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PubMed2004

Evidence for coxsackievirus infection in primary Sjögren's syndrome.

OBJECTIVE: Primary Sjögren's syndrome (SS) is an autoimmune disease characterized by activation of minor salivary gland (MSG) epithelial cells and B and T lymphocytic infiltrates. These findings have long encouraged the hypothesis that a persistent viral infection of the MSG epithelial cells may drive the autoimmune response; however, the identity of that virus has remained elusive. The aim of this study was to test this hypothesis. METHODS: We applied the differential display protocol to MSG RNA samples from patients with primary SS and healthy controls. We then used seminested reverse transcriptase-polymerase chain reaction to amplify the 5'-noncoding region (5'-NCR) of the enteroviral genome in 8 patients with primary SS, 9 patients with secondary SS, and 8 control subjects. Immunohistochemistry was performed to study the expression of the VP1 enteroviral capsid protein in MSG biopsy samples from 12 patients with primary SS, 8 patients with secondary SS, and 16 controls. RESULTS: Differential display analysis yielded a 94-bp fragment of coxsackievirus B4 (CVB4) P2A gene in the primary SS samples. The 5'-NCR was amplified in 7 samples from patients with primary SS and in no samples from patients with secondary SS or controls. The 7 amplified products were sequenced; 4 of the sequences were found to be 98-99% identical to the 5'- NCR of CVB4, and 3 were found to be 97-98% identical to the 5'-NCR of CVA13. Immunohistochemistry for the enteroviral capsid protein VP1 revealed positive staining in epithelial cells and lymphocytic infiltrates in 11 primary SS samples, 1 secondary SS sample, and no control samples. CONCLUSION: We provide evidence that primary SS may be associated with coxsackievirus infection of the MSG epithelial cells and focal lymphocytic infiltrates. Our findings are formulated in a hypothesis concerning the possible role of coxsackieviruses in the induction and maintenance of autoimmunity in primary SS.

Coxsackievirus Infections↗
PubMed1985

Complete sequence of bluetongue virus L2 RNA that codes for the antigen recognized by neutralizing antibodies.

The complete sequence of the RNA which encodes the major outer-shell-neutralizing antigen (VP2) of bluetongue virus serotype 10 was determined from overlapping cDNA clones inserted into pBR322. The segment L2 RNA was 2,926 base pairs long (1.87 X 10(6) daltons) and had, in one strand, an open reading frame capable of coding for a protein that had a calculated size of 111,122 daltons (956 amino acids) and a +11.5 net charge. The coding strands of both the L2 gene and the group-specific L3 gene of bluetongue virus serotype 17 (M. Purdy, J. Petre, and P. Roy, J. Virol. 51:754-759, 1984) had common sequences of some six nucleotides at their 5' termini (namely, GUUAAA...) and eight nucleotides (namely, ...ACACUUAC) at their 3' termini. Both had short 5' noncoding regions with AUG codons at residues 20 to 22 (L2) and 18 to 20 (L3). The sequences flanking these AUG codons were similar (A/GCCAUGG). The 3' noncoding regions were longer (36 nucleotides for L2, 49 nucleotides for L3). The predicted amino acid sequence of the L2, compared with the similarly sized L3 gene product, was rich in cysteine residues and charged amino acids.

Antigens, Viral↗
PubMed1991

Translation of hepatitis A virus RNA in vitro: aberrant internal initiations influenced by 5' noncoding region.

Hepatitis A virus (HAV) RNAs were translated in vitro in rabbit reticulocyte lysates. The pattern of proteins synthesized from full-length HAV RNA was highly complex, consisting of a continuous spectrum of polypeptides ranging from less than 20,000 to greater than 200,000 Da. The pattern was not significantly altered by varying incubation times, ion, or other reaction parameters, or by the addition of HeLa or BS-C-1 cell extracts to the translation reactions. Plasmids engineered with mutations in the 3C coding region produced transcripts which directed the synthesis of the same overall pattern of polypeptide products as those transcribed from wild-type sequences, suggesting that protein processing by 3C did not generate the complex set of protein products. Translation of RNA containing only the P3 coding region of HAV, directly adjacent to the HAV 5' noncoding region, generated a set of protein products which precisely matched a subset of those synthesized from full-length HAV RNA. The translation products of P3 RNA, full-length RNA, and mutant 3C-containing RNAs were analyzed by immunoprecipitation with antisera specific for 3D, VP1, and 2C sequences; several products were subjected to N-terminal sequence analysis. All together, the results demonstrate that translation of HAV RNA in rabbit reticulocyte lysates initiates predominantly at a large number of internal AUG codons, especially those in the P3 coding region. A minor population of products is initiated from sites in the P1 and P2 regions. The latter proteins undergo some proteolytic processing, at unidentified sites, catalyzed by 3C protein sequences. Replacement of the HAV 5' noncoding region with encephalomyocarditis virus 5' end sequences increased initiation at the correct polyprotein start site and both reduced and altered the products generated by internal initiation.

3C Viral Proteases↗
PubMed2006

Construction and mutagenesis of an artificial bicistronic tick-borne encephalitis virus genome reveals an essential function of the second transmembrane region of protein e in flavivirus assembly.

Flaviviruses have a monopartite positive-stranded RNA genome, which serves as the sole mRNA for protein translation. Cap-dependent translation produces a polyprotein precursor that is co- and posttranslationally processed by proteases to yield the final protein products. In this study, using tick-borne encephalitis virus (TBEV), we constructed an artificial bicistronic flavivirus genome (TBEV-bc) in which the capsid protein and the nonstructural proteins were still encoded in the cap cistron but the coding region for the surface proteins prM and E was moved to a separate translation unit under the control of an internal ribosome entry site element inserted into the 3' noncoding region. Mutant TBEV-bc was shown to produce particles that packaged the bicistronic RNA genome and were infectious for BHK-21 cells and mice. Compared to wild-type controls, however, TBEV-bc was less efficient in both RNA replication and infectious particle formation. We took advantage of the separate expression of the E protein in this system to investigate the role in viral assembly of the second transmembrane region of protein E (E-TM2), a second copy of which was retained in the cap cistron to fulfill its other role as an internal signal sequence in the polyprotein. Deletion analysis and replacement of the entire TBEV E-TM2 region with its counterpart from another flavivirus revealed that this element, apart from its role as a signal sequence, is important for virion formation.

Amino Acid Sequence↗
PubMed1994

Genome structure and variability of a virus causing hantavirus pulmonary syndrome.

A previously unrecognized hantavirus (family Bunyaviridae) has recently been detected and shown to be associated with a severe respiratory illness with high mortality, termed hantavirus pulmonary syndrome (HPS). This disease has now been identified throughout the western United States. We present nucleotide sequence characterization of the three RNA segments composing the HPS virus genome and address the question of the apparent emergence of this highly lethal virus. No evidence of genetic reassortment with previously recognized hantaviruses was found, each RNA segment being unique and approximately 30% different at the nucleotide level to the segments of the closest relative, Prospect Hill virus. These findings, together with the observed extensive genetic diversity of HPS viruses and examples of geographic clustering of distinct virus genotypes, suggest that HPS and associated virus have likely existed undetected for many years. The virus genome M segment was determined to be 3696 nucleotides in length and encode G1 and G2 proteins, 652 and 488 amino acids in length. The S segment was found to be 2059 nucleotides in length and to encode a nucleocapsid protein, 428 amino acids in length. S segment analysis also revealed an unusually long noncoding region with numerous repeats and evidence for a potential NSS protein encoded in an overlapping frame.

Amino Acid Sequence↗
PubMed1993

Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism.

The human hepatitis C virus (HCV) contains a long 5' noncoding region (5' NCR). Computer-assisted and biochemical analyses suggest that there is a complex secondary structure in this region that is comparable to the secondary structures that are found in picornaviruses (E.A. Brown, H. Zhang, L.-H. Ping, and S.M. Lemon, Nucleic Acids Res. 20:5041-5045, 1992). Previous in vitro studies suggest that the HCV 5' NCR plays an important role during translation (K. Tsukiyama-Kohara, N. Iizuka, M. Kohara, and A. Nomoto, J. Virol. 66:1476-1483, 1992). Dicistronic and monocistronic expression vectors, in vitro translation, RNA transfections, and deletion mutagenesis studies were utilized to demonstrate unambiguously that the HCV 5' NCR is involved in translational control. Our data strongly support the conclusion that an internal ribosome entry site exists within the 5' noncoding sequences proximal to the initiator AUG. Furthermore, our results suggest that the HCV genome is translated in a cap-independent manner and that the sequences immediately upstream of the initiator AUG are essential for internal ribosome entry site function during translation.

Base Sequence↗
PubMed1993

Regulation of HIV-1 envelope protein synthesis by Tat and Rev in 293 cells.

A plasmid expression vector (B2) with the HIV-1 envelope sequence downstream of the adenovirus type 5 early region 3 promoter could direct the synthesis of envelope protein in the absence of Rev when transfected into 293 cells. We investigated this further using pNL4.3 delta TR, and HIV-1 mutant which lacks the first exon of Tat and Rev and pNL4.3 delta R, an HIV-1 mutant with a premature termination codon in the second coding exon of Rev. In cells transfected with pNL4.3 delta TR and a Tat-expressing vector or with pNL4.3 delta R alone, analysis of RNA revealed the accumulation of cytoplasmic Env mRNA in the absence of Rev. However, envelope protein synthesis was observed in the absence of Rev only in cells transfected with pNL4.3 delta TR and a Tat-expressing vector, not in cells transfected with pNL4.3 delta R. The Env mRNAs synthesized from pNL4.3 delta R can have 536 to 548 nucleotides of 5' non coding sequence, whereas the Env mRNA from pNL4.3 delta TR will have a shortened noncoding sequence of 321 nucleotides. These results indicate that the mRNA sequences 5' to the Env protein initiation codon have a role in Env expression.

Adenoviruses, Human↗
PubMed2006

Gene silencing in vitro and in vivo using intronic microRNAs.

MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) that contain either complete or partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. Numerous miRNAs have been reported to induce RNA interference (RNAi), a posttranscriptional gene-silencing mechanism. Recent evidence also indicates that they are involved in the transcriptional regulation of genome activities. They were first discovered in Caenorhabditis elegans as native RNA fragments that modulate a wide range of genetic regulatory pathways during embryonic development, and are now recognized as small gene silencers transcribed from the noncoding regions of a genome. In humans, nearly 97% of the genome is noncoding DNA, which varies from one individual to another, and changes in these sequences are frequently noted to manifest clinical and circumstantial malfunction. Type 2 myotonic dystrophy and fragile X syndrome were found to be associated with miRNAs derived from introns. Intronic miRNA is a new class of miRNAs derived from the processing of nonproteincoding regions of gene transcripts. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of RNA polymerase (Pol)-II and spliceosomal components for its biogenesis. Several kinds of intronic miRNAs have been identified in C. elegans, mouse, and human cells; however, neither function nor application has been reported. Here, we show for the first time that intron-derived miRNA is not only able to induce RNAi in mammalian cells but also in fish, chicken embryos, and adult mice, demonstrating the evolutionary preservation of this gene regulation system in vivo. These miRNA-mediated animal models provide artificial means to reproduce the mechanisms of miRNA-induced disease in vivo and will shed further light on miRNA-related therapies.

Animals↗
PubMedDate not supplied

Correlation of detectability of hepatitis C virus genome in saliva of elderly Japanese symptomatic HCV carriers with their hepatic function.

The hepatitis C virus (HCV) genome was sought in the saliva of 76 chronic HCV carriers (mean age nearly 60 years) in a rural Japanese town, who had high serum titers of c-100 and anti-core second generation antibodies. In 27 samples (27 cases, 36%), the HCV-RNA genome was detected by the reverse transcriptase - polymerase chain reaction with either of two sets of primers covering two regions of the HCV genome: the 5'noncoding region and the region encompassing the putative envelope (E1). Transaminase values at the time of sampling were higher in the patients with than in those without detectable HCV RNA in saliva (p = 0.04 for alanine aminotransferase, p = 0.04 for aspartate aminotransferase; Wilcoxon test). The prevalence of the positivity was higher by 5'noncoding primers (14/59 vs. 15/68). Our data show that the severity and duration of hepatic dysfunction influence the detectability of the HCV genome in the saliva. This has been a controversial point among investigators.

Adult↗
PubMed1985

Transcription of three c-myc exons is enhanced in chicken bursal lymphoma cell lines.

The chicken c-myc gene, as defined by its homology to the v-myc gene of MC29 virus, is comprised of two exons. Using the techniques of runoff transcription, primer extension, and S1 nuclease protection, we demonstrate that there is a third c-myc exon of approximately equal to 345 base pairs (bp) located 0.7 kbp upstream of the 5' end of the v-myc homology. This first exon is transcribed and present in myc mRNA in normal chicken cells. We also examined RNA from five cell lines derived from avian leukosis virus-induced bursal lymphomas. In all these lines, the level of transcription of the 2.2- to 2.5-kbp myc mRNA is increased 30- to 60-fold over normal cells. The myc mRNA in four of these lines also contains increased levels of the first noncoding exon, and evidence is presented that the long terminal repeat (LTR) in the vicinity of c-myc is functioning as an enhancer of c-myc transcription rather than as a promoter in several of these cell lines. In two cell lines in which the viral LTR has integrated between the first and second exons in the proper orientation for downstream promotion of myc, the LTR does not exhibit promoter function. The pattern of c-myc transcription observed by others in a vast majority of avian leukosis virus-induced neoplasms is not observed in any of the five cell lines examined.

Animals↗
PubMed1983

Nucleotide sequence of the 5' noncoding region and part of the gag gene of mouse mammary tumor virus; identification of the 5' splicing site for subgenomic mRNAs.

We have determined the sequence of the first 1371 nucleotides at the 5' end of the genome of mouse mammary tumor virus using molecularly cloned proviral DNA of the GR virus strain. The most likely initiation codon used for the gag gene of mouse mammary tumor virus is the first one, located 312 nucleotides from the 5' end of the viral RNA. The 5' splicing site for the subgenomic mRNA's is located approximately 288 nucleotides downstream from the 5' end of the viral RNA. From the DNA sequence the amino acid sequence of the N-terminal half of the gag precursor protein, including p10 and p21, was deduced (353 amino acids).

Amino Acid Sequence↗
PubMed1995

Long, nearly identical untranslated sequences at the 3' terminal regions of the genomic RNAs of cherry leafroll virus (walnut strain).

Hybridization analyses of cDNA clones derived from the two genomic RNAs, RNA1 and RNA2, of the walnut strain of the nepovirus cherry leafroll nepovirus (wCLRV) demonstrated a long region of high homology between the two viral RNAs. Subsequent mapping and nucleotide sequencing revealed a long, noncoding, presumably untranslated, region (3' UTR) immediately 5' of the terminal polyadenylate, a region that is almost identical in the two RNAs. This 3' UTR is 1567 nucleotide residues long in RNA1. Homologies of about 80% were found with corresponding regions of genomic RNAs from other strains of CLRV, but not with the corresponding regions of other nepovirus genomic RNAs.

Base Sequence↗
PubMed1988

Isolation and characterization of a variant of human papillomavirus type 11 from a nasal inverting (Schneiderian) papilloma.

We have previously reported the presence of a variant of human papillomavirus (HPV) type 11 in a nasal inverting papilloma [Respler et al., 1987]. In the present study, we have cloned molecularly the DNA of this variant at its unique restriction enzyme Bam HI site into lambda BF101 phage. Restriction enzyme mapping and DNA sequencing revealed that the genome of this virus contained an extra 531 base pair (bp) which was the repeat of most of the noncoding region (ncr) of HPV 11. Insertion of transcriptional control elements, including the repeated sequence, in front of the chloramphenicol acetyltransferase (CAT) gene resulted in a 5- to 30-fold increase in expression in transfected cells, as compared to the constructs containing a single ncr of HPV 11. This increased expression was due to enhanced levels of CAT RNA the synthesis of which is initiated by the viral promoter element.

Acetyltransferases↗
PubMed1992

Intracellular amplification and expression of a synthetic analog of rotavirus genomic RNA bearing a foreign marker gene: mapping cis-acting nucleotides in the 3'-noncoding region.

cDNAs were constructed to encode plus- or minus-sense analogs of gene 9 RNA of porcine rotavirus strain OSU in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene was flanked by the 5'-terminal 44 nucleotides (nt) and 3'-terminal 35 nt of the authentic rotavirus gene. Transfection of plus-sense gene-9-CAT RNA into rotavirus-infected cells resulted in its amplification and in the efficient expression of CAT; this was greatly enhanced by the presence of a 5' cap structure. Amplification was ablated by omitting the rotavirus superinfection or by removing the 3'-terminal 35-nt rotavirus sequence from the RNA. This result indicated that amplification depended both on rotavirus proteins supplied in trans and on cis-acting rotavirus sequences. Minus-sense or double-stranded gene-9-CAT RNA was essentially inactive, indicating that synthetic RNAs can be introduced into the rotavirus replicative cycle in vivo only when provided in the plus sense. However, incorporation of the CAT-bearing RNA into infectious rotavirus was not detected. Two heterologous rotaviruses, the simian RRV and chicken Ch2 strains, efficiently complemented the OSU-based gene-9-CAT RNA, even though the Ch2 strain was only 50%-66% related in the noncoding regions. Mutational analysis of the 35-nt 3'-noncoding region showed that the 3'-terminal 12 or 17 nt were sufficient for reduced (12% or 23%, respectively) levels of amplification, whereas inclusion of the 3'-terminal 19 nt fully restored amplification. Thus, the 3'-terminal cis-acting signals required for amplification include the 7-nt-terminal consensus sequence together with 12 nt of adjoining, less-well-conserved sequence.

Animals↗
PubMed1985

Analyses of the mRNA transcription processes of snowshoe hare bunyavirus S and M RNA species.

The time course of synthesis of snowshoe hare bunyavirus small (S)- and medium (M)-sized viral RNA (vRNA), viral cRNA (vcRNA), and mRNA species was analyzed by using single-stranded DNA probes representing the S- and M-coded gene products. In the presence of puromycin, an inhibitor of protein synthesis, the subgenomic S mRNA species were detected, but not full-length S vcRNA or S vRNA species. No M-related RNA species were identified in puromycin-treated cells. In the absence of puromycin, full-length M and S vRNA, S vcRNA, and subgenomic S mRNA species were observed, as well as apparently full-length M vcRNA species, presumably including the approximately similar-sized M mRNA species. The 5' ends of the S and M mRNA species have been shown to be heterogeneous and some 12 to 17 bases longer than the ends of their corresponding presumptive replicative vcRNA species, in agreement with an earlier report that they represent nonviral primer sequences (D. H. L. Bishop, M. E. Gay, and Y. Matsuoko, Nucleic Acids Res. 11:6409-6418, 1983). The 3' ends of the M and S mRNA species were found to be shorter by some 60 and 100 nucleotides, respectively, than those of their corresponding full-length vcRNA species. Comparison of the 3' noncoding regions of the S and M vcRNA species revealed that there are conserved sequences following the translation termination codons of the two RNA species. One of these conserved sequences is a pyrimidine-rich template sequence that is approximately 20 nucleotides beyond the deduced S mRNA transcription termination site.

Base Sequence↗
PubMed1997

Replication of in vitro tobravirus recombinants shows that the specificity of template recognition is determined by 5' non-coding but not 3' non-coding sequences.

Natural recombinant tobacco rattle tobravirus (TRV) isolates contain sequences from a different tobravirus, pea early browning virus (PEBV). To characterize the sequence requirements for viable recombinant formation hybrid cDNA clones of RNA2 of PEBV and TRV were assembled. Inclusion of 320 nt from the 5' terminus of PEBV or 335 nt from the 5' terminus of TRV in the hybrid RNAs was sufficient to permit their replication by, respectively, PEBV RNA1 or TRV RNA1 regardless of the origin of the 3' terminal region. However, PEBV RNA1 but not TRV RNA1 was sometimes able to support low level replication of RNA2 containing the heterologous 5' terminal region. In vitro translation of PEBV transcripts containing 5' noncoding region deletions supported the hypothesis that in vivo the PEBV coat protein (CP) is expressed from a subgenomic RNA and that, therefore, in the recombinants the CP subgenomic promoter probably is recognized by the replicase of the heterologous virus.

Cloning, Molecular↗
PubMed1995

[A full-size DNA copy of the tick-borne encephalitis virus genome. I. Analysis of the 5'- and 3'-terminal noncoding regions of the genome].

Using reverse transcription and the polymerase chain reaction, cDNA fragments of noncoding regions of the tick-borne encephalitis virus (TBEV) genome were obtained. These fragments were cloned into a pGEM3 vector, and their nucleotide sequences were determined. The heterogeneity of the 3'-terminal untranslated region of the TBEV RNA was revealed. To create a stable full-size DNA copy of the TBEV genome, four cDNA variants differing in length and structure of the 3'-terminal fragment of the viral RNA were cloned into a pBR322-derived vector.

Base Sequence↗
PubMed1992

Molecular cloning and sequence analysis of the mumps virus gene encoding the L protein and the trailer sequence.

We have cloned and determined the nucleotide sequences of the seventh gene of the Miyahara strain of mumps virus (MuV) encoding the L protein. The L gene is 6925 nucleotides in length and contains a single long open reading frame which is capable of coding for a protein of 2261 amino acids with a calculated molecular weight of 256,571 Da. The deduced amino acid sequence of the L protein of MuV showed significant homology with those of six other paramyxoviruses, human parainfluenza type 2 virus, Newcastle disease virus, Sendai virus, measles virus, human parainfluenza type 3 virus, and human respiratory syncytial virus. The predicted MuV L protein contained distinct elements thought to be essential for RNA polymerase activity. A noncoding sequence of 24 nucleotides downstream of the presumed polyadenylation site of the L gene showed significant complementarity with the leader sequence composed of 55 nucleotide at the 3' end of the genomic RNA.

Amino Acid Sequence↗