- Volume 80, Issue 12, 1999
Volume 80, Issue 12, 1999
- Review Article
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- Animal: RNA Viruses
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Functional characterization of a piscine retroviral promoter
Z. Zhang, E. Kim and D. MartineauWalleye dermal sarcoma virus (WDSV) is a retrovirus aetiologically associated with a multifocal skin tumour of walleye. Tumours synchronously develop on 27% of fish and regress seasonally; their severity is influenced by water temperature. To functionally characterize the LTR of WDSV, the LTR was fused to the luciferase reporter gene. WDSV LTR was found to be transcriptionally active in both fish and mammalian cells. WDSV LTR deletion mutants were constructed to identify specific regions that were functionally important in modulating viral gene expression and in temperature responsiveness. The 5′ end 60 bp, which contain a putative ecdysone-response element also present in another fish retrovirus, positively modulated transcription from the WDSV LTR at 25 °C, but not at lower temperatures. A 13 bp region (nt −288 to −275) comprising a putative activator protein-1 element was necessary for maintaining WDSV LTR activity at all temperatures. In marked contrast to the short direct repeats found in mammalian retroviral LTRs, five 5 bp direct repeats (nt −336 and −272) were found to negatively regulate transcription from the WDSV LTR. A region spanning nt −440 to −218 stimulated the activity of a heterologous mammalian promoter in an orientation-dependent manner, modestly in fish cells (1·3- to 2-fold), but markedly (3·7- to 5·1-fold) in mammalian cells. Our results strongly suggest that the putative promoter elements present in the WDSV LTR function differentially in a temperature-specific manner and that complex interactions between these elements modulate WDSV LTR activity in response to temperature changes.
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Transcriptional activation of human TR3/nur77 gene expression by human T-lymphotropic virus type I Tax protein through two AP-1-like elements
More LessThe Tax transactivator of human T-lymphotropic virus type I (HTLV-I) is capable of inducing expression of the human immediate-early TR3/nur77 gene. Deletion and mutation analyses of the TR3/nur77 promoter demonstrated that multiple transcription elements in the 121 bp sequence proximal to the transcription start site are required for full Tax transactivation. Mutations of CArG-like, Ets and RCE motifs in this region severely decreased Tax transactivation. Mutation of either of the two identical AP-1-like elements (NAP 1 and 2) immediately upstream of the TATA box caused around 80% reduction of Tax transactivation. Mutation of both NAP elements blocked Tax-mediated activation totally. These two NAP elements could confer Tax-responsiveness on a heterologous basal promoter. Furthermore, the specific NAP-binding complex was only observed in HTLV-I-infected cells. Formation of this specific NAP-binding complex was correlated directly with Tax expression, as demonstrated in JPX-9 cells upon induction of Tax expression. The specific NAP binding could be competed for by consensus AP-1 and CREB elements, indicating that the NAP-binding proteins probably belong to the AP-1 and CREB/ATF transcription factor families. Supershift analysis with antibodies to both the AP-1 and CREB/ATF transcription factor families revealed that only anti-JunD antibody could partially shift this NAP-binding complex, indicating that JunD is a component of the NAP complex. This work suggests that JunD is involved in Tax-regulated TR3/nur77 expression.
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First seroepidemiological study and phylogenetic characterization of human T-cell lymphotropic virus type I and II infection among Amerindians in French Guiana
More LessWe investigated the serological, epidemiological and molecular aspects of human T-cell lymphotropic virus type I and II (HTLV-I/II) infection in the Amerindian populations of French Guiana by testing 847 sera. No HTLV-II antibodies were detected, but five individuals (0·59%) were seropositive for HTLV-I. Analysis of the nucleotide sequences of 522 bp of the env gene and the compete LTR showed that all of the strains from French Guiana belonged to the cosmopolitan subtype A. The similarities were greater between Amerindian and Creole strains than between Amerindian and Noir-Marron strains or than between Creole and Noir-Marron strains. Phylogenetic analysis showed two clusters: one of strains from Amerindians and Creoles, which belong to the transcontinental subgroup, and the other of strains from Noirs-Marrons, belonging to the West African subgroup. Our results suggest that the Amerindian HTLV-I strains are of African origin.
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Specific passage of simian immunodeficiency virus fromend-stage disease results in accelerated progression to AIDS in rhesus macaques
To determine whether passage of late-stage variants of simian immunodeficiency virus (SIV) would lead to a more virulent infection and rapid disease progression, a study was designed to examine the effects of selective transmission of SIV from late-stage cases of AIDS in Macaca mulatta. In a uniform group of 10 age-matched animals from the same genetic breeding stock infected with SIVB670, it took 7 months before one of the ten animals developed AIDS. Passage of virus taken from this animal immediately prior to death resulted in death of the recipient due to AIDS within 4 months. Again, subsequent passage of virus taken late in disease resulted in an accelerated disease course, with AIDS developing within 2·5 and 1·8 months in two recipients. The fourth passage of virus taken late in disease from the most rapid progressor (1·8 months) resulted in AIDS developing in this recipient within 1 month of infection. During each consecutive passage in vivo, the loss of memory T cells became more acute. Evidence that the virus became more virulent with selective passage of late-stage variants was provided by the markedly increased levels of both plasma antigen and viral RNA. Subsequent in vivo passage from end-stage AIDS selected for a strain of SIV capable of causing the acute development of AIDS as rapidly as 1 month post-infection. The pathology of acute AIDS in these cases closely resembled that seen after a chronic disease course.
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Characterization of aggregates of hepatitis C virus glycoproteins
Hepatitis C virus (HCV) encodes two glycoproteins, E1 and E2, which assemble in oligomeric structures. Studies of HCV glycoprotein assembly using heterologous expression systems have shown that these glycoproteins can follow two pathways: a productive pathway leading to the formation of a non-covalent heterodimer; and a non-productive pathway leading to the formation of large disulfide-linked aggregates. The non-covalent HCV glycoprotein complex is probably the functional complex which plays an active role in the entry process in host cells. The aggregates are believed to be waste products; however, one can imagine that, in infected cells, they could provide HCV glycoproteins with additional functions. To further understand the potential role played by HCV glycoprotein aggregates in HCV infection, a MAb (H14) was produced which specifically recognizes these aggregates but not the non-covalent E1E2 heterodimer. The H14 epitope was shown to be present on both HCV glycoproteins and was sensitive to deglycosylation. An additional characterization of HCV glycoprotein aggregates, with the help of MAb H14, indicates that they share an epitope with a cellular protein called Mac-2 binding protein. The presence of such an epitope on HCV glycoprotein aggregates could potentially lead to the production of autoantibodies recognizing Mac-2 binding protein in HCV-infected patients.
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Human hepatic glyceraldehyde-3-phosphate dehydrogenase binds to the poly(U) tract of the 3′ non-coding region of hepatitis C virus genomic RNA
More LessThe unique poly(U/UC) tract, the middle part of the tripartite 3′ non-coding region (3′NCR) of hepatitis C virus (HCV) genomic RNA, may represent a recognition signal for the HCV replicase complex. In this study, several proteins binding specifically to immobilized ribooligonucleotide r(U)25 mimicking this structure were identified using cytosolic extracts from HCV-negative or -positive liver explants, and a prominent 36 kDa protein was studied further. Competition experiments including homoribopolymers revealed binding affinities in the order: oligo/poly(U)≫(A)≫(C)≫(G). The protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a multifunctional protein known to bind RNA. GAPDH bound efficiently to the full-length HCV RNA and binding to various 3′NCR constructs revealed critical dependence upon the presence of the middle part of the 3′NCR. Polypyrimidine tract-binding protein, described previously to bind the 3′NCR, did not bind efficiently to the middle part of 3′NCR and was captured from liver extracts in considerably smaller quantities.
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Characterization of infectious Murray Valley encephalitis virus derived from a stably cloned genome-length cDNA
More LessAn infectious cDNA clone of Murray Valley encephalitis virus prototype strain 1-51 (MVE-1-51) was constructed by stably inserting genome-length cDNA into the low-copy-number plasmid vector pMC18. Designated pMVE-1-51, the clone consisted of genome-length cDNA of MVE-1-51 under the control of a T7 RNA polymerase promoter. The clone was constructed by using existing components of a cDNA library, in addition to cDNA of the 3′ terminus derived by RT–PCR of poly(A)-tailed viral RNA. Upon comparison with other flavivirus sequences, the previously undetermined sequence of the 3′ UTR was found to contain elements conserved throughout the genus Flavivirus. RNA transcribed from pMVE-1-51 and subsequently transfected into BHK-21 cells generated infectious virus. The plaque morphology, replication kinetics and antigenic profile of clone-derived virus (CDV-1-51) was similar to the parental virus in vitro. Furthermore, the virulence properties of CDV-1-51 and MVE-1-51 (LD50 values and mortality profiles) were found to be identical in vivo in the mouse model. Through site-directed mutagenesis, the infectious clone should serve as a valuable tool for investigating the molecular determinants of virulence in MVE virus.
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Phylogenetic and virulence analysis of tick-borne encephalitis viruses from Japan and far-eastern Russia
We have previously reported that tick-borne encephalitis (TBE) is endemic in a specific area of Hokkaido, Japan. In Oshima, the southern part of Hokkaido, TBE virus was isolated from sentinel dogs, ticks and rodents in 1995 and 1996. To identify when these TBE viruses emerged in Hokkaido, the times of divergence of TBE virus strains isolated in Oshima and far-eastern Russia were estimated. TBE virus was isolated in Khabarovsk in 1998 and the nucleotide sequences of viral envelope protein genes of isolates from Oshima and Khabarovsk were compared. From the synonymous substitution rate of these virus strains, the lineage divergence time of these TBE virus strains was predicted phylogenetically to be about 260–430 years ago. Furthermore, the virulence of TBE virus isolates from Oshima and Khabarovsk were compared in a mouse model. The results showed that the isolates possessed very similar virulence in mice. This report provides evidence that the Oshima strains of TBE virus in Hokkaido emerged from far-eastern Russia a few hundred years ago and this explains why these strains possess virulence similar to the TBE viruses isolated in Russia.
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Vaccination of cattle with a DNA plasmid encoding the bovine viral diarrhoea virus major glycoprotein E2
More LessBovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle that is ubiquitously distributed worldwide. In this study, cattle were immunized by intramuscular injections with plasmid DNA expressing the BVDV type 1 major glycoprotein E2. Animals either received injections of naked DNA (N-DNA) or DNA in cationic liposomes (L-DNA). Both DNA preparations induced virus-specific neutralizing antibodies in vaccinates, although the response was much lower in N-DNA-immunized animals. N-DNA-vaccinated animals also showed virus-specific lymphocyte proliferation responses to type 1, live BVDV in vitro, whereas L-DNA vaccination induced no such responses. After 16 weeks, DNA-vaccinated and mock-vaccinated animals were challenged with a USDA-certified BVDV type 1 strain. Four significant observations were made: (1) N-DNA-vaccinated calves showed limited protection from virus challenge, (2) L-DNA-vaccinated animals did not show any signs of protection, (3) the challenge induced strong memory responses in the production of serum neutralizing antibodies to both genotypes (type 1 and 2 of BVDV), and (4) the challenge induced a mucosal memory response in nasal secretions of both L- and N-DNA-vaccinated animals.
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Mapping the binding domains on decay accelerating factor (DAF) for haemagglutinating enteroviruses: implications for the evolution of a DAF-binding phenotype
More LessDecay accelerating factor (DAF) functions as a cell attachment receptor for a wide range of human enteroviruses, the interaction accounting for the haemagglutination phenotype exhibited by many members of this family. Haemagglutination inhibition assays using purified truncated soluble DAF (sDAF) receptors and short consensus repeat (SCR) domain-specific antibodies have been used to determine the domain(s) of DAF to which the viruses bind. Further sDAF-mediated virus neutralization and biosensor analysis have been used to confirm the virus-binding domains of DAF. Of the four distinct clusters of human enteroviruses, three contain representatives that bind DAF. The majority of DAF-binding enteroviruses occupy the ‘CBV-like’ cluster, and require SCR domains 2–4 for DAF binding. In contrast, the DAF-binding representatives of the ‘ENV70-like’ and ‘PV-like’ clusters require SCR1 for DAF interaction. These studies confirm that DAF binding is a widespread characteristic amongst phylogenetically divergent clusters within the enteroviruses and suggest that the ability to bind DAF may have evolved more than once within this group of viruses.
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Comparative analysis of virus–host cell interactions of haemagglutinating and non-haemagglutinating strains of coxsackievirus B3
More LessDecay-accelerating factor (DAF/CD55), and coxsackievirus–adenovirus receptor (CAR) have been identified as cellular receptors for coxsackie B viruses (CBV). To elucidate the interplay of DAF and CAR on the cell surface, virus–receptor interactions of two coxsackieviruses of serotype B3 (non-haemagglutinating CBV3 and haemagglutinating CBV3-HA strain) were analysed. Binding assays revealed clear differences between these viruses with regard to their interactions with DAF and CAR. However, only the combination of anti-DAF and anti-CAR antibodies resulted in complete inhibition of virus binding for both strains. In plaque-reduction assays, anti-DAF antibodies had no effect, whereas CAR-specific antibodies significantly reduced productive infection of HeLa cells by both viruses. Interestingly, a synergistic inhibitory effect of anti-DAF and anti-CAR antibodies was also observed with regard to infection. These findings support the model of preferential interactions of both strains of CBV3 with closely associated DAF and CAR proteins on HeLa cells, despite displaying clear differences in their binding phenotypes.
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La protein is required for efficient translation driven by encephalomyocarditis virus internal ribosomal entry site
More LessTranslation of internal ribosomal entry site (IRES)-dependent mRNAs is mediated by RNA-binding proteins as well as canonical translation factors. In order to elucidate the roles of RNA-binding proteins in IRES-dependent translation, the role of polypyrimidine tract-binding protein (PTB) and La protein in encephalomyocarditis virus (EMCV) IRES-dependent translation was investigated. PTB was required for efficient EMCV IRES-driven translation but, intriguingly, an excess of PTB suppressed it. Such a translational suppression by surplus PTB was relieved by addition of La protein. A possible role for La protein in IRES-dependent translation is discussed.
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Transmission of Eurasian avian H2 influenza virus to shorebirds in North America
More LessInfluenza A virus of the H2 subtype caused a serious pandemic in 1957 and may cause similar outbreaks in the future. To assess the evolution and the antigenic relationships of avian influenza H2 viruses, we sequenced the haemagglutinin (HA) genes of H2 isolates from shorebirds, ducks and poultry in North America and derived a phylogenetic tree to establish their interrelationships. This analysis confirmed the divergence of H2 HA into two geographical lineages, American and Eurasian. One group of viruses isolated from shorebirds in North America had HA belonging to the Eurasian lineage, indicating an interregional transmission of the H2 gene. Characterization of HA with a monoclonal antibody panel revealed that the antigenicity of the Delaware strains differed from the other avian strains analysed. The data emphasizes the importance of avian influenza surveillance.
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Bunyavirus superinfection and segment reassortment in transovarially infected mosquitoes
More LessRapid evolution of bunyaviruses may occur by RNA segment reassortment between closely related viruses. Reassortment between viruses occurs in dually infected mosquitoes when two different viruses are simultaneously ingested or when the second virus is ingested within 2 days of the first virus. By 3 days after oral infection, interference to superinfection occurs, thus limiting the potential for evolution. Aedes triseriatus mosquitoes can also be transovarially infected (TI+) with LaCrosse (LAC) virus. In these studies, the potential for oral superinfection of TI+ mosquitoes was assessed. Approximately 20% of mosquitoes TI+ with either a temperature-sensitive LAC virus or wild-type (wt) LAC virus became superinfected by ingesting blood meals containing wt LAC or snowshoe hare (SSH) viruses. LAC virus TI+ mosquitoes superinfected with SSH virus were detected by blot hybridization or RT–PCR. Viruses from these mosquitoes were plaque purified and genotyped using RT–PCR. Reassortant genomes were detected in 2·3% of the viruses genotyped, and 4·0% of the genomes tested were diploid for one genome segment.
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Molecular characterization of human group C rotavirus genes 6, 7 and 9
More LessGenes 6, 7 and 9 of human group C rotavirus ‘Bristol’ strain, encoding non-structural proteins (NSP) 3, 1 and 2, respectively, were cloned and sequenced. Human group C rotavirus genome segment 6 is 1350 bp and contains a single ORF of 1231 nucleotides (encoding 402 amino acids). Genome segment 7 is 1270 bp and encodes a protein of 394 amino acids and genome segment 9 is 1037 bp and encodes a 312 amino acid protein. The human group C rotavirus genes 6, 7 and 9 showed 78, 67 and 88% sequence identity, respectively, to the corresponding porcine group C rotavirus genes. The derived protein sequences were compared with those of the porcine ‘Cowden’ group C and mammalian group A rotavirus strains. The human group C rotavirus NSP1 protein sequence is one amino acid longer than the porcine group C equivalent. In common with group A and porcine group C rotaviruses, the human group C rotavirus NSP1 protein has a zinc finger motif. Human group C rotavirus NSP2 has two hydrophobic heptad repeat regions, a basic, RNA-binding domain and a basic, proline-rich region. Human group C rotavirus NSP3 has both single- and double-stranded RNA-binding domains and several hydrophobic heptad repeat regions, one of which forms a leucine zipper. This work completes the molecular characterization of the non-structural proteins of a human group C rotavirus. Phylogenetic analysis of all the non-structural genes of group A, B and C rotaviruses suggests that these viruses have diverged at a constant rate from a common ancestor.
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- Animal: DNA Viruses
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Mutations in the conserved carboxy-terminal hydrophobic region of glycoprotein gB affect infectivity of herpes simplex virus
More LessGlycoprotein gB is the most highly conserved glycoprotein in the herpesvirus family and plays a critical role in virus entry and fusion. Glycoprotein gB of herpes simplex virus type 1 contains a hydrophobic stretch of 69 aa near the carboxy terminus that is essential for its biological activity. To determine the role(s) of specific amino acids in the carboxy-terminal hydrophobic region, a number of amino acids were mutagenized that are highly conserved in this region within the gB homologues of the family Herpesviridae. Three conserved residues in the membrane anchor domain, namely A786, A790 and A791, as well as amino acids G743, G746, G766, G770 and P774, that are non-variant in Herpesviridae, were mutagenized. The ability of the mutant proteins to rescue the infectivity of the gB-null virus, K082, in trans was measured by a complementation assay. All of the mutant proteins formed dimers and were incorporated in virion particles produced in the complementation assay. Mutants G746N, G766N, F770S and P774L showed negligible complementation of K082, whereas mutant G743R showed a reduced activity. Virion particles containing these four mutant glycoproteins also showed a markedly reduced rate of entry compared to the wild-type. The results suggest that non-variant residues in the carboxy-terminal hydrophobic region of the gB protein may be important in virus infectivity.
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Characterization of the DNA polymerase loci of the novel porcine lymphotropic herpesviruses 1 and 2 in domestic and feral pigs
More LessTwo novel porcine gammaherpesviruses, porcine lymphotropic herpesviruses 1 and 2 (PLHV-1 and -2), have been detected by amplification of short DNA polymerase (DPOL) sequences from blood and spleen of domestic pigs while searching for unknown herpesviruses in pigs as possible risk factors in xenotransplantation. In the present study, the DPOL genes of the two viruses and the open reading frames (ORFs) that follow in the downstream direction were amplified by PCR-based genome walking from adaptor-ligated restriction fragment libraries of porcine spleen samples. The sequences determined for the two PLHVs exhibited a very low G+C content (37 mol%) and a marked suppression of the CpG dinucleotide frequency. The DPOL proteins encoded were 95% identical and showed a close relationship (60% identity) to the DPOL protein of a ruminant gammaherpesvirus, alcelaphine herpesvirus 1 (AlHV-1). This was confirmed by phylogenetic analyses of the conserved regions of the two PLHV DPOL proteins. The PLHV ORFs downstream of DPOL exhibited 83% identity to each other and ≫50% similarity to ORF A5, the position equivalent of AlHV-1. From these data, the PLHVs can be firmly classified to the subfamily Gammaherpesvirinae. To find a natural reservoir for the PLHVs, organs of feral pigs were screened with five different PCR assays, targetting either the DPOL gene or 3′-flanking sequences. In all samples, PLHV sequences were detected that originated predominantly from PLHV-2, suggesting the possibility of virus transfer between feral and domestic pig populations.
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The human herpesvirus-8 ORF 57 gene and its properties
Human herpesvirus-8 (HHV-8) is a γ2 lymphotropic herpesvirus associated with Kaposi’s sarcoma, a major neoplasm of AIDS patients, and with other AIDS-related neoplasms. The HHV-8 ORF 57 gene is conserved throughout the herpesvirus family and has a herpes simplex virus type 1 homologue, IE63 (also termed ICP27), which is an essential regulatory protein and acts at both transcriptional and post-transcriptional levels. We show that, contrary to the published HHV-8 sequence, which predicts a protein of 275 amino acids, the ORF 57 gene is spliced, contains a single intron and encodes a protein of 455 amino acids. For several gammaherpesviruses examined, the upstream coding exon is 16–17 amino acids in length and is rich in methionine residues. When ORF 57 was fused to the gene for enhanced green fluorescent protein (EGFP), the fusion protein exhibited a punctate nuclear distribution that co-localized with the cellular splicing factor SC-35. Unlike the IE63–EGFP fusion protein, ORF 57–EGFP did not shuttle from the nucleus to the cytoplasm in the presence of actinomycin D. However, ORF 57–EGFP was capable of shuttling from a transfected monkey nucleus to a recipient mouse nucleus in an interspecies heterokaryon assay. These data indicate that HHV-8 ORF 57 and IE63 possess certain common properties.
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Characterization of the transcriptional repressor RBP in Epstein–Barr virus-transformed B cells
More LessRBP, a transcriptional repressor, is intricately involved in Epstein–Barr virus (EBV) transformation of human B cells. The EBV nuclear proteins EBNA-2, -3, -4 and -6 all utilize RBP to regulate the transcription of both cellular and viral genes. This study investigates the isoforms of the RBP protein in Burkitt’s lymphoma (BL) cells and in EBV-transformed lymphoblastoid cell lines (LCLs). Two-dimensional gel electrophoresis showed the presence of two different cellular isoforms of RBP; the molecular masses and isoelectric points of these two isoforms corresponded to RBP-Jκ and RBP-2N. Fractionation studies and green fluorescent protein (GFP)-tagged expression studies demonstrated that both RBP isoforms were located predominantly in the cell nucleus. Interestingly, GFP-tagged RBP-Jκ showed diffuse, uniform nuclear staining, whereas GFP-tagged RBP-2N showed a discrete nuclear pattern, demonstrating differences between the two isoforms. Within the nuclear fraction of EBV-negative BL cells, RBP existed both in a free form and bound to chromatin, whereas in LCLs the intranuclear RBP was predominantly chromatin-bound. Expression of the EBV latent proteins was found to lead to the sequestering of RBP from the cytoplasm into the cell nucleus and to an increase in the chromatin-bound forms of RBP.
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Dissociation of patching by latent membrane protein-1 of Epstein–Barr virus from its stimulation of NF-κB activity
More LessAlterations were made in the amino terminus and the first two transmembrane-spanning regions of the latent membrane protein-1 (LMP-1) of Epstein–Barr virus. These mutant proteins were tested for their abilities to patch and to stimulate NF-κB activity. A subset of these derivatives retains the wild-type topology of LMP-1 in the plasma membrane, but has lost the ability to patch. Deletion of residues 9–20 of LMP-1, which contain potential SH3-binding motifs, abrogates patching of LMP-1. However, mutation of the prolines within these motifs, which eliminates binding of LMP-1 to SH3 domains in vitro, does not prevent patching by LMP-1. Deletion of the first two transmembrane regions of LMP-1 does prevent it patching. Some of the derivatives of LMP-1 which do not patch do stimulate NF-κB activity. Patching by LMP-1 appears to be a higher-order assemblage of protein that is compatible with the stimulation of NF-κB activity but is not necessary for this signalling.
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Human papillomavirus type 16 E6 variants in cervical carcinoma: relationship to host genetic factors and clinical parameters
More LessInfection with human papillomavirus type 16 (HPV-16) confers a high risk for the development of cervical neoplasia. Variants of this virus may interact differentially with host genetic factors, possibly altering the disease course. Thus, HPV-16 E6 variants may differ in their ability to degrade p53 whereas the polymorphic p53 alleles may provide more or less susceptible substrates for the viral oncogene product. Also, E6 variants may differ in immunogenicity by generating different peptides for presentation by polymorphic HLA molecules to specific T cells. This study examines HPV-16 E6 sequence variation in cervical carcinomas from the UK and its relationship to polymorphism of HLA and p53 and to clinical parameters. Sequence analysis of the HPV-16 E6 ORF from 77 tumour biopsies detected the viral prototype sequence in 38% of cases. The most common variation detected was a T to G transition at base pair 350, resulting in an amino acid change from a leucine to a valine. Overall, the frequencies of 350T and 350G sequences were similar (49·4% and 50·6% respectively). Other mutations of lower frequencies were detected together with and independently of 350G. HPV-16 E6 sequence variation at base pair 350 did not correlate with HLA genotype or clinical outcome. There was no difference in the distribution of p53 proline and arginine alleles between HPV-16-positive cervical carcinoma patients and local controls, and no influence on clinical outcome; however, there was a trend for an increased frequency of p53 arginine homozygotes among the 350T carcinoma patients.
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Identification of a new promoter in the early region of the human papillomavirus type 16 genome
More LessTranscription of the human papillomavirus type 16 (HPV-16) genome is controlled by several promoters; the P97 promoter is considered to be the main one. An additional promoter has been identified within the E7 ORF as well as an antisense promoter just upstream of the L2 ORF. The significance of these promoters for early and late gene expression and their activity related to cell differentiation is not known in detail. Identification of two new, previously undescribed transcription start sites at nt 542 just upstream of the E7 ORF and at nt 611 within the E7 ORF is reported. The promoter responsible for the start site at nt 542 (P542) was active in SiHa, HeLa and C33A cells. Very low promoter activity was found upstream of the nt 611 start site. The E7 protein has previously been shown to be synthesized from a polycistronic mRNA encoding both the E6 and E7 proteins under the control of the P97 promoter. The data reported in the present paper suggest that promoter P542 may control synthesis of the E7 oncoprotein from a monocistronic mRNA.
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Failure of viral oncoproteins to target the p53-homologue p51A
More LessThe p51/p63/KET proteins were identified based on their strong homology to the tumour suppressor p53 and a related set of proteins termed p73. All these protein species were shown to activate transcription from at least some p53-responsive promoters. To evaluate a possible role of the transcriptionally active splicing variant p51A/p63γ in tumour suppression, we determined whether viral oncoproteins that inactivate p53 might also target p51A. Neither the large T-antigen of simian vacuolating virus 40 (SV40) nor the E6 protein from human papillomavirus type 18 were found to inhibit p51A-mediated transcription, whereas they strongly suppress the activity of p53. Further, SV40 T-antigen directly interacts with p53 but not detectably with p51A. Finally, a cytoplasmic mutant (K128A) of SV40 T-antigen relocalizes p53 from the nucleus to the cytoplasm, but p51A remains in the nucleus when coexpressed with cytoplasmic T-antigen. These results strongly suggest that the inhibitory effect of these viral oncoproteins is specific for p53 and does not measurably affect p51A. Thus, unlike p53, p51A does not appear to be a necessary target in virus-induced cell transformation and may not exert a role comparable to p53 in tumour suppression.
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Induction of apoptosis after switch-on of the hepatitis B virus X gene mediated by the Cre/loxP recombination system
The HBx protein of hepatitis B virus is a multifunctional protein that is implicated in the pathogenesis of hepatocellular carcinoma by regulating gene transcription, causing cell proliferation and, as shown recently, inducing cell death. However, analysis of the effects of HBx in stable cultured cell clones has been hampered because only cell lines that adapted to the effects of HBx were selected during the establishment of cell clones. Here, we describe a system in which transcription of the X gene of hepatitis B virus is switched on by the use of the site-specific Cre recombinase. Two human liver cell lines, HLF and HepG2, were used, the former with a mutant p53 allele and the latter with wild-type p53. The stable cell clones isolated, which carried the X gene in a transcriptionally silent state, were infected with recombinant adenovirus carrying Cre recombinase. Ninety-six hours after adenovirus infection, cell clones that expressed HBx had undergone TUNEL-positive cell death with characteristics of apoptosis. Apoptosis was induced despite concomitant inactivation of the p53 protein as a result of its cytoplasmic translocation by HBx. In contrast, neither the X gene-carrying cells infected with wild-type adenovirus nor various control cells infected with Cre-expressing adenovirus exhibited apoptosis. These results indicate that the expression of HBx protein leads to liver cell apoptosis independently of the p53 pathway. The significance of HBx-induced apoptosis in natural infection is unclear, but it may contribute to the development of hepatitis and serve to spread progeny virus to neighbouring cells while evading the host immune responses.
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Atypical nucleoprotein complexes mediate CRE-dependent regulation of the early promoter of minute virus of mice
More LessThe P4 promoter of the parvovirus minute virus of mice (MVMp) directs transcription of the genes encoding non-structural proteins. We have previously shown that functional upstream CRE elements contribute to both the ras oncogene-dependent activation of promoter P4 and its down-modulation by known activators of cyclic AMP-dependent protein kinase A (PKA). In the present work, the nucleoprotein complexes formed with the P4 CRE elements were characterized with regard to their polypeptide constituents and the nucleotides taking part in the interaction. Atypical interactions, both at the protein–protein and protein–DNA level, were observed, which may be a reflection of the divergence of the parvoviral CREs from the usual consensus. The CRE-mediated regulation of promoter P4 by PKA and Ras is discussed in light of these findings.
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- Insect
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Sequence and organization of the Spodoptera exigua multicapsid nucleopolyhedrovirus genome
The nucleotide sequence of the DNA genome of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV), a group II NPV, was determined and analysed. The genome contains 135611 bp and has a G+C content of 44 mol%. Computer-assisted analysis revealed 139 ORFs of 150 nucleotides or larger; 103 have homologues in Autographa californica MNPV (AcMNPV) and a further 16 have homologues in other baculoviruses. Twenty ORFs are unique to SeMNPV. Major differences in SeMNPV gene content and arrangement were found compared with the group I NPVs AcMNPV, Bombyx mori (Bm) NPV and Orgyia pseudotsugata (Op) MNPV and the group II NPV Lymantria dispar (Ld) MNPV. Eighty-five ORFs were conserved among all five baculoviruses and are considered as candidate core baculovirus genes. Two putative p26 and odv-e66 homologues were identified in SeMNPV, each of which appeared to have been acquired independently and not by gene duplication. The SeMNPV genome lacks homologues of the major budded virus glycoprotein gene gp64, the immediate-early transactivator ie-2 and bro (baculovirus repeat ORF) genes that are found in AcMNPV, BmNPV, OpMNPV and LdMNPV. Gene parity analysis of baculovirus genomes suggests that SeMNPV and LdMNPV have a recent common ancestor and that they are more distantly related to the group I baculoviruses AcMNPV, BmNPV and OpMNPV. The orientation of the SeMNPV genome is reversed compared with the genomes of AcMNPV, BmNPV, OpMNPV and LdMNPV. However, the gene order in the ‘central’ part of baculovirus genomes is highly conserved and appears to be a key feature in the alignment of baculovirus genomes.
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- Plant
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Nonstructural proteins of Tobacco rattle virus which have a role in nematode-transmission: expression pattern and interaction with viral coat protein
More LessRNA 2 of Tobacco rattle virus isolate PpK20 encodes the viral coat protein (CP) and two nonstructural proteins of 40 kDa (‘40K protein’) and 32·8 kDa (‘32·8K’). The 40K protein is required for transmission of the virus by the vector nematode Paratrichodorus pachydermus whereas the 32·8K protein may be involved in transmission by other vector nematode species. An antiserum was raised against the 40K protein expressed in E. coli and used to study the expression and subcellular localization of this protein in infected Nicotiana benthamiana plants. The time-course of the expression of the 40K protein in leaves and roots was similar to that of CP and both proteins were similarly distributed over the 1000 g pellet, 30000 g pellet and 30000 g supernatant fractions of leaf and root homogenates. Using the yeast two-hybrid system, a strong interaction between CP subunits was observed and weaker interactions between CP and the 32·8K protein and between CP and the 40K protein were detected. A deletion of the C-terminal 19 amino acids of CP interfered with the CP–40K interaction but not with CP–32·8K or CP–CP interactions, whereas a C-terminal deletion of 79 amino acids interfered with CP–40K and CP–32·8K interactions but not with the CP–CP interaction. As the C terminus of CP is known to be involved in nematode-transmission of tobraviruses, the data support the hypothesis that interactions between CP and RNA 2-encoded nonstructural proteins play a role in the transmission process.
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Context of the coat protein DAG motif affects potyvirus transmissibility by aphids
More LessPrevious work with tobacco vein mottling virus (TVMV) has established that a highly conserved three amino acid motif, asp-ala-gly (DAG), located near the N terminus of the coat protein (CP), is important for aphid transmission. However, several other potyviruses which have motifs other than DAG are aphid-transmissible. Creation of these motifs in TVMV through site-directed mutagenesis failed to render TVMV aphid-transmissible from infected plants, and the creation of a putative complementary motif in the helper component did not restore transmissibility. In an isolate of tobacco etch virus (TEV) that contains two consecutive DAG motifs separated by a single ala, transmissibility was abolished or reduced by mutations affecting the first motif, whereas mutations in the second motif had little or no effect. In a TEV mutant made non-transmissible due to an altered first motif, substitution of val for ala in the position immediately before the second DAG restored transmissibility, whereas changing val to ala in the location prior to the first DAG resulted in reduced TEV transmissibility. In contrast, a val to ala change in the position preceding the single DAG motif of TVMV did not affect transmission. Creation of another DAG motif at the beginning of the TVMV CP core, in a position where certain other potyviruses have a second DAG motif, did not restore transmissibility. Our results suggest that the mere presence of a DAG motif does not guarantee transmissibility and that the context in which the DAG or equivalent motif is found plays a role in the process.
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Bacteriophage-like particles associated with the gene transfer agent of Methanococcus voltae PS
More LessThe methanogenic archaeobacterium Methanococcus voltae (strain PS) is known to produce a filterable, DNase-resistant agent (called VTA, for voltae transfer agent), which carries very small fragments (4400 bp) of bacterial DNA and is able to transduce bacterial genes between derivatives of the strain. Examination by electron microscopy of two preparations of VTA that were concentrated and partially purified by different methods showed virus-like particles with isometric heads, about 40 nm in diameter, and with 61 nm long tails. These particles co-sedimented with the minute bacteriophage ϕX174 in a sucrose density gradient.
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