequences derived from their respective 5' noncoding regions in vivo. Specific RNA binding by poliovirus 3C was found to be dependent on the integrity of stem-loop d of the RNA cloverleaf structure located at the 5' end of poliovirus genomic RNA. In contrast, mutation of stem-loop b did not prevent this in vivo interaction. However, mutation of stem-loop b, which serves as the RNA binding site for a cellular co-factor important for efficient poliovirus replication, did significantly attenuate the efficiency of 3C RNA binding in vivo and 3CD RNA binding in vitro. This in vivo protein:RNA binding assay was also used to identify several residues in 3C that are critical for RNA binding, but dispensable for 3C proteinase activity. The mammalian cell-based RNA binding assay described in this study may have considerable potential utility in the future detection or analysis of in vivo RNA/protein interactions unrelated to the 3C/RNA interaction described here.

Base Sequence↗
PubMed1990

Expression of the herpes simplex virus type 1 glycoprotein C gene requires sequences in the 5' noncoding region of the gene.

The role of the 5' noncoding region of the herpes simplex virus type 1 glycoprotein C (gC) gene in viral gene expression was investigated with recombinant herpesviruses that contained the bacterial beta-galactosidase gene under the control of the gC promoter-regulatory region. Each of these viruses had the same DNA sequences from the start of gC transcription upstream to -114 but had variable segments of the downstream 140-base-pair sequence that is between the start of gC transcription and translation. Analysis of beta-galactosidase expression and mRNA synthesis from these viruses demonstrated the importance of DNA sequences from the start of gC transcription downstream to +38 for optimal expression from the gC promoter.

Animals↗
PubMed1993

[Molecular cloning and sequencing of hepatitis C virus gene from human blood].

To clone and characterize hepatitis C virus strain in China, we extracted RNA from the pooled plasma of persons with HCV infection in Hunan, China. HCV gene was amplified and cloned by RT-PCR and gene recombinant techniques. A total of 2,307 nucleotides of the complementary DNA clones of HCV (HCV-Hun), including the clone of 5' noncoding region (311bp), core region (340bp), envelope region (397bp), NS1 region (634bp), NS3 region (245bp), and NS5 region (380bp) were isolated and identified. The homologies in nucleotide sequence between HCV-Hun and HCV-US or HCV-J were 98.1% and 98.7% respectively in 5' noncoding region; 92.6% and 97.4% in core region; 74.3% and 88.7% in envelope region; 83.5% and 91.0% in NS1 region; 82.9% and 94.7% in NS3 region; and 74.8% and 90.1% in NS5 region. The similar results were seen in deduced amino acid sequence homologies between above HCV stains. These findings indicate that HCV-Hun were more mimic to HCV-J than HCV-US, and there were considerable heterogeneity in hepatitis C virus genomes isolated from different areas of the world.

Cloning, Molecular↗
PubMed1986

Nucleotide sequence of the 26 S mRNA of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and deduced sequence of the encoded structural proteins.

A cDNA clone containing all of the 26 S mRNA coding region of the RNA genome of Venezuelan equine encephalitis (VEE) virus, virulent strain Trinidad donkey (TRD), has been constructed and sequenced. The nucleotide and deduced amino acid sequences of the 26 S RNA of VEE virus conform to the general organization of the alphavirus subgenomic mRNA. Excluding the poly(A) tail, the VEE 26 S RNA is 3913 nucleotides long with a protein coding region of 3762 nucleotides. Codon usage in the translated region is nonrandom and correlates well with that reported for Sindbis (SIN), Semliki Forest (SF), and Ross River (RR) alphaviruses. Highly conserved sequences of 19 to 22 nucleotides representing putative replicase recognition sites occur at the 26 S RNA junction region of the 42 S genomic RNA and at the 3' terminus immediately preceding the poly(A) tail. The conserved sequence at the 26 S/42 S junction region of VEE virus differs from that of other alphaviruses in that an ochre termination codon (UAA) is substituted for a GGU (Gly) codon present in the other viruses. The 5' and 3' noncoding regions (30 and 121 nucleotides, respectively) of the VEE 26 S RNA are shorter than has been reported for several other alphaviruses. The approximate transmembrane domains of the VEE E1 and E2 envelope glycoproteins have been identified. VEE E1 contains a single asparagine-linked glycosylation site, whereas E2 has three such sites, all of which are apparently glycosylated. The deduced amino acid sequence of the VEE polyprotein shows an overall homology of 44 to 46% with the precursor polyproteins of SIN, SF, and RR viruses. VEE virus capsid, E1, and E2 structural proteins show 43 to 46%, 50 to 53%, and 36 to 41% homology, respectively, with the cognate proteins of SIN, SF, and RR viruses.

Amino Acid Sequence↗
PubMed1989

Sequence analyses and structural predictions of double-stranded RNA segment S1 and VP7 from United States prototype bluetongue virus serotypes 13 and 10.

The nucleotide sequence of segment S1 and the deduced amino acid sequence of VP7 from bluetongue virus (BTV) serotype 13 was determined. Sequences were obtained by use of standard dideoxy DNA sequencing and by direct sequencing of genomic double-stranded RNA (dsRNA). The dsRNA was sequenced with a new dideoxy protocol that produces 300 to 350 bases per set of reactions. Segment S1 is 1156 bp long and contains one long open reading frame capable of coding for 349 amino acids. The protein, VP7, is rather hydrophobic, and has a calculated molecular weight of 38,619 and a net charge of +1.5 at pH 7.0. Segment S1 of BTV-13 has 79.6% of its nucleotides conserved when compared with segment S1 of BTV-10. While most of these differences occur at the third codon position of the open reading frame, the differences between the 89-base-long, 3' noncoding regions occur predominantly in pockets at positions 1092-1098, 1112-1114, and 1125-1129. Potential stem-loop structures encompassing the stop codon of the open reading frame are proposed for both serotypes. Comparisons of VP7 from BTV-13 and BTV-10 indicate that 93.7% of the amino acid residues are conserved, including a single lysine at position 255. Secondary structure predictions infer an eight-stranded beta-barrel structure between residues 150 and 250. This putative beta-barrel may serve as a target for the development of drugs to combat bluetongue disease. Comparable structures detected in the core proteins of single-stranded RNA viruses from both plants and animals suggest that these viruses and BTV had a common origin.

Amino Acid Sequence↗
PubMed2000

Detection and sequencing of measles virus from peripheral mononuclear cells from patients with inflammatory bowel disease and autism.

It has been reported that measles virus may be present in the intestine of patients with Crohn's disease. Additionally, a new syndrome has been reported in children with autism who exhibited developmental regression and gastrointestinal symptoms (autistic enterocolitis), in some cases soon after MMR vaccine. It is not known whether the virus, if confirmed to be present in these patients, derives from either wild strains or vaccine strains. In order to characterize the strains that may be present, we have carried out the detection of measles genomic RNA in peripheral mononuclear cells (PBMC) in eight patients with Crohn's disease, three patients with ulcerative colitis, and nine children with autistic enterocolitis. As controls, we examined healthy children and patients with SSPE, SLE, HIV-1 (a total of eight cases). RNA was purified from PBMC by Ficoll-paque, followed by reverse transcription using AMV; cDNAs were subjected to nested PCR for detection of specific regions of the hemagglutinin (H) and fusion (F) gene regions. Positive samples were sequenced directly, in nucleotides 8393-8676 (H region) or 5325-5465 (from noncoding F to coding F region). One of eight patients with Crohn disease, one of three patients with ulcerative colitis, and three of nine children with autism, were positive. Controls were all negative. The sequences obtained from the patients with Crohn's disease shared the characteristics with wild-strain virus. The sequences obtained from the patients with ulcerative colitis and children with autism were consistent with being vaccine strains. The results were concordant with the exposure history of the patients. Persistence of measles virus was confirmed in PBMC in some patients with chronic intestinal inflammation.

Adult↗
PubMed2002

The complete genome sequence of Perina nuda picorna-like virus, an insect-infecting RNA virus with a genome organization similar to that of the mammalian picornaviruses.

Perina nuda picorna-like virus (PnPV) is an insect-infecting RNA virus with morphological and physicochemical characters similar to the Picornaviridae. In this article, we determine the complete genome sequence and analyze the gene organization of PnPV. The genome of PnPV consists of 9476 nucleotides (nts) excluding the poly(A) tail and contains a single large open reading frame (ORF) of 8958 nts (2986 codons) flanked by 473 and 45 nt noncoding regions on the 5' and 3' ends, respectively. Northern blotting did not detect the presence of any subgenomic RNA. The PnPV genome codes for four structural proteins (CP1-4), and determination of their N-terminal sequences by Edman degradation, showed that all four are located in the 5' region of the genome. The 3' part of the PnPV genome contains the consensus sequence motifs for picornavirus RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) in that order from the 5' to the 3' end. In all of these characters, the genome organization of PnPV resembles the mammalian picornaviruses and two other insect picorna-like viruses, infectious flacherie virus (IFV) of the silkworm and Sacbrood virus (SBV) of the honeybee. In a phylogenetic tree based on the eight conserved domains in the RdRp sequence, PnPV formed a separate cluster with IFV and SBV, which suggests that these three insect picorna-like viruses might constitute a novel group of insect-infecting RNA viruses.

Amino Acid Sequence↗
PubMed1988

Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of dengue type 2 virus, Jamaica genotype: comparative analysis of the full-length genome.

The sequence of the 5'-end of the genome of dengue 2 (Jamaica genotype) virus has been previously reported (V. Deubel, R. M. Kinney, and D. W. Trent, 1986, Virology 155, 365-377). We have now cloned and sequenced the remaining 75% of the genomic RNA that encodes the nonstructural proteins. The complete genome is 10,723 bases in length with a single open reading frame extending from nucleotides 97 to 10,269 encoding 3391 amino acids. The 3'-noncoding extremity presents a stem- and loop-structure and contains a repeated oligonucleotide sequence. Comparisons of the nucleotide sequences of the genomes of dengue 2 viruses of different topotypes reveal 90-95% similarity, with 64-66% similarity evident between dengue viruses of different serotypes. The amino acid sequence of the polyprotein of dengue 2 Jamaica virus shows 97, 68, 50, and 44% similarity with those of other dengue 2, dengue 1, or dengue 4, West Nile, and yellow fever viruses, respectively. Despite amino acid sequence divergence, the hydrophobic profile of the flavivirus proteins is highly conserved. Proteins NS1, NS3, and NS5 are the most conserved. Conserved amino acid stretches present in all flavivirus proteins may be involved in common essential biological functions.

Amino Acid Sequence↗
PubMed2006

Multitypic hepatitis C virus infection identified by real-time nucleotide sequencing of minority genotypes.

The prevalence of concurrent multitypic hepatitis C virus (HCV) infection is uncertain. A sensitive and specific approach to identifying minority HCV genotypes in blood is presented. Following serum extraction and reverse transcription PCR to amplify cDNA originating from the viral 5' noncoding region, the amplified product mixture was treated with genotype-specific restriction endonuclease to digest the dominant genotype. Residual amplicons were subjected to PCR cloning and then to real-time DNA sequencing using a Pyrosequencer to identify the remaining genotypes. Dilution experiments showed that minority genotypes may be detected when they represent 1:10,000 of the total population and in serum specimens with viral loads as low as 1,000 IU/ml. Of 37 patients with bleeding disorders and 44 injecting drug users, infection by more than one HCV genotype was found in 7 (19%) and 4 (9%) patients, respectively. The low rate of detection in people at high risk of repeated HCV infection suggests that multitypic HCV carriage is uncommon.

5' Untranslated Regions↗
PubMed1990

Specific detection of enteroviruses in clinical samples by molecular hybridization using poliovirus subgenomic riboprobes.

Enteroviruses were specifically detected in crude clinical specimens or in cell cultures in which the viruses were amplified by dot hybridization by using poliovirus type 1-derived, subgenomic radiolabeled cRNA probes (riboprobes). The sensitivity of this test varied from 2.5 to 33%, when clinical specimens without cell culture were examined, and was about 85% in cell culture lysates. The specificity of the test was 90 to 100%. The riboprobe corresponding to the 5'-noncoding sequence specifically detected the majority of enteroviruses (56 of 57 tested); the riboprobe derived from the VPI capsid region hybridized with the three poliovirus serotypes and with some coxsackieviruses type A and with echovirus type 7. Echovirus 22 did not hybridize with any riboprobe. In stool specimens, nasal aspirates, and cerebrospinal fluids from patients with meningitis, only one type of virus was identified in different clinical samples from the same patient by the seroneutralization test. Hybridization allowed the detection of enteroviral RNAs easily in stool specimens and nasal aspirates but with a low efficiency in cerebrospinal fluids without amplification of the viruses in cell cultures.

Capsid↗
PubMed1989

Restricted variability of a 17 nucleotide stretch within the 5'-noncoding region of poliovirus genome.

The outbreak of poliomyelitis in Finland in 1984 was caused by a wild strain of poliovirus 3 with uncommon molecular and antigenic properties. We prepared a synthetic oligonucleotide probe complementary to nucleotides 494-510 in the 5'-noncoding part of the genome of a representative strain of the outbreak. This short nucleotide stretch was found to be relatively well conserved within the outbreak and uncommon among 82 independent poliovirus isolates. It may thus be a useful marker for screening isolates to identify those requiring more detailed genetic comparison. The sequences of the corresponding region of the genome are known for 32 separate poliovirus strains and 3 coxsackie B virus strains and show 6 fully conserved nucleotides that could assume a constant hairpin-loop position in a hypothetical secondary structure of the RNA. This could explain the persistence of a particular 17 nucleotide sequence for 40 years in nature in this highly variable region of the poliovirus genome.

Animals↗
PubMed1996

Phylogenetic analysis of hepatitis C virus isolates from hemodialysis patients.

A high prevalence of hepatitis C virus (HCV) infection has been reported in hemodialysis patients. Main risk factors for transmission are previous blood transfusions and possibly nosocomial infections within the dialytic environment. In the present study 224 hemodialysis patients from the same department were tested for the presence of anti-HCV antibodies and HCV-RNA. The presence of anti-HCV in hemodialysis patients was correlated with a history of more than 10 blood transfusions (P = 0.001) and with a duration of hemodialysis treatment for more than 10 years (P = 0.001). The issue of possible patient-to-patient infection was addressed by sequence analysis of all HCV-RNA positive hemodialysis patients (N = 14) together with a control panel of HCV isolates from 56 unrelated non-hemodialysis patients with hepatitis C from the same geographical area. Subsequent phylogenetic analysis of nucleotide sequences obtained from the 5'-noncoding region and the nonstructural NS-5 region of the HCV genome revealed that only two hemodialysis patients were infected by a highly related HCV isolate. The remaining HCV-RNA positive hemodialysis patients including those without previous blood transfusions were all infected by phylogenetically-distant HCV isolates, providing evidence against a nosocomial transmission route. The data of the present study show that molecular epidemiological techniques are important to investigate the issue of nosocomial infection. In our hemodialysis unit patient-to-patient infection appears uncommon and draws attention towards other possible (such as, blood products such as human serum albumin, immunoglobulins) or even yet unrecognized transmission routes.

Adult↗
PubMed1992

Detection of HCV RNA in saliva, urine, seminal fluid, and ascites.

Approximately half of the patients with type C hepatitis do not have a history of parenteral exposure. The route of nonparenteral infection remains unknown. To evaluate the possible role of body fluids, the existence of hepatitis C virus (HCV) RNA in saliva, urine, seminal fluid, and ascites was examined by "nested" polymerase chain reaction (PCR). Amplification of the HCV 5' noncoding sequences was carried out. The amplified product was confirmed by Southern blot hybridization and restriction endonuclease digestion. Among 34 patients with chronic liver disease who were positive for anti-HCV and serum HCV RNA, the prevalence of HCV RNA in body fluids was 100% (7/7) in ascites, 48% (15/31) in saliva, 24% (4/17) in seminal fluid, and 7% (2/29) in urine. The body fluids collected from 3 healthy subjects and 5 patients with chronic liver disease who were positive for anti-HCV but negative for serum HCV RNA were all negative for HCV RNA. Hence, the potential infectivity of body fluids in patients testing negative for serum HCV RNA can probably be discounted. Conversely, the presence of HCV RNA in saliva and seminal fluid of patients positive for serum HCV RNA suggests sexual and household contact as likely modes of nonparenteral transmission of type C hepatitis. Furthermore, the high prevalence of HCV RNA in ascites and saliva may have important implications in medical and dental practice.

Ascitic Fluid↗