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PubMed1998

Nucleic acid sequence-based amplification, a new method for analysis of spliced and unspliced Epstein-Barr virus latent transcripts, and its comparison with reverse transcriptase PCR.

Nucleic acid sequence-based amplification (NASBA) assays were developed for direct detection of Epstein-Barr virus (EBV) transcripts encoding EBV nuclear antigen 1 (EBNA1), latent membrane proteins (LMP) 1 and 2, and BamHIA rightward frame 1 (BARF1) and for the noncoding EBV early RNA 1 (EBER1). The sensitivities of all NASBAs were at least 100 copies of specific in vitro-generated RNA. Furthermore, 1 EBV-positive JY cell in a background of 50,000 EBV-negative Ramos cells (the relative sensitivity) was detected by using the EBNA1, LMP1, and LMP2 NASBA assays. The relative sensitivity of the EBER1 NASBA was 100 EBV-positive cells, which was probably related to the loss of small RNA molecules during the isolation. The BARF1 and LMP2 NASBAs were evaluated on clinical material. BARF1 expression was found in 6 of 7 nasopharyngeal carcinomas (NPC) but in 0 of 22 Hodgkin's disease (HD) cases, whereas LMP2 expression was found in 7 of 7 NPCs and in 17 of 22 HD cases. For detection of EBNA1 transcripts in HLs (n = 12) and T- and B-cell non-Hodgkin's lymphomas (n = 3 and n = 2, respectively), NASBA was compared with reverse transcriptase (RT) PCR. Two samples were positive only with NASBA, and two other samples were positive only with RT-PCR; for all other samples, the RT-PCR and NASBA results were in agreement. We conclude that NASBA is suitable for sensitive and specific detection of the above-mentioned EBV transcripts, regardless of their splicing patterns and the presence of EBV DNA. The EBNA1, LMP2, and BARF1 NASBAs developed in this study proved to be reliable assays for detection of the corresponding transcripts in EBV-positive clinical material.

Base Sequence↗
PubMed1993

NF-IL6 represses early gene expression of human papillomavirus type 16 through binding to the noncoding region.

The expression of human papillomavirus type 16 (HPV16) early genes, including E6 and E7 transforming genes, is regulated by several cellular factors binding to the noncoding region (NCR), such as the glucocorticoid receptor, NF-I, and AP1, all of which are positive regulators. We demonstrated that the nuclear factor for interleukin 6 expression (NF-IL6) specifically binds to the HPV16 NCR ranging from nucleotides 7007 to 7766 and represses the early gene expression of HPV16. The responsive element in HPV16 NCR was determined within the region ranging from nucleotides 7454 to 7766. In this region, many binding sites for other cellular transactivators, such as NF-I and AP1, have been detected. Interestingly, three of seven binding sites for NF-I and two of two binding sites for AP1 in this region overlap with the putative NF-IL6 binding sites identified by computer analysis. Competition experiments with the oligonucleotides containing such NF-I or AP1 sites indicated that NF-IL6 certainly binds to them. Furthermore, in a chloramphenicol acetyltransferase assay using mutant NF-IL6 expression vectors, the DNA binding domain of NF-IL6 was shown to be necessary for repression, whereas the functional domain was not. These findings indicate that repression may be caused by competition with other transcriptional activators, such as NF-I and AP1. Thus, NF-IL6 may play a significant role in the regulation of viral transcription as a part of the host's resistance to viral infection.

Acute-Phase Reaction↗
PubMed2006

Analysis of the 5' noncoding region versus the NS5b region in genotyping hepatitis C virus isolates from blood donors in France.

The 5' noncoding region (5' NCR) of the hepatitis C virus (HCV) has become the standard for genotyping even though several reports show that its use can result in classification errors. The purpose of this study was to perform genotyping based on sequence analysis of the NS5b region in a set of 357 HCV strains isolated from blood donors in France in 2002 and 2003. Results were compared with those previously obtained using 5' NCR analysis, and HCV subtype distribution was reevaluated. Twenty-six of 120 strains (approximately 22%) initially identified as genotype 1b by 5' NCR region sequence analysis were reclassified as genotype 1a by NS5b region sequence analysis. Similarly, 14 of 23 strains (approximately 61%) initially identified as 2a/2c were reclassified as non-2a and non-2c subtypes, and 12 of 22 strains (approximately 45%) initially identified as 4c/4d subtypes were reclassified as non-4c and non-4d subtypes. Sequence analysis of the NS5b region also revealed 5 putative new subtype 2 variants and 2 putative new subtype 4 variants. Although these findings demonstrated full agreement between 5' NCR and NS5b sequence analysis with regard to type classification, genotyping based on phylogenetic analysis of the NS5b region is more accurate for subtype determination than genotyping based on analysis of the 5' NCR. Sequence analysis of the NS5b region is mandatory for epidemiologic studies.

5' Untranslated Regions↗
PubMed1995

Mechanism of translation initiation on hepatitis C virus RNA.

In vitro translation experiments involving reticulocyte and HeLa cell lysates have demonstrated that translation initiation on hepatitis C virus (HCV) RNA occurs by entry of ribosomes to the internal sequence (internal ribosomal entry site [IRES]) within the 5' noncoding region (5'NCR). Specific binding factors that bind to the HCV IRES were examined by UV cross-linking tests. A protein p57 (polypyrimidine tract binding protein [PTB]), known to be a binding factor that binds to picornavirus IRES, was found to bind to the HCV IRES. To investigate functions of this PTB in IRES-dependent translation initiation, hyperimmune antibodies to PTB (anti-PTB) were prepared by immunizing rabbits with purified recombinant PTB produced in E. coli. Anti-PTB IgGs were purified and used to deplete PTB in a cell-free protein synthesis system prepared from HeLa cells. In vitro translation experiments using PTB-depleted lysates suggested that host factor dependency of IRES is different in individual IRESs. As we showed previously, function of the IRES typical of group II HCV is more efficient than the IRES typical of group I HCV in a cell-free protein synthesis system from HeLa cells. Recombinant HCV IRESs were constructed and tested for their function in the in vitro system. Most recombinant IRESs showed an initiation activity of the more efficient typical group II IRES level or higher. This observation, together with the fact that group I HCV is the most common isolate of HCV worldwide, suggests that the efficiency of the IRES function of HCV may be generally suppressed in nature, and that the replication ability of HCV that permits its persistent infection in humans may require a delicate balance at a molecular level.

Animals↗
PubMed2004

Transcriptional regulation of TT virus: promoter and enhancer regions in the 1.2-kb noncoding region.

Since the discovery of TT virus (TTV) in 1997, its mechanism of transcriptional control has remained unsolved. Molecular analysis points at the 1.2-kb noncoding region (NCR) as being responsible for transcriptional control. The 5' terminus of TTV mRNA was located at nt 114 using the primer extension method (nt 114 will be referred to as position +1). This employed the PE1 primer, designed to start approximately 100 nt downstream of the predicted initiation site. Overall promoter and enhancer activity of the NCR was analyzed using dual luciferase assays in K562, Jurkat, U937, A549, HepG2, Huh7, and HeLaS3 cells. Of those tested, K562 showed the highest relative luciferase activity of 31.1, and activity in HepG2 (14.6) was significantly higher than that in Huh7 (2.8). Fragments of <250 nt length, spanning the NCR, were inserted into a luciferase vector possessing an SV40 promoter. Fragments F5(-542/-311) and F6(-310/-197) showed promoter-enhancing activities of >6.0 by insertion not only in the sense orientation, but also both in the antisense orientation and downstream of the luciferase gene. The 5' deletion of NCR from -1201 to -370 resulted in no significant decrease in the level of luciferase activity. A gradual decrease in the activity of the 5'-deletion mutants from position -370 through -155 was consistent with the loss of enhancer binding sites detected during fragment analysis. A further deletion at position -76 completely abolished luciferase expression, indicating that region -154/-76 contains the critical regulatory element for functioning of the TTV promoter.

5' Untranslated Regions↗
PubMed2004

Hepatitis C virus (HCV) genotypes in the Caribbean island of Martinique: evidence for a large radiation of HCV-2 and for a recent introduction from Europe of HCV-4.

Molecular epidemiological studies of hepatitis C virus (HCV) in the Caribbean may help to specify the origin and spread of HCV infection. Indeed, the Caribbean population is intermixed from European and African origins and geographically close to the American continent. We characterized HCV genotypes in the Caribbean island of Martinique. HCV genotypes were analyzed by sequencing or reverse hybridization in the 5' noncoding region (5'NC) in 250 HCV-monoinfected and 85 HCV-human immunodeficiency virus (HIV)-coinfected patients. In addition, sequencing in the nonstructural 5B (NS5B) gene was required to determine the subtype or to perform phylogenetic analysis in selected samples. Genotypes 1 to 6 were found, respectively, in 84.4, 6.8, 5.2, 2.8, 0.4, and 0.4% of 250 HCV-monoinfected patients and in 71.7, 7.1, 15.3, 5.9, 0, and 0% of 85 HCV-HIV-coinfected patients. HCV-1b was found in 66.4% of the HCV-monoinfected patients and was associated with blood transfusion, whereas HCV-1a was detected in 41.2% of the HCV-HIV-coinfected patients and was associated with intravenous drug use (IVDU). The HCV-3 strains belonged to subtype 3a and were linked to IVDU. Phylogenetic analyses were focused on HCV-2 and HCV-4, which are common in Africa. Two opposite patterns were evidenced. NS5B sequences from 19 HCV-2 isolates were affiliated with many different subtypes described either in Europe or in West Africa, suggesting an ancient radiation. In contrast, seven of the nine HCV-4 NS5B sequences ranged within HCV-4a and HCV-4d clusters spreading in continental France by the IVDU route. Epidemiological data demonstrate the recent introduction of HCV-4a and -4d subtypes into the Caribbean.

Adult↗
PubMed1984

Separation of sequences defining basal expression from those conferring alpha gene recognition within the regulatory domains of herpes simplex virus 1 alpha genes.

The genes of herpes simplex virus 1 form three major groups--alpha, beta, and gamma--whose expression is coordinately regulated and sequentially ordered in a cascade fashion. To determine how the infected cell differentiates between these gene groups, alpha-regulated chimeric genes were constructed in earlier studies by fusing the structural sequences of the thymidine kinase (TK) gene, a beta gene, to the 5' noncoding sequences of alpha genes. These studies showed that (i) one or more structural components of the virion act in trans to increase alpha gene expression and (ii) the 5' noncoding sequences of alpha genes contain cis-acting domains that promote gene expression and confer alpha-gene regulation. These two domains could be moved independently, but the regulatory domain required a promoter for its function. We report here the properties of three sequences containing features common to the regulatory regions of all alpha genes. Sequence 1, containing (G + C)-rich inverted repeats, increased the basal level of TK expression when fused 5' to either the alpha gene 4 promoter or the truncated beta TK promoter. The effect was to some extent orientation dependent. Moreover, sequence 1 restored beta regulation to the truncated beta TK promoter but did not confer alpha-specific regulation on any of the chimeric genes tested. Sequences 2 (49 base pairs) and 3 (29 base pairs), containing an (A + T)-rich homolog from alpha gene 27 and alpha gene 0, respectively, restored alpha-specific regulation to the alpha promoter gene but only sequence 2 conferred alpha regulation on the truncated beta promoter gene. Our results indicate that (i) in natural beta TK the promoter and regulatory domains overlap, (ii) sequence 1 determines basal level of expression and substitutes for a promoter component that is essential for beta but not alpha regulation, and (iii) conversion of a gene with a promoter into an alpha gene requires two elements. Sequence 2 may contain both whereas sequence 3 contains only one.

Amino Acid Sequence↗
PubMed1993

At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide.

In a previous study we sequenced the 5' noncoding (NC) region of 44 isolates of hepatitis C virus (HCV) and identified heterogeneous domains that provided evidence for additional genetic groups of HCV not previously recognized. In this study we have determined the complete nucleotide sequence of the putative envelope 1 (E1) gene in 51 HCV isolates from around the world and found that they could be grouped into at least 12 distinct genotypes. The E1 gene sequence of 8 of these genotypes has not been reported previously. Although the genetic relatedness of HCV isolates determined by the previous analysis of the 5' NC region predicted the relationships observed in the E1 gene, analysis of the 5' NC sequence alone did not accurately predict all HCV genotypes. The nucleotide and amino acid sequence identities of the E1 gene among HCV isolates of the same genotype were in the range of 88.0-99.1% and 89.1-98.4%, respectively, whereas those of HCV isolates of different genotypes were in the range of 53.5-78.6% and 49.0-82.8%, respectively. The latter differences are similar to those found when comparing the envelope gene sequences of the various serotypes of the related flaviviruses as well as other RNA viruses. We found that some genotypes of HCV were widely distributed around the world, whereas others were identified only in discreet geographical regions. Four genotypes were identified exclusively in Africa and comprised the majority of HCV isolates on that continent. The E1 gene was exactly 576 nucleotides in length in all 51 HCV isolates with no in-frame stop codons. Analysis of the predicted E1 protein identified several conserved domains that may be important for maintaining its biological function: (i) eight invariant cysteine residues, (ii) three potential N-linked glycosylation sites, (iii) a domain of nine amino acids (GHRMAWDMM), and (iv) an amino acid doublet (GV) near the putative cleavage site at the C terminus of the protein. In conclusion, the discovery of at least 12 genotypes of HCV has important implications for HCV diagnosis and vaccine development.

Amino Acid Sequence↗
PubMed2003

Variations in transcription termination signals of human respiratory syncytial virus clinical isolates affect gene expression.

Human respiratory syncytial virus (HRSV) has a single-stranded, negative-sense RNA genome with 10 genes encoding 11 proteins. Sequences at the beginning of the HRSV genes are highly conserved; however, the gene end sequences vary around a semiconserved consensus sequence, and the nontranscribed intergenic regions vary in both length and sequence. The regions at the junctions between HRSV genes (the gene end sequence of an upstream gene, intergenic region, and the gene start sequence of a downstream gene) contain elements required for efficient termination of the upstream gene and transcription of the downstream gene. Previous studies have examined variation in the HRSV coding sequences, but none have systematically analyzed the noncoding transcriptional control regions for variability. We determined the gene start and gene end sequences of each of the 10 HRSV genes from 14 clinical isolates for variations from the sequence of the prototype A2 strain. No changes were found in any of the gene start sequences. Eight of the 10 gene end sequences, however, contained variations. Several of these, a U(4)-tract instead of a U(6)- or U(5)-tract at the M and SH gene ends, respectively, (U(4)A) and an A-to-G change at position four in the G gene end (A4G), were predicted to affect termination and were examined for their effects on transcription. The changes were found to inhibit transcriptional termination, resulting in increased polycistronic readthrough and correspondingly reduced initiation of the downstream monocistronic mRNA. Viruses with the A4G variant G gene end sequence produced less F protein than those with A2-like G gene end sequences. Examination of additional G gene end sequences available in GenBank revealed that the observed A4G variation was restricted to one phylogenetic lineage of HRSV. All viruses examined within this lineage possessed this variant G gene end sequence. The data presented show that the gene end sequences of naturally occurring HRSV clinical isolates vary from those of the prototypic A2 strain and that certain of these changes inhibit efficient transcriptional termination and downstream gene expression.

Gene Expression Regulation↗
PubMed2003

Clinical evaluation of two methods for genotyping hepatitis C virus based on analysis of the 5' noncoding region.

We compared the performance characteristics of a standardized direct sequencing method (TRUGENE HCV 5'NC; Visible Genetics Inc., Toronto, Ontario, Canada) and a reverse hybridization line probe assay (INNO-LiPA HCV II; Bayer Corp., Tarrytown, N.Y.) for genotyping of hepatitis C virus (HCV). Both methods are based on detection of sequence heterogeneity in the 5' noncoding (5'NC) region. Concordance between the genotyping methods was assessed by testing 172 samples representing the six major genotypes. Sequence analysis of the more phylogenetically informative nonstructural 5B (NS5B) region was also done with 148 (86%) samples to confirm the accuracy of and resolve discrepancies between the 5'NC genotyping results. The sensitivities of the methods were assessed by using the 5'NC amplicon from both the qualitative and quantitative AMPLICOR HCV tests (Roche Diagnostics Corp., Indianapolis, Ind.). The ability of the methods to detect mixed-genotype infections was determined with mixtures of two different genotypes at relative concentrations ranging from 1 to 50%. Both 5'NC methods were able to genotype 99.4% of the samples with type agreement for 99.5% and subtype agreement for 68.2% of the samples. No or ambiguous subtype results were found by the line probe assay for 16.5% and by the TRUGENE 5'NC test for 17.1% of the samples. Discrepancies occurred between the line probe assay and NS5B results at the type level for 1.4% of the samples and at the subtype level for 14.2% of the samples. Discrepancies also occurred between the TRUGENE 5'NC and NS5B results at the type level for 2% of the samples and at the subtype level for 8.1% of the samples. We also found two distinct strains of HCV classified as type 2 by analysis of the 5'NC region that were type 1 by analysis of the NS5B region. The sensitivities of the two 5'NC genotyping methods were comparable and dependent on the amplification test used ( approximately 10(3) IU/ml with the qualitative HCV RNA tests and approximately 10(5) IU/ml with the quantitative HCV RNA tests). Genotype mixtures were successfully identified at a relative concentration of 5% by the line probe assay and 10% by the TRUGENE 5'NC test. In conclusion, the performance characteristics of the 5'NC methods were similar and both methods produced accurate results at the genotype level but neither method should be used for subtyping.

5' Untranslated Regions↗
PubMed1996

Detection and clinical features of hepatitis C virus type 6 infections in blood donors from Hong Kong.

The genotype distribution of hepatitis C virus (HCV) was investigated in 212 viraemic blood donors from Hong Kong. A subset of the samples was investigated using three different genotyping assays to establish the accuracy of each in this population. These assays were restriction fragment length polymorphism (RFLP) of amplified 5' noncoding region (5'NCR) sequences, RFLP of the core region, and a serotyping assay using peptides from two antigenic regions of NS4. Genotypes detected in Hong Kong blood donors were 1a (6.2%), 1b (58.8%), 2a (1.4%), 2b (1.4%), 3a (1.9%), and 6a (27.0%). All genotyping assays produced concordant results. No evidence was obtained for the presence of type 6 group variants recently identified in Southeast Asia, other than type 6a. A serotyping assay based upon the detection of type-specific antibody to epitopes in NS4 produced similar results to the genotyping assays (98% concordance), but a reduced sensitivity (75%) compared with genotyping methods. Sequence variation in NS4 was not the cause of the reduced rate of detection of type 6 antibody in this population. Eighty-four percent donors infected with type 6a were male, compared to 75% donors infected with type 1b. The median alanine transaminase (ALT) level in type 6 infected donors was lower than in type 1b, (43.8 and 51.1 U/l, respectively) although these values were not statistically significant (P = 0.094). There was no significant difference between the ages of donors infected with types 1b and 6a. Risk factors for HCV infection in the blood donors included blood transfusion, intravenous drug abuse, and tattooing. A significantly greater number of donors infected with HCV-6a reported a history of drug abuse (66%) than donors infected with HCV-1b (7%).

Adolescent↗
PubMed2005

Size heterogeneity in the 3' noncoding region of South American isolates of yellow fever virus.

The 3' noncoding region (3' NCR) of flaviviruses contains secondary and tertiary structures essential for virus replication. Previous studies of yellow fever virus (YFV) and dengue virus have found that modifications to the 3' NCR are sometimes associated with attenuation in vertebrate and/or mosquito hosts. The 3' NCRs of 117 isolates of South American YFV have been examined, and major deletions and/or duplications of conserved RNA structures have been identified in several wild-type isolates. Nineteen isolates (designated YF-XL isolates) from Brazil, Trinidad, and Venezuela, dating from 1973 to 2001, exhibited a 216-nucleotide (nt) duplication, yielding a tandem repeat of conserved hairpin, stem-loop, dumbbell, and pseudoknot structures. YF-XL isolates were found exclusively within one subclade of South American genotype I YFV. One Brazilian isolate exhibited, in addition to the 216-nt duplication, a deletion of a 40-nt repeated hairpin (RYF) motif (YF-XL-DeltaRYF). To investigate the biological significance of these 3' NCR rearrangements, YF-XL-DeltaRYF and YF-XL isolates, as well as other South American YFV isolates, were evaluated for three phenotypes: growth kinetics in cell culture, neuroinvasiveness in suckling mice, and ability to replicate and produce disseminated infections in Aedes aegypti mosquitoes. YF-XL-DeltaRYF and YF-XL isolates showed growth kinetics and neuroinvasive characteristics comparable to those of typical South American YFV isolates, and mosquito infectivity trials demonstrated that both types of 3' NCR variants were capable of replication and dissemination in a laboratory-adapted colony of A. aegypti.

Aedes↗
PubMed2003

[Activity of specific deoxyribozymes to cleave hepatitis C virus RNA in vitro].

OBJECTIVE: To analyze the cleavage activity of two deoxyribozymes targeting at hepatitis C virus (HCV) RNA in vitro and evaluate their prospects of antiviral therapy. METHODS: Two specific sequences containing 5' ...A / U... 3' in HCV 5'-noncoding region and 5'-fragment of C region (5'-NCR-C) were selected as the target sites, and with the active region of 5'GGCTAGCTACAACGA3', two phosphorothioate deoxyribozymes (TDRz) named as TDRz-127 and TDRz1 were synthesized. HCV RNA 5'-NCR-C was transcribed in vitro from plasmid pHCV-neo which was completely linearized with restriction endonuclease Nar I, and its 5'-end phosphoric acid was deleted by calf intestinal alkaline phosphatase (CIP), then radiolabelled with T4 polynucleotide kinase and gamma-32P-ATP. Under the conditions such as pH 7.5 and a 10 mmol/L Mg2+ concentration, TDRz-127 and TDRz1 were separately (a 5 micromol/L final concentration) or combinedly (each 2.5 micromol/L) mixed with the substrate RNA (200 nmol/L). After denaturation and then renaturation, the reaction systems were incubated in 37 degrees C, and aliquots were removed to terminate the reaction at intended time points. The cleavage products were separated with 8% denaturated polyacrylamide gel electrophoresis and displayed by autoradiography. Finally, the optical density of each product band was measured with Gel Documentation-Analyzing Systems for calculating the percentages of cleaved HCV 5'-NCR-C. RESULTS: After reaction for 15, 30, 45, 60, 75 and 90 min under the adopted conditions, about 8.3%, 16.1%, 24.3%, 26.2%, 29.4% and 31.1% of HCV 5'-NCR-C was cleaved by TDRz-127 respectively; 7.4%, 13.0%, 15.6%, 18.7%, 19.4% and 20.3% by TDRz1; and 15.1%, 29.6%, 37.8%, 39.1%, 41.5%, 42.6% by combining the two TDRzs. CONCLUSIONS: Cleavage percentage of both TDRz-127 and TDRz1 increases with the time, and the effect of combining the two TDRzs is better than that of anyone.

5' Untranslated Regions↗
PubMed1992

Late promoter of human papillomavirus type 8 and its regulation.

Human papillomavirus type 8 (HPV8) belongs to the HPV types associated with skin carcinomas of patients with epidermodysplasia verruciformis (EV). Its noncoding regulatory sequences (NCR) were shown to drive the expression of the reporter gene chloramphenicol acetyltransferase (cat) in transient assays with human epithelial cells (HT3 cells). This constitutive activity could be enhanced by coexpression of the HPV8 transactivator protein E2. The analysis of 5' deletions of the NCR showed that the EV-specific sequence motif M33 and the neighboring AP1 site are essential for the promoter activity, whereas 44 nucleotides located immediately upstream of M33 are strongly inhibitory. The same effects were observed in simian virus 40-immortalized fetal keratinocytes (SV61 cells) and spontaneously immortalized skin keratinocytes (HaCaT cells). By using primer extension and RNase protection analyses two promoters could be identified within the HPV8 NCR. A nested set of weak signals, corresponding to start sites between positions 175 to 179, represented the previously described E6 promoter. The vast majority of transcripts was initiated at position 7535 and shown to undergo processing at an NCR-internal splice donor (positions 1 to 8). The promoter P7535 is similar to late promoters of other skin-associated papillomaviruses as far as localization, transcript structure, and sequence characteristics are concerned. To confirm that P7535-initiated transcripts proceed indeed to the L1 gene for the major capsid protein, viral mRNAs from an HPV8-induced lesion of a patient with EV were characterized by RNase protection and sequence analysis of polymerase chain reaction-amplified cDNAs. The NCR leader (positions 7535 to 4) appeared in two messages with three exons each. The third exon started with the second ATG codon of L1 in both cases; the short central exons from the 3' part of the early coding region were defined by a common splice acceptor site (position 3303) and different splice donor sites (positions 3443 and 3704).

Adult↗
PubMed1993

Identification of a large bent DNA domain and binding sites for serum response factor adjacent to the NFI repeat cluster and enhancer region in the major IE94 promoter from simian cytomegalovirus.

The major immediate-early (MIE) transactivator proteins of cytomegaloviruses (CMV) play a pivotal role in the initiation of virus-host cell interactions. Therefore, cis- and trans-acting factors influencing the expression of these proteins through their upstream promoter-enhancer regions are important determinants of the outcome of virus infection. S1 nuclease analysis and in vitro transcription assays with the MIE (or IE94) transcription unit of simian CMV (SCMV) (Colburn) revealed a single prominent mRNA start site associated with a canonical TATATAA motif. This initiator region lies adjacent to a 2,400-bp 5'-upstream noncoding sequence that encompasses a newly identified 1,000-bp (A+T)-rich segment containing intrinsically bent DNA (domain C), together with the previously described proximal cyclic AMP response element locus (domain A) and a tandemly repeated nuclear factor I binding site cluster (domain B). Deleted MIE reporter gene constructions containing domain A sequences only yield up to 4-fold stronger basal expression in Vero cells than the intact simian virus 40 promoter-enhancer region, and sequences from position -405 to -69 (ENH-A1) added to a minimal heterologous promoter produced a 50-fold increase of basal expression in an enhancer assay. In contrast, neither the nuclear factor I cluster nor the bent DNA region possessed basal enhancer properties and neither significantly modulated the basal activity of the ENH-A1 segment. A second segment of domain A from position -580 to -450 was also found to possess basal enhancer activity in various cell types. This ENH-A2 region contains three copies of a repeated element that includes the 10-bp palindromic sequence CCATATATGG, which resembles the core motif of serum response elements and proved to bind specifically to the cellular nuclear protein serum response transcription factor. Reporter gene constructions containing four tandem copies of these elements displayed up to 13-fold increased basal enhancer activity and 18-fold tetradecanoyl phorbol acetate responsiveness in U937 cells, but an ENH-A2 DNA segment encompassing two of the core serum response transcription factor binding sites failed to respond to serum induction in NIH 3T3 cells. Although there are overall similarities in the organizations of both the MIE enhancers and MIE transcription units among human CMV, SCMV, and murine CMV, the specific arrangements of repetitive motifs are quite different, and the bent DNA and ENH-A2 domains appear to be unique to SCMV.

Base Sequence↗
PubMed1999

Annual and seasonal variation of tick-borne encephalitis virus (TBEV) prevalence in ticks in selected hot spot areas in Germany using a nRT-PCR: results from 1997 and 1998.

The prevalence of tick-borne encephalitis virus (TBEV) in Ixodes ricinus tick populations in endemic areas of Germany with the highest TBE risk is unknown. Annual and seasonal differences in TBEV prevalence have also not been studied. Against this background, in May 1997 we started a systematic virus surveillance programme in ticks collected in locations known to have a high incidence of autochthonous TBE cases. These were 5 locations in Baden-Württemberg (Black Forest) and 8 locations in Bavaria (surrounding Passau). Field-collected ticks were randomly assigned to pools of 10 adults or 20 nymphs, respectively. The tick pools were tested for the presence of TBEV-RNA using a newly developed, sensitive nested reverse transcriptase polymerase chain reaction assay (nRT-PCR). The primer pairs were selected from the 5'-terminal noncoding region, a highly conserved part of the virus. The specificity was tested by computer homology searches of sequences, as well as by sequencing of the first and the second amplificates, by Southern blot hybridisation with a DIG-labelled oligonucleotide probe, and by restriction enzyme analysis. The method has proved to be very sensitive, with a detection limit of 20 fg of TBEV RNA per PCR run, or a single positive tick. Based on biostatistical considerations a sample size of at least 1000 ticks per estimation point was chosen. The estimated TBEV prevalence and confidence intervals (CI) were calculated from the nRT-PCR results of pooled samples (10 adults or 20 nymphs) using appropriate formulae for pooled testing. In order to identify the estimated TBEV prevalence as well as to assess the influence of annual and seasonal factors on TBEV prevalence, ticks were sampled twice a year (May and September) in 1997 and 1998 at exactly identical sites. These sites were selected because they were known to have had the highest incidence of autochthonous TBE cases during the previous 10 years. On sampling days, relevant local meteorological data were also noted. In total, 8500 I. ricinus ticks were investigated in this study, 4270 (3540 nymphs, 730 adults) from the Black Forest habitats, and 4230 (3680 nymphs, 550 adults) from the Bavarian locations. In the foci near Freiburg (Black Forest), the estimated virus prevalence was relatively high in the whole tick population, during 1997 with only slight seasonal differences [3.4% (confidence interval, CI, 2.3-4.8%) in May and 2.9% (CI 1.7-4.5%) in September]. In contrast, in 1998, in the same foci the estimated TBEV prevalence was considerably lower [1.1% (CI 0.5-2.0%) in May and 0.6% (CI 0.2-1.4%) in September]. Thus, while the seasonal differences again remained low, the annual variation was marked. In the Bavarian foci in 1997, the estimated virus prevalence of the whole tick population studied was lower than in the Black Forest foci and the seasonal fluctuations were low: in May 1997 0.9% (CI 0.4-1.8%) of the ticks were positive, in September 1.1% (CI 0.5-1.9%). In 1998, in May 2.0% (CI 1.1-3.3%) of the ticks were positive, and in September 1.1% (CI 0.5-2.1%). For the whole study period, every 50th to 100th I. ricinus nymph or adult in the Passau region was calculated to give a positive signal in the nRT-PCR. The TBEV prevalence data indicate that residents and visitors of areas in Germany known for high endemic activity take a significant risk of contracting TBEV infection, if bitten by ticks. In addition, the data suggest that annual fluctuations may exist in the whole tick population studied. Seasonal fluctuations of the virus prevalence in ticks were small.

Adult↗