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PubMed1996

Comparative study of three methods for genotyping hepatitis C virus strains in samples from Spanish patients.

Hepatitis C virus (HCV) genotypes may be investigated by a variety of laboratory methods that target different parts of the HCV genome and have various degrees of technical difficulty. Since the choice of a particular method is difficult, we compared the performance of (i) a type-specific PCR with type-specific primers from the core region, (ii) molecular hybridization of the PCR-amplified 5' noncoding region to type-specific probes, and (iii) identification of type-specific antibodies against epitopes of nonstructural region 4 by enzyme-linked immunosorbent assay (ELISA). One hundred fifty-one patients with biopsy-proved chronic hepatitis and HCV RNA in serum were investigated. The HCV genotype was identified in 99%, 100%, and 85% of the cases by type-specific PCR, probe hybridization, and ELISA, respectively. The type-specific PCR disclosed infection with type 1a in 3%, type 1b in 74%, and type 3a in 4% of the cases and suggested infection with two or more HCV types, including 2a/2c and 2b, in the remaining 18%. Apparently mixed infections were more prevalent in patients with past intravenous drug use (P < 0.001), but cloning and sequencing of PCR products did not confirm a mixed infection in any of the four cases investigated. Concordant results were obtained by the three procedures with virtually all of the samples in which the type-specific PCR revealed a single HCV genotype. Type-specific hybridization and ELISA usually recognized the genotype producing the strongest DNA band in samples in which type-specific PCR suggested a mixed infection. In conclusion, the three procedures evaluated in this study are reliable for investigation of HCV genotype. Type-specific PCR provides information about HCV subtypes, but a mixed infection detected with this method should be interpreted with caution.

Enzyme-Linked Immunosorbent Assay↗
PubMed2006

Evaluation of the invader assay for genotyping hepatitis C virus.

The Invader 1.0 assay (Invader HCV Genotyping Assay, version 1.0; Third Wave Technologies, Inc., Madison, WI) has been developed for the rapid differentiation of hepatitis C virus (HCV) genotypes 1 to 6 based on sequence variation within the HCV 5' noncoding (NC) region. In the present study, we evaluated the compatibility of Invader 1.0 with the COBAS MONITOR (COBAS AMPLICOR HCV MONITOR Test, version 2.0; Roche Molecular Systems, Inc., Branchburg, NJ), COBAS AMPLICOR (COBAS AMPLICOR Hepatitis C Virus Test, version 2.0; Roche Molecular Systems, Inc.), and COBAS TaqMan (COBAS TaqMan HCV Test; Roche Molecular Systems, Inc.) assays. The minimum HCV RNA titers required for successful HCV genotyping (>/=90% success rate) were 1,000 IU/ml for COBAS MONITOR, 100 IU/ml for COBAS AMPLICOR, and 10 IU/ml for COBAS TaqMan. Invader 1.0 results obtained from unpurified COBAS TaqMan amplification products of 111 retrospectively selected clinical serum specimens (genotypes 1 to 6, with virus titers ranging from 15.1 to 2.1 x 10(7) IU/ml) showed 98% concordance with results obtained from the TRUGENE HCV 5' NC Genotyping Kit (Bayer HealthCare LLC, Tarrytown, NY), used in conjunction with COBAS AMPLICOR. Although the assay is sensitive, accurate, and easy to perform, additional optimization of the Invader 1.0 interpretive software (Invader Data Analysis Worksheet) may be necessary to reduce potential misidentification of HCV genotypes in low-titer specimens. In summary, Invader 1.0 is compatible with a variety of commercially available PCR-based HCV 5' NC region amplification assays and is suitable for routine HCV genotyping in clinical laboratories.

5' Untranslated Regions↗
PubMed2004

[Hepatitis c virus genotype research by ABC programs of 5'-NCR restriction endonuclease digestion].

OBJECTIVE: In order to fully understand hepatitis c virus (HCV) genotype 3b, 1a, 2b and 6a infection in China, We built HCV 5'-noncoding region (5'-NCR) of different genotypes and subtypes. METHODS: The classification HCV into variable genotypes (subtypes) was carried on by programs A, B and C A. Using a combination of three restriction endonuclease BHH' (BsrB I, Hae II, Hinf I) digestions at the same time. The distinct genotypes were classified into 5 groups: genotype 1 (1a, 1b), 6a, 2 (2a, 2b), genotype 3 (3a, 3b), genotype4 (4a). B. With regard to genotype 1, we could distinguish subtype 1a from 1b using BstU I digestion. C. Using restriction endonuclease Hae III, genotype 2a, 2b, 3b, 4a, 6a are differentiated respectively. RESULTS: (1) HCV genotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 6a are fully discriminated by comparison with the genotypes regular samples. (2) Of the 93 patients, HCV genotype distribution in China was 66.67% for 1b, 18.28% for 2a, 3.23% for 1b/2b, 3b, 2b respectively. 2.15% for 2a/2b, 1b/2a respectively. 1.08% for 1a. CONCLUSION: This research indicated that adoption of HCV 5'-NCR A B C restriction endonuclease digestions techniques, might be sensitive and efficient to detect HCV and discriminate HCV genotype (subtypes) 1a to 6a.

5' Untranslated Regions↗
PubMed2004

Functional analysis of the noncoding regions of the Uukuniemi virus (Bunyaviridae) RNA segments.

The role of the variable portion of the noncoding regions (NCRs) of the three Bunyaviridae RNA segments (L, M, S) in transcription, replication, and packaging was studied using the recently developed plasmid-driven RNA polymerase I minigenome system for Uukuniemi (UUK) virus, genus Phlebovirus (11), as a model. Comparison of the different segments showed that all NCRs were sufficient to mediate transcription/replication of a minigenome but demonstrated decreased promoter strength in the order M > L > S. Chimeric minigenomes with flanking NCRs from different genome segments revealed that the number of total base pairs within the inverted, partially complementary ends was important for transcription and replication. Point mutations increasing the base-pairing potential produced increased reporter expression, indicating that complementarity between the 5' and 3' ends is crucial for promoter activity. The role of the intergenic region (IGR) located between the two open reading frames of the ambisense UUK virus S segment was analyzed by inserting this sequence element downstream of the reporter genes. The presence of the IGR was found to enhance reporter expression, demonstrating that efficient transcription termination, regulated by the IGR, is important for optimal minigenome mRNA translation. Finally, genome packaging efficacy varied for different NCRs and was strongest for L followed by M and S. Strong reporter gene activity was still observed after seven consecutive cell culture passages, indicating a selective rather than random genome-packaging mechanism. In summary, our results demonstrate that the NCRs from all three segments contain the necessary signals to initiate transcription and replication as well as packaging. Based on promoter strength, M-segment NCRs may be the preferred choice for the development of reverse genetics and minigenome rescue systems for bunyaviruses.

Animals↗
PubMed2000

Human La antigen is required for the hepatitis C virus internal ribosome entry site-mediated translation.

The 5'-noncoding region (5'-NCR) of the hepatitis C virus (HCV) RNA genome serves as an internal ribosome entry site (IRES) and mediates translation initiation in a cap-independent manner. Previously, we reported the interaction between La antigen and the HCV IRES, which appeared to occur in the context of initiator AUG. It was further shown that HCV IRES-mediated translation was stimulated in the presence of human La antigen. In this study, we have defined the cis- and trans-acting elements responsible for La-5'-NCR interactions and established the dependence of the HCV IRES efficiency on cellular La antigen. During the La-IRES interaction, initiator AUG but not the neighboring codons was found to be the direct target of La binding. The C terminus effector domain-dependent modulation of La binding to the HCV IRES is demonstrated by deletion and substitution mutagenesis of the protein. An RNA systematic evolution of ligands by exponential enrichment (SELEX), generated against La protein that selectively binds La in HeLa lysates and competes for the protein binding to the 5'-NCR, was used to demonstrate the requirement of La for the HCV IRES function in the context of mono- and dicistronic mRNAs. Sequestration of La antigen by the RNA SELEX in HeLa translation lysates blocked the HCV and poliovirus IRES-mediated translation in vitro. The functional requirement of La protein for the HCV IRES activity was further established in a liver-derived cell line and in an add-back experiment in which the inhibited IRES was rescued by recombinant human La. These results strongly argue for the novel role of La protein during selection of the initiator AUG and its participation during internal initiation of translation of the HCV RNA genome.

5' Untranslated Regions↗
PubMedDate not supplied

[Sequence, necessary for initiating RNA synthesis, in the 3'-noncoding region of the classical swine fever virus genome].

Classical swine fever virus (CSFV) is the causative agent of swine fever, which represents an economically important disease in hogs. We previously made a prediction about the recognition sites of replication initiation of CSFV by using the information content method, and it was predicted that the 21 nucleotides located at 3' end of the CSFV genome 3'UTR were essential to CSFV replication. In this paper, we experimentally studied these 21 nucleotides by site-directed mutagenesis. It was found that the 3'UTRs with the 21 nucleotides had the function of initiating RNA synthesis, while the 3'UTRs without the 21 nucleotides did not. The 21 nucleotides alone, without the rest of 3'UTR, were able to initiate RNA synthesis, though with a slump. It was demonstrated that the 21 nucleotides were essential to the replication of CSFV genome. The other part of 3'UTR was also required for sufficient RNA synthesis. It is highly likely that the 21 nucleotides were the necessary site for the CSFV genome replication initiation, and that the elements required for sufficient RNA synthesis were in the other part of 3'UTR. It was assumed that the CSFV replicase bound to the site and initiated the replication of the CSFV genome. In the 21 nucleotides, it was found that the mutation of position 216 and destruction of the 3' terminus in the 3'UTR precluded initiation of RNA synthesis, that the mutation of position 212 did not affect the capacity for initiation of RNA synthesis but attenuated the synthesis of RNA. Among the four mutants of 3'UTR at position 219, three produced the 3'UTR without initiation of RNA synthesis, and the other one produced the 3'UTR with initiation of less RNA synthesis. Therefore, it could be concluded that T216 was the most important while T212 was the least important, and that G219 and C228 were also important for RNA synthesis. The normal base component within the 21 nucleotides was essential to sufficient RNA synthesis.

3' Untranslated Regions↗
PubMed2001

Early detection of acute rhinovirus infections by a rapid reverse transcription-PCR assay.

The development of a rhinovirus (RV)-RNA-specific reverse transcription (RT)-PCR assay is complicated by the close homology between the RV and enterovirus (EV) genomes in the highly conserved 5'-noncoding region, which is chosen for primer design in most RT-PCR assays. We have developed a sensitive, rapid, and RV-specific nested RT-PCR assay and have used it to test nasopharyngeal aspirates from 556 patients presenting with acute respiratory tract infections. RV RNA was detected by nested RT-PCR not only in all of 52 samples that were RV positive by virus isolation methods but also in 124 of 367 samples that were negative by virus isolation methods and enzyme-linked immunosorbent assay (ELISA). In addition, in 23 of 137 samples that were positive for a different respiratory virus by virus isolation and/or ELISA, RV RNA was detected by RT-PCR. EVs, adenoviruses, respiratory syncytial viruses, coronaviruses, and influenza and parainfluenza viruses, including clinical isolates as well as stock viruses, were not amplified in our RV-specific RT-PCR assay, indicating that this assay was highly specific. The processing time was less than 2 days for the RT-PCR, as opposed to up to 2 weeks for virus isolation. These results indicate that nested RT-PCR is more sensitive than conventional methods for the detection of RV in patients experiencing acute respiratory tract infections and represents the only reliable tool for the early laboratory diagnosis of RV infections. This is especially important in light of new opportunities for therapy currently being developed.

Adolescent↗
PubMed1990

Molecular analysis of structural protein genes of the Yamagata-1 strain of defective subacute sclerosing panencephalitis virus. IV. Nucleotide sequence of the fusion gene.

The full-length cDNA corresponding to the mRNA of the fusion (F) protein of the Yamagata-1 strain of subacute sclerosing panencephalitis (SSPE) virus was cloned, and its complete nucleotide sequence was determined. The F gene was composed of 2369 nucleotides and contained a single large coding region, which is located between two noncoding regions. The 5'-terminal noncoding region consisted of 584 nucleotides comprising 44.9% cytosine, and had several inverted repetitious sequences. The 3'-terminal noncoding region had a relatively low homology of 91.7% with the MV. The coding region was expanded for nucleotides 585-2189, which encoded 534 amino acids with a molecular weight of 57,963. The homology of the amino acid sequence of the F protein between the MV and SSPE virus was 96.27%, and the positions of cysteine and proline were almost identical in the two viruses. The functional domains of SSPE-virus F protein closely resembled those of MV F protein, including the cleavage site, a signal sequence, the fusion-related stretch, the transmembrane region, and four potential glycosylation sites. Four antigenic epitopes on the MV F protein were also conserved on the SSPE-virus F protein. However, deletion of one nucleotide (position 2155) of the SSPE virus was found when compared with the MV, and shifted the coding frame, causing the substitutions of 27 C-terminal amino acids of the MV F protein with 11 different residues. The variations of the C-terminal region of the F protein were observed with two other SSPE viruses, suggesting that this may be a common property of SSPE virus that differs from MV.

Amino Acid Sequence↗
PubMed2002

Characterization of interactions between RTA and the promoter of polyadenylated nuclear RNA in Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8.

RTA (replication and transcription activator; also referred to as ORF50, Lyta, and ART), an immediate-early gene product of Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8, disrupts latency and drives lytic replication. RTA activates the expression of polyadenylated nuclear (PAN) RNA (also known as T1.1 or nut-1) of KSHV. This novel noncoding PAN RNA is the most abundant lytic transcript of KSHV; therefore, studying PAN RNA expression serves as a model system for understanding how RTA transactivates target genes during lytic replication. The RTA-responsive element of the PAN promoter (pPAN RRE) was previously identified, and our data suggested direct binding of full-length RTA to the pPAN RRE. Here, we present a detailed analysis of specific interactions between RTA and the PAN promoter. We expressed and purified the DNA-binding domain of RTA (Rdbd) to near homogeneity and measured its affinity for the pPAN RRE. In electrophoretic mobility shift assays (EMSAs), the dissociation constant (K(d)) of Rdbd on the pPAN RRE was determined to be approximately 8 x 10(-9) M, suggesting a strong interaction between RTA and DNA. The specificity of RTA binding to the PAN promoter was confirmed with supershift assays. The Rdbd binding sequences on the PAN promoter were mapped within a 16-bp region of the pPAN RRE by methylation interference assays. However, the minimal DNA sequence for Rdbd binding requires an additional 7 bp on both sides of the area mapped by interference assays, suggesting that non-sequence-specific as well as sequence-specific interactions between RTA and DNA contribute to high-affinity binding. To better understand the molecular interactions between RTA and the PAN promoter, an extensive mutagenesis study on the pPAN RRE was carried out by using EMSAs and reporter assays. These analyses revealed base pairs critical for both Rdbd binding in vitro and RTA transactivation in vivo of the PAN promoter. The results from methylation interference, deletion analysis, and mutagenesis using EMSAs and reporter assays were closely correlated and support the hypothesis that RTA activates PAN RNA expression through direct binding to DNA.

Base Sequence↗
PubMed1982

Structural features of the herpes simplex virus alpha gene 4, 0, and 27 promoter-regulatory sequences which confer alpha regulation on chimeric thymidine kinase genes.

Previous studies have shown that herpes simplex virus genes form three groups, alpha, beta, and gamma, whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Chimeric genes constructed by fusion of the coding and 5' nontranslated leader sequences of the thymidine kinase (TK) gene to the sequences upstream from the site of initiation of transcription of alpha genes 4 and 27 are regulated as alpha genes and are induced in cells converted to TK+ phenotype by infection with TK- virus. In alpha gene 4 (S. Mackem and B. Roizman, Proc. Natl. Acad. Sci. U.S.A. 79:4917-4921, 1982), both the promoter and the regulatory region are separable and movable. The promoter permits expression but not induction when fused to TK in the noncoding leader region of the gene. The regulator, when fused to the promoter of an expressible but noninducible portion of the natural beta TK, renders the gene inducible as an alpha gene; it consists of multiple regulatory units acting cumulatively. In this paper, we report on the precise site of initiation of transcription of alpha gene 0 within the inverted b sequences of the L component of viral DNA. We also report the following. (i) The chimeric gene consisting of the coding and 5' nontranslated leader regions of the TK gene fused to portions of the domain of alpha gene 0 extending largely upstream from the site of initiation of transcription of alpha gene 0 was regulated in the same fashion as the alpha 4- and alpha 27-TK chimeras. The regulatory region in the alpha gene 0 is largely upstream from nucleotide - 140. (ii) The promoter-regulatory regions of alpha genes 0, 4, and 27 share TATA sequences, A + T-rich (consensus) sequences occurring in regulating regions of alpha genes 0 and 4 in more than one copy, and multiple G + C-rich inverted repeats. The relation of these sequences to the function of the promoter-regulatory regions of the alpha genes is discussed.

Animals↗
PubMed2005

Roles of the polypyrimidine tract and 3' noncoding region of hepatitis C virus RNA in the internal ribosome entry site-mediated translation.

Hepatitis C virus (HCV) genome contains a 3'noncoding region (3'NCR) consisting of a variable region, a polypyrimidine tract (polyU/UC) and the X region. To examine the roles of 3'NCR and polyU/UC tract in the internal ribosome entry site (IRES)-mediated translation process, a variety of 3'NCRs containing different lengths of polyU/UC tract were obtained from HCV infected patients and cloned respectively to the downstream of the firefly luciferase coding gene linked to HCV 5'NCR and 30 nucleotides of core gene (containing IRES element). The results of in vitro translation in rabbit reticulocyte lysate (RRL) and cell transfection assay in mammalian cells showed that the IRES-mediated translation efficiency could be enhanced by the full-length of 3'NCR of HCV RNA. However, contradictory results were observed when the role of polyU/UC tract in the IRES-mediated translation was studied. While the IRES-mediated translation efficiency was inhibited by the presence of polyU/UC tract in in vitro translation experiments, transfection of these expression cassettes into hepatic cell line showed that polyU/UC tract enhanced IRES-mediated translation efficiency in vivo. Cellular-fraction complement experiments showed that cellular factors were required for the enhancement by the polyU/UC tract. Further antibody blocking assay and UV cross-linking assay suggested the correlation of IRES-mediated translation with host factors, including the La protein. The data above also indicated that the modulations of the IRES-mediated translation by the HCV 3'NCR and the polyU/UC tract were in a length-independent manner.

3' Untranslated Regions↗
PubMed1996

Cell proteins bind specifically to West Nile virus minus-strand 3' stem-loop RNA.

The first 96 nucleotides of the 5'noncoding region (NCR) of West Nile virus (WNV) genomic RNA were previously reported to form thermodynamically predicted stem-loop (SL) structures that are conserved among flaviviruses. The complementary minus-strand 3' NCR RNA, which is thought to function as a promoter for the synthesis of plus-strand RNA, forms a corresponding predicted SL structure. RNase probing of the WNV 3' minus-strand stem-loop RNA [WNV (-)3' SL RNA] confirmed the existence of a terminal secondary structure. RNA-protein binding studies were performed with BHK S100 cytoplasmic extracts and in vitro-synthesized WNV (-)3' SL RNA as the probe. Three RNA-protein complexes (complexes 1,2, and 3) were detected by a gel mobility shift assay, and the specificity of the RNA-protein interactions was confirmed by gel mobility shift and UV-induced cross-linking competition assays. Four BHK cell proteins with molecular masses of 108, 60, 50, and 42 kDa were detected by UV-induced cross-linking to the WNV (-)3' SL RNA. A preliminary mapping study indicated that all four proteins bound to the first 75 nucleotides of the WNV 3' minus-strand RNA, the region that contains the terminal SL. A flavivirus resistance phenotype was previously shown to be inherited in mice as a single, autosomal dominant allele. The efficiencies of infection of resistant cells and susceptible cells are similar, but resistant cells (C3H/RV) produce less genomic RNA than congenic, susceptible cells (C3H/He). Three RNA-protein complexes and four UV-induced cross-linked cell proteins with mobilities identical to those detected in BHK cell extracts with the WNV (-)3' SL RNA were found in both C3H/RV and C3H/He cell extracts. However, the half-life of the C3H/RV complex 1 was three times longer than that of the C3H/He complex 1. It is possible that the increased binding activity of one of the resistant cell proteins for the flavivirus minus-strand RNA could result in a reduced synthesis of plus-strand RNA as observed with the flavivirus resistance phenotype.

Animals↗
PubMed2003

Cloning and nucleotide analysis of segment A gene of infectious bursal disease virus detected in Korea.

A strain of infectious bursal disease virus (IBDV) was detected from bursal tissues of chicks which suffered from infectious bursal disease (IBD) in Chinju, Korea and provisionally named as Chinju strain. A full-length cDNA clone for segment A gene of the virus was constructed, and complete nucleotide sequence of the gene including noncoding region was determined and analyzed by comparison with that of other IBDV strains. The segment A gene of Chinju strain consisted of 3,269 nucleotides including 862 adenine (26.4%), 917 cytosine (28.0%), 854 guanine (26.1%) and 636 thymine (19.5%). There were regions for two open reading frames (ORFs), ORF1 encoding the VP5 with ATG codon at nucleotides 98-100 and ORF2 encoding the polyprotein of VP2, VP4 and VP3 in the nucleotides 132-3,170. In deduced translation the ORF2 encoded 1,012 amino acids. The full nucleotide sequence of segment A gene and amino acid sequence of ORF2 of the Chinju strain showed 98-99% homology with those of the very virulent IBDVs (vvIBDVs) such as HK46, OKYM, UK661, UPM97/ 61, D6948 and BD3/99. In phylogenetic analysis of nucleotide and amino acid sequences, the Chinju strain was also related closely to the vvIBDVs. Hence, it was suggested that the Chinju strain is a vvIBDV. The nucleotide and amino acid sequences of the Chinju strain with pertinent information can be useful for the development of genetically engineered vaccines and diagnostic reagents against vvIBDV.

Amino Acid Sequence↗
PubMed1997

Rapid diagnosis of dengue viremia by reverse transcriptase-polymerase chain reaction using 3'-noncoding region universal primers.

A reverse transcriptase-polymerase chain reaction (RT-PCR) method was developed as a rapid diagnostic test of dengue viremia. To detect dengue viruses in serum or plasma specimens, a pair of universal primers was designed for use in the RT-PCR. Using these primers, the 3'-noncoding region of dengue virus types 1, 2, 3, and 4 could be amplified, but not those of other flaviviruses, such as West Nile virus, Japanese encephalitis virus, and yellow fever virus, or the alphavirus Sindbis virus. The sensitivity of the RT-PCR assay was similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture. Combining a silica method for RNA isolation and RT-PCR dengue virus could be detected in a 6-hr assay. In a preliminary study using this method, we detected dengue virus in 38 of 39 plasma specimens from which dengue virus had been isolated by mosquito inoculation. We then applied this method for detecting dengue viremia to 117 plasma samples from 62 children with acute febrile illnesses in a dengue-endemic area. We detected dengue viremia in 19 of 20 samples obtained on the day of presentation, which had been confirmed as acute dengue infection by mosquito inoculation and antibody responses. The overall sensitivity of this method was 91.4% (32 of 35; 95% confidence interval [CI] = 82.2-100%). The results from testing plasma samples from febrile nondengue patients showed a specificity of 95.4% (42 of 44; 95% CI = 89.3-100%).

Acute Disease↗
PubMed1995

Differentiation-dependent transcription of the epidermodysplasia verruciformis-associated human papillomavirus type 5 in benign lesions.

Human papillomavirus type 5 (HPV 5) induces cutaneous lesions and persists in skin carcinomas of patients with epidermodysplasia verruciformis (EV). We investigated the expression pattern of HPV 5 in biopsies from benign skin lesions of EV patients by cDNA analysis and in situ hybridization. Nine different cDNAs could be generated from total RNA of one of these lesions by reverse transcription and PCR amplification with HPV 5-specific primers. We could identify two major splice donors: one was found in the E6-proximal part of the noncoding region (NCR), and the other just downstream of the first ATG codon of ORF E1. Each of the characterized transcripts was processed at one or the other donor site and the two corresponding leader exons were found in combination with both 3'-early and late exons. Two transcripts appear to be specific for EV-associated papillomaviruses: one species might encode an E1--E2C fusion protein, and the other mRNA (NCR/E2) is probably encoding for the full-length E2 protein. According to the results of the cDNA analysis, riboprobes were designed for in situ hybridization experiments to study the cell differentiation-dependent expression of the different exons. Only the E7/E1 and E4 probes led to strong signals almost throughout the epithelium. The signals generated by the 5'-E2 and E1 probe increased with cell differentiation and were mainly confined to the nucleus. The NCR, E6, E7, L2, and L1 probes yielded more or less strong signals in the terminally differentiated epidermal layers. The difference in the cell differentiation-dependent expression of the 5'-early region exon (probe E7/1) and L2/L1 exons may point to a differentiation-dependent processing of transcripts.

Base Sequence↗
PubMed1999

Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray.

Although most eukaryotic mRNAs need a functional cap binding complex eIF4F for efficient 5' end- dependent scanning to initiate translation, picornaviral, hepatitis C viral, and a few cellular RNAs have been shown to be translated by internal ribosome entry, a mechanism that can operate in the presence of low levels of functional eIF4F. To identify cellular mRNAs that can be translated when eIF4F is depleted or in low abundance and that, therefore, may contain internal ribosome entry sites, mRNAs that remained associated with polysomes were isolated from human cells after infection with poliovirus and were identified by using a cDNA microarray. Approximately 200 of the 7000 mRNAs analyzed remained associated with polysomes under these conditions. Among the gene products encoded by these polysome-associated mRNAs were immediate-early transcription factors, kinases, and phosphatases of the mitogen-activated protein kinase pathways and several protooncogenes, including c-myc and Pim-1. In addition, the mRNA encoding Cyr61, a secreted factor that can promote angiogenesis and tumor growth, was selectively mobilized into polysomes when eIF4F concentrations were reduced, although its overall abundance changed only slightly. Subsequent tests confirmed the presence of internal ribosome entry sites in the 5' noncoding regions of both Cyr61 and Pim-1 mRNAs. Overall, this study suggests that diverse mRNAs whose gene products have been implicated in a variety of stress responses, including inflammation, angiogenesis, and the response to serum, can use translational initiation mechanisms that require little or no intact cap binding protein complex eIF4F.

Cysteine-Rich Protein 61↗
PubMed1996

The human immunodeficiency virus type 1 5' packaging signal structure affects translation but does not function as an internal ribosome entry site structure.

The role of the RNA secondary structure in the 5' packaging signal region of human immunodeficiency virus type 1 (HIV-1) in initiating translation of gag mRNA has been investigated both in vitro and in the presence of cellular cofactors in vivo. Heat denaturation of the structure and mutagenic deletion both lead to an increase in levels of translated products, indicating that the structure is a significant inhibitor of translation. The proximity of the gag AUG to the packaging signal structure suggested that it might function as an internal ribosome entry site. However, in both a cell-free system and eukaryotic cells, translation will initiate at a novel upstream initiation codon introduced within the 5' noncoding region. This codon is utilized exclusively, resulting in gag protein products with an extra 11 amino acids at the amino terminus, which, when expressed in T lymphocytes, are confined intracellularly, probably because of the lack of an N-terminal glycine myristoylation signal. Deletion of the secondary structure abolishes gag production even in the presence of tat and rev in trans. Using dicistronic constructs containing the HIV-1 5' leader cloned between two heterologous open reading frames, we were unable to detect any significant expression of the second open reading frame that would have been supportive of an internal ribosome entry site mechanism. Using mutant proviruses either lacking the entire packaging signal structure region or containing the introduced upstream initiation codon in long-term replication studies, we were unable to detect reverse transcriptase activity in culture supernatants. The 5' packaging signal structure of HIV-1 does not serve as an internal ribosome entry site. The translation of gag is consistent with ribosomal scanning. However, the packaging signal structure causes significant translational inhibition.

Amino Acid Sequence↗
PubMed1996

Risk factors for the transmission of hepatitis C.

BACKGROUND: Due to the availability of testing for antibodies to the hepatitis C virus (anti-HCV) and with the use of the polymerase chain reaction (RT-PCR) to detect HCV-RNA, more sensitive and specific measures can be applied to assess routes of HCV transmission. The aim of the present study was to investigate possible risk factors for transmission of HCV. METHODS: 160 consecutive patients with chronic hepatitis C (mean age 47.1 +/- 14.1 yr) attending a hepatology out-patient clinic were interviewed to identify transmission risk factors. Genotyping of HCV isolates was performed by direct sequencing of RT-PCR products in the 5'-noncoding and the NS-5 region. RESULTS: The risk factors of HCV infection were as follows: transfusion of blood or blood products 34.4%, intravenous drug abuse 20.6%, heterosexual contact 3.8%, occupational risk 1.9% and tattoo 0.6%. In 62/160 (38.7%) the route of transmission remained unknown. In one HCV-infected couple we analyzed the nucleotide sequences of the NS-5 region of the respective HCV isolates and found almost complete sequence homology (> 97%). The majority of patients with post-transfusional or unknown mode of transmission were infected with genotype HCV-1a and -1b, while in 6/10 patients with previous i.v. drug abuse, genotype HCV-3a was present. We found no evidence that the mode of disease acquisition influences the course of liver disease. CONCLUSION: The majority of patients with chronic hepatitis C have a classical parenteral transmission risk factor. In our study, no source of HCV acquisition was identified in 38.7% of patients. It may well be that the major factors in these "sporadic" HCV infections are variations on the known risk factors. However, since the proportion of these cases is rather high, further attention should be on alternative and as yet unclear transmission routes.

Adolescent↗