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PubMed2003

Structural determinants of insert retention of poliovirus expression vectors with recombinant IRES elements.

Although picornaviruses provide attractive vectors for expression of foreign genes, poor genetic stability restricts their use for immunization purposes. A new prototype vector was generated to increase foreign insert retention, by shifting of the initiation codon to a cryptic AUG within the internal ribosomal entry site (IRES) and replacement of IRES domain VI with foreign ORFs. Using our strategy to replace regulatory noncoding sequences with unrelated foreign genetic material, we generated stable poliovirus-based expression vectors with robust long-term expression of foreign ORFs. Our studies revealed that size and predicted secondary structure formed by the heterologous sequences govern long-term retention and efficiency of expression of foreign inserts replacing IRES structures. These observations indicate that, with certain limitations imposed by structural preferences, foreign sequences can functionally replace IRES substructures in stable picornavirus immunization vectors.

Base Sequence↗
PubMed1989

Avian retroviral vectors derived from avian defective leukemia virus: role of the translational context of the inserted gene on efficiency of the vectors.

We have constructed retroviral vectors derived from the genome of avian erythroblastosis virus ES4 (AEV ES4). The neo selectable gene was substituted for the original v-erbA or v-erbB oncogenes of AEV, either in the same or in a different reading frames. Recombinant retrovirus were rescued and used to infect chicken embryo fibroblasts or quail QT6 cells. When the neo gene was inserted in the same reading frame as the original oncogene, we obtained (1) a high level of expression of the neo gene, (2) a balanced ration of both genomic and subgenomic RNAs, and (3) high titer recombinant viruses. Conversely, when the neo gene was inserted in a reading frame different from that of the original oncogene, we observed (1) a very low level of expression of the neo protein, (2) a predominance of the viral transcript used as translational template for the neo protein synthesis, and (3) low titer recombinant viruses. One of the vectors was used to transfer a human delta-globin gene into avian cells in culture without detectable rearrangement of this gene, but exhibited a deletion within the conserved noncoding region located between the two original oncogenes. Our data provide information for further construction of double expression vectors. Furthermore, three of the vectors would provide helpful tools to identify genetic elements of the virus genome involved in splicing regulation.

Alpharetrovirus↗
PubMed1993

Translational control by influenza virus. Selective translation is mediated by sequences within the viral mRNA 5'-untranslated region.

In cells infected by influenza virus type A, host cell protein synthesis declines rapidly and dramatically, while influenza viral protein synthesis occurs efficiently throughout infection. Previously, we had shown that the selective translation of influenza viral mRNAs in infected cells occurred in a cap-dependent manner and was due at least in part to structures inherent in the mRNAs. Using chimeras containing the noncoding and coding regions of cellular and viral mRNAs, we can now report that the selective translation is mediated by sequences within the 5'-untranslated regions (UTR) of the viral mRNAs. Polysome analysis confirmed that a 45-nucleotide sequence contained in the 5'-UTR of the influenza viral nucleocapsid protein was necessary and sufficient to allow the host cell translational machinery to discriminate between viral and cellular mRNAs. In reciprocal experiments in which the 5'-UTR of the cellular mRNA-secreted embryonic alkaline phosphatase replaced the nucleocapsid protein 5'-UTR, viral protein synthesis was inhibited in virus-infected cells, resembling host protein synthesis. Finally, we demonstrated that the 5'-UTR of another influenza viral mRNA, that encoding the nonstructural protein, also conferred resistance to the shutoff of protein synthesis in influenza virus-infected cells.

Animals↗
PubMed1989

Sequence of cowpea chlorotic mottle virus RNAs 2 and 3 and evidence of a recombination event during bromovirus evolution.

The genomic sequence of cowpea chlorotic mottle virus (CCMV) was completed by sequencing biologically active cDNA clones of CCMV RNA2 (2774 bases) and RNA3 (2173 bases). While only the central core of the encoded 94-kDa CCMV 2a protein contains features conserved among known and putative RNA replication proteins from many viruses, both flanking regions of CCMV 2a show substantial similarity to the corresponding protein of the related brome mosaic virus (BMV). The 3a proteins of CCMV and BMV, implicated as contributors to the distinct host specificities of the two viruses, show lower levels of conservation but are still discernibly related throughout. Major differences occur in the organization of noncoding sequences in CCMV and BMV RNA3. With respect to an otherwise similar region preceding the BMV 3a gene, the CCMV RNA3 5' noncoding sequence contains a clearly bounded 111-base insertion that must reflect a sequence rearrangement in evolution of at least one of the two viruses. The presence of a subgenomic promoter-like sequence near the end of the novel CCMV sequence makes the organization of genes in CCMV RNA3 reminiscent of the 3' end of tobacco mosaic virus RNA, suggesting that CCMV or its 3a gene might have been derived from an ancestor with fewer genomic RNAs. Sequence similarities between the CCMV and BMV RNA3 intercistronic regions include the subgenomic mRNA promoter and an oligo(A), but not an intercistronic segment required for BMV RNA3 amplification, implying that replication signals on the two RNA3s may be organized quite differently.

Amino Acid Sequence↗
PubMed1996

Levels of hepatitis C virus in blood donors infected with different viral genotypes. International HCV Collaborative Study Group.

The level of hepatitis C virus (HCV) in the serum of 337 blood donors infected with different viral genotypes was investigated by branched DNA assay. Viral genotype was deduced by restriction analysis of the virus 5'-noncoding region. Samples included genotypes 1a, 1b, 2a, 2b, 3,4,5, and 6. Multivariate analysis revealed that the ranges of HCV levels were similar for all viral genotypes and subtypes (P=.18), with the possible exception of genotype 4. Virus levels were significantly lower in female than in male subjects (P<.001) but did not correlate with donor age (P=.06) or genotype or with donor age, sex, or country. These results indicate a similar replicative capacity in vivo for different HCV genotypes and clarify the influence of host and virus factors on disease severity and responsiveness to interferon treatment.

Blood Donors↗
PubMed1996

Hepatitis C virus in body fluids after liver transplantation.

Recurrence of hepatitis C virus (HCV) after liver transplantation is common and is associated with high blood levels of HCV RNA. Higher blood levels of HCV may promote body fluid expression of the virus. We tested 152 body fluid specimens from 33 patients with chronic hepatitis C, 21 of whom had undergone prior liver transplantation. All patients had hepatitis C viremia, as determined by a reverse-transcription polymerase chain reaction (PCR) to the 5' noncoding region. The virus was quantitated in serum by the branched chain DNA assay (bDNA). Body fluids (33 sputum, 33 saliva, 33 urine, 32 tear, 9 vaginal, and 12 semen samples) were analyzed using PCR for HCV RNA. Serum HCV RNA by bDNA in the posttransplantation group was 255 +/- 229 x 10(5) compared with 50 +/- 56 x 10(5) eq/mL in the patients who did not undergo transplantation (P = .01). All urine, tear, and semen specimens were negative for HCV RNA. Five of 21 (24%) posttransplantation patients had detectable HCV RNA using PCR in oral secretions compared with 0 of 12 patients who did not undergo transplantation (not statistically significant). However, 5 of 11 patients with serum HCV RNA by bDNA results greater than 150 x 10(5) eq/mL had positive RNA in oral secretions compared to 0 of 22 patients with bDNA less than 150 x 10(5) eq/mL (P = .01). Posttransplantation patients were more likely to have bDNA levels exceeding 150 x 10(5) eq/mL (11 of 21 v 1 of 12, P = .03). Patients within the first year of transplantation were particularly prone to viral RNA levels exceeding 150 x 10(5) eq/mL (8 of 9 v 3 of 12, P = .01). We conclude that HCV RNA can occasionally be detected using PCR in oral secretions after liver transplantation. This is more likely during the first year posttransplantation when blood levels of HCV RNA often exceed 150 x 10(5) eq/mL by the branched chain DNA assay. Whether or not these observations represent an increased risk of transmission of infection during the early posttransplantation period is not certain.

Adult↗
PubMed1999

Biological consequences of deletions within the 3'-untranslated region of flaviviruses may be due to rearrangements of RNA secondary structure.

It was previously reported that deletions introduced into the 3'-untranslated region (3'-UTR) of dengue type 4 (DEN 4) virus (Men, R., Bray, M., Clark, D., Chanock, R.M., Lai, C.J., 1996. DEN 4 virus mutants containing deletions in the 3'-noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in Rhesus monkeys. J. Virol. 70, 3930-3937), tick-borne encephalitis (TBE) virus (Mandl, C.W., Holzmann, H., Meixner, T., Rauscher, S., Stadler, P.F., Allison, S.L. , Heinz, F.X., 1998. Spontaneous and engineered deletions in the 3'-noncoding region of TBE virus: construction of highly attenuated mutants of a flavivirus. J. Virol. 72, 2132-2140) and subgenomic replicons of Kunjin virus (Khromykh, A.A., Westaway, E.G., 1997. Subgenomic replicons of the flavivirus Kunjin: construction and applications. J. Virol. 71, 1497-1505) altered the infectivity of the mutants and reduced the efficiency of RNA replication. Here, these deletions were superimposed onto the models of secondary structure we constructed previously and the folding of the modified 3'-UTR sequences was simulated. The analysis showed that most of the deletions disrupted or reshaped conserved elements of secondary structure and that the biological effects of these deletions are likely to represent structural rearrangements in the 3'-UTR, rather than the loss of sequence motifs. The analysis also suggested that the overall structural integrity of the flaviviral 3'-UTR is essential for optimal performance of its promotor function, although two distinct parts can be defined: the most 3'-terminal structures and sequences which may be critical for the initiation of minus-strand RNA synthesis, and more proximal structures and sequences that possibly function as enhancers of viral RNA replication. The functional significance of certain structural elements and their possible effect on the efficiency of viral replication in different cells are also discussed.

3' Untranslated Regions↗
PubMed1986

Properties of intracellular bovine papillomavirus chromatin.

Episomal nucleoprotein complexes of bovine papillomavirus type 1 (BPV-1) in transformed cells were exposed to DNase I treatment to localize hypersensitive regions. Such regions, which are indicative for gene expression, were found within the noncoding part of the genome, coinciding with the origin of replication and the 5' ends of most of the early mRNAs. However, there were also regions of hypersensitivity within the structural genes. These intragenic perturbations of the chromatin structure coincide with regulatory sequences at the DNA level. One of these regions maps in close proximity to a Z-DNA antibody-binding site which is located near the putative BPV-1 enhancer sequence.

Animals↗
PubMed1988

Sequence analysis of the 3'-end of the feline coronavirus FIPV 79-1146 genome: comparison with the genome of porcine coronavirus TGEV reveals large insertions.

The genetic information, carried on mRNA 6 of feline infectious peritonitis virus (FIPV) strain 79-1146, was determined by sequence analysis of cDNA clones derived from the 3' end of the FIPV genome. Two ORFs were found, encoding polypeptides of 11K (ORF-1) and 22K (ORF-2). The FIPV sequence was compared to the 3' end sequence of transmissible gastroenteritis virus (TGEV). ORF-1 has a homologous counterpart (ORF-X3) in the TGEV genome; both ORFs are located at the same position relative to the nucleocapsid gene. However, as a result of an in-frame insertion or deletion, ORF-1 is 69 nucleotides larger than ORF-X3. A similar event has occurred immediately downstream of ORF1: a 624-nucleotide segment, containing the complete ORF-2, is absent in the TGEV sequence. Most sequence similarity (98.5%) was found in the 3' noncoding sequences. ORF-X3 and ORF-1 are preceded by the sequence AACTAAAC, which is assumed to be the transcription-initiation signal in FIPV and TGEV (P.A. Kapke and D.A. Brian (1986) Virology 151, 41-49). By S1 nuclease analysis, the 5' end of FIPV RNA 6 was mapped immediately upstream of this sequence. A 700-nucleotide TGEV-specific RNA was found by cross-hybridization with an FIPV 3' end probe, suggesting that TGEV ORF-X3 is also carried on a separate mRNA. The differences at the 3' ends of the FIPV and TGEV genomes may be the result of RNA recombination events.

Amino Acid Sequence↗
PubMed1991

Improvement of avian leukosis virus (ALV)-based retrovirus vectors by using different cis-acting sequences from ALVs.

Production and expression of double-expression vectors which transduce both Neo(r) and lacZ genes and are based on the structure of avian leukosis virus were enhanced by using cis-acting sequences (long terminal repeats and noncoding sequences) from Rous-associated virus-1 and Rous-associated virus-2 rather than those of avian erythroblastosis virus previously used in our constructs. Polyclonal producer cells obtained after transfection of these vectors into the Isolde packaging cell line gave rise to titers as high as 3 x 10(5) lacZ CFU/ml, whereas it was possible to isolate clones of producer cells giving rise to titers of more than 10(6) resistance focus-forming units per ml.

Animals↗
PubMed1999

Determining the nucleotide sequence and capsid-coding region of himetobi P virus: a member of a novel group of RNA viruses that infect insects.

We determined the complete genome sequence of Himetobi P virus (HiPV), an insect picorna-like virus, which was isolated from the small brown planthopper, Laodelphax striatellus. The genome of HiPV consists of 9,275 nucleotides excluding the poly (A) tail, and contains two large open reading frames (ORFs), which were separated by a 176-nucleotide noncoding region. The deduced amino acid sequence of the first ORF contains core motifs of picornaviral helicase, protease, and RNA-dependent RNA polymerase. The capsid protein-coding region was mapped onto the second ORF by determining the N-terminal amino acid sequences of the capsid proteins. Subgenomic RNA for the capsid protein gene was not detected in the infected tissue. The capsid protein precursor gene of HiPV lacks an AUG initiation codon at the expected position and the upstream sequence of the gene is predicted to form several stem-loop structures, suggesting that the precursor is produced by internal ribosome entry site (IRES) mediated-translation, as occurs in Plutia stali intestine virus (PSIV). These characteristics of the HiPV genome are similar to those of a new group of RNA viruses consisting of Drosophila C virus (DCV), Rhopalosiphum padi virus (RhPV), and PSIV.

Amino Acid Sequence↗
PubMed1993

Detection of HCV RNA using reverse transcription and nested polymerase chain reaction in chronic non-A, non-B liver diseases in Korea.

To investigate the interrelationship between the anti-HCV prevalence and the HCV RNA positivity in chronic non-A, non-B (NANB) liver diseases in Korea (n = 137), we performed RT-nested PCR using 4 primers in the 5'-noncoding region of HCV genome. The overall HCV RNA positive rates were 62.8%; 87.7% (57/65) of anti-HCV positive cases and 40.3% (29/72) of anti-HCV negative cases. Four patterns according to the combination of anti-HCV and HCV RNA assays could be observed; 41.6% in [anti-HCV+/HCV RNA+], 5.8% in [anti-HCV+/HCV RNA], 21.2% in [anti-HCV+/HCV RNA], and 31.4% in [anti-HCV+/HCV RNA]. Our results suggested that HCV may be still related to chronic NANB liver diseases negative for anti-HCV, and other unknown agents, so-called non-B non-C, might be accounted for patients negative for both, although a low concentration of HCV RNA, or the presence of HCV RNA or HBV DNA in liver tissue should be excluded.

Hepacivirus↗
PubMed2005

Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells.

MicroRNAs (miRNAs) are an endogenously encoded class of small RNAs that have been proposed to function as key posttranscriptional regulators of gene expression in a range of eukaryotic species, including humans. The small size of miRNA precursors makes them potentially ideal for use by viruses as inhibitors of host cell defense pathways. Here, we demonstrate that the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an array of 11 distinct miRNAs, all of which are expressed at readily detectable levels in latently KSHV infected cells. Individual KSHV miRNAs were expressed at up to 2,200 copies per cell. The KSHV miRNAs are expressed from what appears to be a single genetic locus that largely coincides with an approximately 4-kb noncoding sequence located between the KSHV v-cyclin and K12/Kaposin genes, both of which are also expressed in latently infected cells. Computer analysis of potential mRNA targets for these viral miRNAs identified a number of interesting candidate genes, including several mRNAs previously shown to be down-regulated in KSHV-infected cells. We hypothesize that these viral miRNAs play a critical role in the establishment and/or maintenance of KSHV latent infection in vivo and, hence, in KSHV-induced oncogenesis.

Base Sequence↗
PubMed1992

Detection of hepatitis C virus RNA in serum of patients with chronic hepatitis C treated with interferon-alpha.

We tested serial serum samples for hepatitis C virus RNA from patients undergoing treatment for chronic hepatitis C with interferon-alpha using an assay that combined reverse transcription and polymerase chain reaction. The subjects studied were 20 patients with chronic hepatitis who had serum antibody to hepatitis C virus (anti-C100-3). Before therapy, hepatitis C virus RNA was detected in 18 (90%) and 20 (100%) patients using primer sets derived from the NS3 region or the 5'-noncoding region of hepatitis C virus, respectively. Hepatitis C virus RNA became undetectable in all patients whose ALT level fell into the normal range during therapy. However, hepatitis C virus RNA reappeared in all patients whose ALT levels rose again after therapy, usually before the relapse. In patients whose ALT levels did not become normal, hepatitis C virus RNA did not disappear during therapy. Thus therapy with interferon-alpha appears to be beneficial in chronic hepatitis C because of its suppressive effects on hepatitis C virus replication. Detection of hepatitis C virus RNA in serum is useful for evaluating the antiviral effect of interferon.

Adult↗
PubMed1986

The nucleotide sequence of the mRNA encoding the fusion protein of measles virus (Edmonston strain): a comparison of fusion proteins from several different paramyxoviruses.

Membrane fusion is the primary cytopathic effect observed in cells infected with measles virus. The viral protein responsible for this process has previously been defined as the fusion (F) protein. Fusion is activated by the proteolytic cleavage of a precursor molecule (F0) to yield two disulfide-linked polypeptides (F1 and F2). In this paper the mRNA for the membrane fusion protein has been cloned and the resulting cDNAs were sequenced. A mRNA composed of 2377 nucleotides was found to contain one open reading frame which could potentially code for a protein of 550 amino acids. This corresponding gene product was identified as the fusion protein through use of antibodies directed against a synthetic peptide which was constructed from the deduced amino acid sequence. A long and rather G-C rich 5' terminus was found on the mRNA and this noncoding region may play some role in regulation of protein synthesis at the translational level. Protein sequence data derived from the cDNA clones revealed a highly conserved F1 amino terminus which is characteristic of most paramyxoviruses. Very little amino acid homology (except for the conservation of the F1 terminus and 9 cysteines) was evident when the sequence was compared to other paramyxovirus fusion proteins. However an overall hydrophobic nature was characteristic of all the F proteins and hydrophobicity plots for the fusion proteins of 4 different paramyxoviruses were very similar. Computer analysis was also employed to analyze the secondary structure of the measles virus F protein. Large stretches of alpha helix were characteristic of the regions which purportedly interact with membranes. The functional domains of the F protein and their similarity to those of the influenza hemagglutinin protein are discussed in this communication. We concluded that the distribution of hydrophobic regions capable of spanning biological membranes determines the fusogenic nature of the F protein.

Amino Acid Sequence↗
PubMed1992

Hepatitis C virus replication during acute infection in the chimpanzee.

The events following experimental infection of 2 chimpanzees with the H strain of hepatitis C virus (HCV) were studied by quantitating the levels of HCV RNA in liver and serum. Serum and liver samples were tested every 1-3 weeks for up to 32 weeks. The genomic and antigenomic strands of HCV RNA were individually detected in liver and serum by strand-specific reverse transcription followed by polymerase chain reaction (PCR) using nested primers specific for the 5' noncoding region of the HCV genome and were quantitated by end-point dilution of the nucleic acid extract. Both genomic and antigenomic strands were detected in liver and serum within 1 week after inoculation and approximately 1 week before the development of elevated levels of alanine aminotransferase (ALT) activity. Changes in levels of antigenomic strand paralleled those of the genomic strand in both serum and liver. Titers of HCV RNA in serum and liver generally correlated with changes in ALT levels.

Acute Disease↗
PubMed2005

Radiation leukemia virus common integration at the Kis2 locus: simultaneous overexpression of a novel noncoding RNA and of the proximal Phf6 gene.

Retroviral tagging has been used extensively and successfully to identify genes implicated in cancer pathways. In order to find oncogenes implicated in T-cell leukemia, we used the highly leukemogenic radiation leukemia retrovirus VL3 (RadLV/VL3). We applied the inverted PCR technique to isolate and analyze sequences flanking proviral integrations in RadLV/VL3-induced T lymphomas. We found retroviral integrations in c-myc and Pim1 as already reported but we also identified for the first time Notch1 as a RadLV common integration site. More interestingly, we found a new RadLV common integration site that is situated on mouse chromosome X (XA4 region, bp 45091000). This site has also been reported as an SL3-3 and Moloney murine leukemia virus integration site, which strengthens its implication in murine leukemia virus-induced T lymphomas. This locus, named Kis2 (Kaplan Integration Site 2), was found rearranged in 11% of the tumors analyzed. In this article, we report not only the alteration of the Kis2 gene located nearby in response to RadLV integration but also the induction of the expression of Phf6, situated about 250 kbp from the integration site. The Kis2 gene encodes five different alternatively spliced noncoding RNAs and the Phf6 gene codes for a 365-amino-acid protein which contains two plant homology domain fingers, recently implicated in the Börjeson-Forssman-Lehmann syndrome in humans. With the recent release of the mouse genome sequence, high-throughput retroviral tagging emerges as a powerful tool in the quest for oncogenes. It also allows the analysis of large DNA regions surrounding the integration locus.

Amino Acid Sequence↗
PubMed1989

Reversion of the attenuated and temperature-sensitive phenotypes of the Sabin type 3 strain of poliovirus in vaccinees.

Isolates of type 3 poliovirus from vaccine-recipients were characterized in terms of virulence, sensitivity of growth to high temperatures, and differences in genome structure from the Sabin type 3 vaccine strain. These included point mutations in the region of the genome coding for the structural proteins and in the 5' noncoding region, and the presence of type 1 or type 2 poliovirus genomic sequences resulting from intertypic recombination. Isolates from healthy vaccinees resembled those from vaccine-associated cases of poliomyelitis in all of these properties. Suppression of the temperature-sensitive phenotype was strictly correlated with reversion to virulence in nonrecombinant type 3 strains. Recombinant isolates were more attenuated than expected, even when they had lost all mutations known to attenuate the type 3 vaccine strain.

Base Sequence↗