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Nucleotide sequence at the junction between the coding region of the adenovirus 2 hexon messenger RNA and its leader sequence.

We have determined a 139-base-pair sequence of adenovirus 2 DNA that is located immediately leftwards of the cleavage site for endonuclease Sma I at position 51.1. The established sequence includes the hexon AUG initiator codon, located 75--77 nucleotides leftwards of this cleavage site, and codons for the first 26 amino acids of the hexon polypeptide. By the use of purified hexon mRNA as a template and separated strands of small restriction enzyme fragments as specific primers, the complete 5' noncoding region of the hexon mRNA was synthesized and part of its sequence was determined. The tripartite leader sequence of the hexon mRNA starts 39 nucleotides upstream from the initiator AUG triplet and the total length of the 5' noncoding part of the hexon mRNA was estimated to be 235 nucleotides. The sequence at the junction of the leader sequence permits the formation of secondary structures that may be of importance for the splicing reaction.

Adenoviruses, Human↗

Mutations in the nonconserved noncoding sequences of the influenza A virus segments affect viral vRNA formation.

Influenza A virus replication and packaging is mediated by cis-acting signals, which are located at the 3' and the 5' end of the viral segments. The terminal residues can be divided into conserved and nonconserved residues. We have constructed a mutant influenza A/WSN/33 virus, which contains multiple mutations in the nonconserved residues of the neuraminidase (NA) segment. This virus shows a segment-specific reduction of the genomic RNA content in the infected cell and in the progeny virus. Further mutants and revertant viruses revealed that it was not possible to define specific residues, which were responsible for the reduction of the NA-specific RNA. Thus, it appears that an efficient vRNA formation is dependent on the synergistic effect of the terminal sequences.

Animals↗

Predicted stem-loop structures and variation in nucleotide sequence of 3' noncoding regions among animal calicivirus genomes.

Caliciviruses are nonenveloped with a polyadenylated genome of approximately 7.6 kb and a single capsid protein. The "RNA Fold" computer program was used to analyze 3'-terminal noncoding sequences of five feline calicivirus (FCV), rabbit hemorrhagic disease virus (RHDV), and two San Miguel sea lion virus (SMSV) isolates. The FCV 3'-terminal sequences are 40-46 nucleotides in length and 72-91% similar. The FCV sequences were predicted to contain two possible duplex structures and one stem-loop structure with free energies of -2.1 to -18.2 kcal/mole. The RHDV genomic 3'-terminal RNA sequences are 54 nucleotides in length and share 49% sequence similarity to homologous regions of the FCV genome. The RHDV sequence was predicted to form two duplex structures in the 3'-terminal noncoding region with a single stem-loop structure, resembling that of FCV. In contrast, the SMSV 1 and 4 genomic 3'-terminal noncoding sequences were 185 and 182 nucleotides in length, respectively. Ten possible duplex structures were predicted with an average structural free energy of -35 kcal/mole. Sequence similarity between the two SMSV isolates was 75%. Furthermore, extensive cloverleaflike structures are predicted in the 3' noncoding region of the SMSV genome, in contrast to the predicted single stem-loop structures of FCV or RHDV.

Base Sequence↗

Specificity in the association of tomato black ring virus satellite RNA with helper virus.

The satellite RNAs (sat-RNAs) associated with some isolates of tomato black ring virus (TBRV) consist of single-stranded molecules of about 1375 nucleotides, encoding a nonstructural protein of 48K which has been shown to be involved in the replication of the sat-RNA. The TBRV sat-RNAs are also dependent for their replication and for their encapsidation on the helper virus. To characterize the nature of the association between sat-RNA and helper virus, transcripts of sat-RNA from TBRV isolates C and L (respectively, of serotypes G and S) have been prepared and inoculated onto Chenopodium quinoa leaves or protoplasts. Transcript of the TBRV sat-RNA C is efficiently multiplied when coinoculated with the genomic RNAs of TBRV isolate G (used instead of TBRV isolate C, because isolate G was depleted of sat-RNA), but does not multiply with TBRV isolate L. On the other hand, transcript of the sat-RNA L is able to multiply with the cognate helper virus and, less efficiently, with grapevine chrome mosaic virus (another nepovirus, 80% similar to TBRV), but does not multiply with TBRV G. The specificity of the association resides at the level of sat-RNA replication. Analysis of the multiplication of chimeric sat-RNAs, obtained by exchanging different regions between the two sat-RNAs C and L, showed that the 5' and the 3' noncoding regions of the sat-RNA, although important for replication, are not implicated in specificity. The results suggest that the determinants of the specificity are contained in the 48K sat-RNA-encoded protein.

Base Sequence↗

The nucleotide sequence and genome organization of the RNA2 and RNA3 segments in broad bean mottle virus.

Complete nucleotide sequences of broad bean mottle virus (BBMV) genomic RNAs 2 and 3 were determined. They consist of 2811 and 2293 nucleotides, respectively. Both RNAs are caped and, unlike in other tricornaviruses, both initiate with an A residue. BBMV RNA2 is monocistronic and encodes an 815 amino acid 2a protein, whereas RNA3 is dicistronic, encoding for a 295 amino acid 3a protein and for the 190 amino acid coat protein. A central, 423 amino acid 2a protein core region is highly homologous among the three bromoviruses, whereas both N- and C-termini are more heterologous. Most of the homologies among 3a proteins are concentrated within the N-termini two-thirds of the molecule that is predominantly hydrophobic, whereas the C-terminal one-third contains a large number of charged amino acids. The homologies among coat proteins are clustered within several mostly hydrophobic, or neutral, domains. The 5' noncoding region of the RNA2 has 110 nucleotides, whereas that of RNA3 contains 330 nucleotides. As in cowpea chlorotic mottle virus, but unlike in Brome mosaic virus, the 5' noncoding region includes subgenomic promoter-like sequences. The BBMV RNA3 intercistronic region also has subgenomic promoter sequences and contains a long poly(A) stretch. At the 3' end, BBMV RNAs 2 and 3 have 257 and 236 noncoding nucleotides, respectively.

Base Sequence↗

Internal binding of eucaryotic ribosomes on poliovirus RNA: translation in HeLa cell extracts.

Translation initiation on poliovirus mRNA in poliovirus-infected cells has been shown to occur by internal binding of ribosomes to the 5' noncoding region (J. Pelletier and N. Sonenberg, Nature [London] 334:320-325, 1988). Here we show that internal ribosome binding can occur in HeLa cell extracts in vitro. Internal binding to the 5' noncoding region of poliovirus mRNA in a bicistronic context was independent of the upstream open reading frame and did not require poliovirus proteins.

HeLa Cells↗

Structural and functional characterization of the unusually short long terminal repeats and their adjacent regions of a novel endogenous avian retrovirus.

We have cloned the long terminal repeats and their flanking regions from four different proviruses belonging to a large, highly conserved, novel family of avian endogenous retroviruses. This family, termed the endogenous avian retrovirus (EAV) family, is distinct from the previously characterized avian endogenous and exogenous retroviruses. We have analyzed the sequences of the long terminal repeats and their adjacent noncoding viral sequences, including the gag leader region and the 3' noncoding region, of several different members of the EAV family and have found that the regulatory region of these novel viruses contains several unique features. The LTRs of the EAV proviruses are extremely short (243 bp long) but contain all of the essential regulatory features of longer avian retrovirus LTRs. The gag leader region and the 3' noncoding region of the novel EAVs are only weakly related to those of other avian retroviruses. Northern blot hybridization analysis of RNA from Line-0 chicken embryos reveals several transcripts derived from the EAV proviruses. Primer extension analysis indicates that all transcripts initiated from 5' proviral LTRs are initiated at the predicted +1 position within the EAV LTRs. The relative shortness, sequence divergence from other known LTRs, and the retention of the transcriptional integrity of the EAV LTRs make these LTRs an interesting model system for LTR function and for study of the potential involvement of such highly conserved retroviral elements in development.

Animals↗

Genome cloning and analysis of the large RNA segment (segment A) of a naturally avirulent serotype 2 infectious bursal disease virus.

The genome of infectious bursal disease virus (IBDV) of serotype 2 (strain OH) has been cloned, and 3171 nucleotides of genome segment A cDNA sequence have been determined for the first time. Sequence homology of OH-IBDV with the most distant serotype 1 IBDV at the nucleotide level is 83.1%, and the amino acid sequence homology of the polyprotein is 89.6%. Alignment of the polyprotein amino acid sequences showed the hypervariable region in VP2 to be 151-152 amino acid residues long in IBDV. A second variable region, 37 amino acid residues long, was identified in the N-terminal third of the IBDV VP2 molecule. IBDV strains, like the IPNV strains, also contain inverted repeats that may form stem-and-loop structures in the 5' noncoding sequences. These inverted repeats are variable between the two IBDV serotypes, particularly at the AT basepairs.

Animals↗

Base-pair formation between noncapped influenza virus in vitro transcripts and 18S rRNA in rabbit reticulocyte 80S ribosome-mRNA complexes detected by psoralen photoreaction.

RNA-RNA interactions between 18S ribosomal RNA and noncapped influenza cRNA were detected by psoralen photochemical cross-linking in reticulocyte 80S ribosome-cRNA complexes. In vitro transcripts of type A influenza virus synthesized by endogenous RNA polymerase with adenylyl-(3'----5')-guanosine primer formed 80S complexes with rabbit reticulocyte ribosomes. The extent of the complex formation by these noncapped cRNAs was less than that by the m7G-capped reovirus in vitro transcripts, but the former RNAs in the 80S complexes were cross-linked to 18S rRNA as efficiently as the latter RNAs by photoreaction with an RNA cross-linking agent, 4'-aminomethyl-4,5',8-trimethylpsoralen. These results suggested that mRNA with or without the cap structure on the 5'-terminal can form complexes with the ribosomes in a eukaryotic cell-free translation system by base-pair formation with 18S rRNA. Correspondingly, sequences capable of forming extensive base-pairs including four- to five-base complementarities were found between the 3'-terminal of rabbit reticulocyte 18S ribosomal RNA and the 5'-noncoding regions of either influenza virus transcripts or reovirus mRNA.

Animals↗

Development of a recombinant RNA technique for the construction of chimeric RNA with a long poly(C) tract.

The murine cardioviruses and bovine aphthoviruses are distinguished from other (+) strand RNA viruses by their long poly(C) tract in the 5'-noncoding region. The presence of this poly(C) tract has long hampered the construction of full-length cDNA with the complete poly(C) tract, because long poly(dC-dG) homopolymer-containing plasmids are difficult to amplify in bacterial systems. To overcome this problem, we constructed a chimeric RNA by joining the poly(C) region of the viral RNA to the 5'-truncated RNA transcript of the encephalomyocarditis (EMC) virus cDNA. The non-chimeric, recombinant EMC virus with a short poly(C) tract produces recombinant progeny virus, but this is not pathogenic in vivo. On the other hand, the EMC viral RNA chimera with the complete poly(C) tract produces recombinant progeny virus that is pathogenic in vivo. This method of viral RNA construction will be invaluable for functional studies of other cardioviruses and aphthoviruses, as well as for recombinant RNA manipulations.

Animals↗

The R region found in the human foamy virus long terminal repeat is critical for both Gag and Pol protein expression.

It has been suggested that sequences located within the 5' noncoding region of human foamy virus (HFV) are critical for expression of the viral Gag and Pol structural proteins. Here, we identify a discrete approximately 151-nucleotide sequence, located within the R region of the HFV long terminal repeat, that activates HFV Gag and Pol expression when present in the 5' noncoding region but that is inactive when inverted or when placed in the 3' noncoding region. Sequences that are critical for the expression of both Gag and Pol include not only the 5' splice site positioned at +51 in the R region, which is used to generate the spliced pol mRNA, but also intronic R sequences located well 3' to this splice site. Analysis of total cellular gag and pol mRNA expression demonstrates that deletion of the R region has little effect on gag mRNA levels but that R deletions that would be predicted to leave the pol 5' splice site intact nevertheless inhibit the production of the spliced pol mRNA. Gag expression can be largely rescued by the introduction of an intron into the 5' noncoding sequence in place of the R region but not by an intron or any one of several distinct retroviral nuclear RNA export sequences inserted into the mRNA 3' noncoding sequence. Neither the R element nor the introduced 5' intron markedly affects the cytoplasmic level of HFV gag mRNA. The poor translational utilization of these cytoplasmic mRNAs when the R region is not present in cis also extended to a cat indicator gene linked to an internal ribosome entry site introduced into the 3' noncoding region. Together these data imply that the HFV R region acts in the nucleus to modify the cytoplasmic fate of target HFV mRNA. The close similarity between the role of the HFV R region revealed in this study and previous data (M. Butsch, S. Hull, Y. Wang, T. M. Roberts, and K. Boris-Lawrie, J. Virol. 73:4847--4855, 1999) demonstrating a critical role for the R region in activating gene expression in the unrelated retrovirus spleen necrosis virus suggests that several distinct retrovirus families may utilize a common yet novel mechanism for the posttranscriptional activation of viral structural protein expression.

Gene Expression Regulation, Viral↗

Nucleotide sequence 5' of the chicken c-myc coding region: localization of a noncoding exon that is absent from myc transcripts in most avian leukosis virus-induced lymphomas.

We have determined the nucleotide sequence of the 2.2-kilobase-pair region upstream of the chicken c-myc coding exons. Using RNA blot analysis, we have localized a noncoding exon to a region that is separated from the c-myc coding sequences by an intron of 700-800 base pairs. In most avian leukosis virus-induced lymphomas proviral integration has occurred within, or downstream of, the first exon, thus presumably displacing the regulatory sequences that normally control c-myc expression. More than 70% of the integration sites were clustered in a 250-base-pair region in the first intron, immediately preceding the coding sequences. Sequences from the upstream noncoding exon were absent from the myc transcripts in these lymphomas; RNA transcripts from the normal c-myc allele were not expressed at detectable levels.

Animals↗

The role of mRNA 5'-noncoding and 3'-end sequences on 40S ribosomal subunit recruitment, and how RNA viruses successfully compete with cellular mRNAs to ensure their own protein synthesis.

Since the elaboration of the scanning model to explain eukaryotic translation initiation, alternative hypotheses have gained support. Cap and 5' end-independent recruitment of the 40S ribosomal subunit conferred by the presence of an internal ribosome entry segment (IRES) in the 5'UTR of the mRNA is widely accepted, and has been formally and definitively proven for a picornavirus. However, the mechanism of IRES function remains essentially a black box. Using the complex viral IRESes as model systems, approaches taken to shed light on the mystery include systematic comparisons and molecular genetic analyses. The hypothesis that actively translated mRNAs are circular, rather than linear, molecules is based on rather indirect evidence. This model has invoked a revision of the image of 40S ribosomal subunit recruitment, to include recycling from the mRNA 3'- to the 5'-end in addition to true de novo 5'-end directed entry. Biochemical and genetic studies are used to define the network of interactions necessary for efficient ribosome recruitment. This has lent weight to the concept of mRNA 5'-3' cross-talk and clarified the mechanics of how this enhances translation efficiency. These refinements and revisions to the model of translation initiation form the core of this review, with current knowledge being considered from the perspective on how host-cell translation could yield to selective viral translation via the phenomenon of translational shut-off.

5' Untranslated Regions↗

The 3'-terminal consensus sequence of rotavirus mRNA is the minimal promoter of negative-strand RNA synthesis.

We used an in vitro template-dependent replicase assay (D. Chen, C. Zeng, M. Wentz, M. Gorziglia, M. Estes, and R. Ramig. J. Virol. 68:7030-7039, 1994) to identify the cis-acting signals required for replication of a genome segment 9 template from the group A rotavirus strain OSU. The replicase phenotypes for a panel of templates with internal deletions or 3'-terminal truncations indicated that no essential replication signals were present within the open reading frame and that key elements were present in the 5' and 3' noncoding regions. Chimeric constructs containing portions of viral sequence ligated to a nonviral backbone were generated to further map the regions required for in vitro replication of segment 9. The data from these constructs showed that the 3'-terminal seven nucleotides of the segment 9 mRNA provided the minimum requirement for replication (minimal promoter). Analysis of additional chimeric templates demonstrated that sequences capable of enhancing replication from the minimal promoter were located immediately upstream of the minimal promoter and at the extreme 5' terminus of the template. Mutational analysis of the minimal promoter revealed that the 3'-terminal -CC residues are required for efficient replication. Comparison of the replication levels for templates with guanosines and uridines at nucleotides -4 to -6 from the 3' terminus compared with levels for templates containing neither of these residues at these positions indicated that either or both residues must be present in this region for efficient replication in vitro.

Animals↗

A chimeric plasmid from cDNA clones of poliovirus and coxsackievirus produces a recombinant virus that is temperature-sensitive.

We have inserted a 405-nucleotide fragment from the 5' noncoding region of the coxsackievirus B3 genome into an infectious cDNA copy of the poliovirus RNA genome. Transfection of plasmid DNA containing this hybrid genome construct into cultured monkey cells produced infectious virus. Recombinant virus stocks displayed a temperature-sensitive phenotype for growth at 37 degrees C. We found that there is a dramatic reduction in the level of viral proteins and viral RNAs in HeLa cells infected with the recombinant at 37 degrees C compared to that obtained at 33.5 degrees C. Thus, insertion of a portion of the coxsackievirus genome into the poliovirus genome produces a temperature-sensitive recombinant virus. That this substitution occurs in a region of the poliovirus genome that, to date, has not been shown to have any coding function suggests that RNA sequences involved in replicase recognition or ribosome binding may contribute to the temperature-sensitive phenotype of the recombinant virus.

Animals↗

[Diagnosis of type C chronic hepatitis using PCR technology].

Many studies have suggested that examination of a patient's serum for viral RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) is the most sensitive and specific serological means of determining chronic HCV infection. Moreover, technique to quantify HCV RNA in serum using PCR technology has been developed, and diagnosis of HCV genotype by PCR with mixed primers has been used for diagnosis of type C hepatitis. Herein, we discuss the laboratory technique and significance of various PCR methods. HCV RNA can be detected by amplification technique of RT-PCR. Primers in the 5'-noncoding region are frequently used to detect HCV RNA, because the RNA sequence in this region is the most conserved. We usually perform 30 cycles of amplification with outer primers and another 30 cycles with inner primers (nested RT-PCR). Using this technique, HCV RNA was detected in 96% of anti-HCV-positive chronic hepatitis patients. This PCR technique gives us important information for the diagnosis of type C chronic hepatitis indicating HCV infection. The competitive PCR technique is usually used to quantify HCV RNA. This method is based on complification of the target RNA with known amounts of synthetic mutant RNA. The mutant RNA should be amplified by the same primers for amplifying target RNA, and should be distinguished from target RNA by the size of the amplified DNA product by making a deletion or restriction site artificially. Quantifying HCV RNA before interferon therapy is useful to predict the effectiveness of the therapy; patients with a large amount of HCV RNA tend to have a poor outcome.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗