- Volume 75, Issue 11, 1994
Volume 75, Issue 11, 1994
- Animal
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Cervical/vaginal dysplasias of Transgenic Mice Harbouring Human Papillomavirus Type 16 E6-E7 Genes
More LessHalf of the female transgenic mice harbouring human papillomavirus type 16 (HPV-16) E6-E7 genes under control of the mouse mammary tumour virus promoter, developed malignant tumours, including salivary gland carcinomas, lymphomas and skin histiocytomas. Although the E6-E7 genes are aetiological factors for human anogenital carcinoma, the transgenic mice produced no tumours in the anogenital tract. We investigated cytological and histological changes in the anogenital tract of the same transgenic mice. Seventeen (77 %) of 22 transgenic mice developed dysplastic and/or hyperplastic changes in the cervix and vagina. HPV-16 E6-E7 mRNA signals were observed in the genital lesions, while they were not detected in the normal cervices and vaginas of transgenic mice and control mice by RNA in situ hybridization analysis. RNA/PCR analysis using poly(A)+ RNA showed that only a full- length E6-E7 RNA was expressed in three scraped cell samples from dysplasia, whereas full-length and spliced E6-E7 transcripts were in three cell samples from dysplasia/hyperplasia. These results suggest that expression ofboth E6 and E7 genes of HPV-16 is important for inducing dysplastic and hyperplastic changes in the genital epithelium.
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DNA-binding Studies of the Epstein-Barr Virus Nuclear Antigen 2 (EBNA-2): Evidence for Complex Formation by Latent Membrane Protein Gene Promoter-binding Proteins in EBNA-2-positive Cell Lines
The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA- 2) protein is essential for the immortalization of human primary B cells by EBV. EBNA-2 trans-activates cellular and viral genes like CD23, c-fgr, latent membrane protein 1 (LMP1) and terminal protein 1 (TP1). Transactivation of the TP1 promoter and of the BamHl C promoter has already been investigated in detail and appears to be mediated via protein-protein interactions and not by direct binding of EBNA-2 type A (of EBV type 1) to the DNA. EBNA-2 is able to trans-activate the expression of the LMP gene in several cell lines. Various reports have delineated the cis-acting elements of the LMP promoter through which EBNA-2 mediates transactivation. To determine whether EBNA-2 also trans- activates the LMP promoter by protein-protein interactions, we performed a series of gel retardation assays and competition experiments with LMP promoter fragments of different sizes. We determined that the protein-binding region on the LMP promoter was within a 42 bp fragment encompassing nucleotides −135 to − 176 relative to the LMP transcriptional start site. None of the DNA fragments investigated indicated interaction of EBNA-2 with the DNA via protein- protein interactions. No significant differences between EBNA-2-positive and EBNA-2-negative nuclear extracts could be seen in the gel retardation assay under conditions that clearly showed binding of EBNA-2A to the TP1 promoter. However, analysis of sucrose gradient fractions in the gel retardation assay provided evidence that the LMP promoter-binding proteins form a complex of higher Mr in EBNA-2-positive cell extracts. These complexes were destroyed by detergent. We deduce from these results that EBNA-2-positive cells might indeed contain specific complexes bound to the LMP promoter which are, however, too labile to be detected in a standard gel retardation assay.
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The Human Cytomegalovirus UL100 Gene Encodes the gC-II glycoproteins Recognized by Group 2 Monoclonal Antibodies
More LessIn human cytomegalovirus (HCMV) the envelope glycoprotein complexes designated gC-II contain two immunologically and biochemically distinct glycoproteins. Monoclonal antibodies (MAbs) recognizing the gC-II glycoproteins have been divided into two groups based on the M r of the glycoproteins they recognize. We have now identified the HCMV UL100 gene as the gene encoding the gC-II glycoprotein recognized by the Group 2 MAbs. To do this, gC-II complexes were immunoaffinity purified and cleaved with cyanogen bromide (CNBr). CNBr peptides were separated by reverse phase high performance liquid chromatography (RPHPLC). Amino acid sequences which matched sequences found in the protein encoded by the HCMV UL100 gene were obtained from three purified peptides. To confirm the assignment we made synthetic peptides using amino acid sequence from the carboxyl terminus of the protein encoded by the UL100 gene. These peptides were used to make murine antibodies. The anti-UL100 antibodies immunoprecipitated gC-II complexes and were reactive with gC-II glycoproteins recognized by Group 2 MAbs in Western blotting. Several overlapping UL100 fusion proteins were expressed in E. coli. Only one of these fusion proteins was recognized by gC-II Group 2 MAbs. None of these UL100 fusion proteins were recognized by gC-II Group 1 MAbs. These data showed that the UL100 gene encoded the gC-II glycoprotein recognized by the Group 2 MAbs and that the epitope recognized by these antibodies was located between amino acids 315 to 372 at the carboxyl terminus.
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Absence of Varicella-zoster Virus (VZV) Glycoprotein V Does not Alter Growth of VZV in vitro or Sensitivity to Heparin
More LessVaricella-zoster virus (VZV) encodes at least five glycoproteins, gpl to gpV. VZV gpV, M r 100K to 110K, is the product of VZV open reading frame (ORF) 14. VZV gpV is homologous to herpes simplex virus gC and pseudorabies virus gill. To determine whether gpV is required for viral replication, we inserted a stop codon after the fifteenth codon of the ORF 14 gene in a cosmid containing the gene. Transfection of human melanoma cells with the cosmid containing the mutant ORF14 gene and three other cosmids resulted in the production of infectious VZV. Immunoprecipitation indicated that the mutant virus did not express gpV. VZV that did not express gpV grew at the same rate as parental virus and was inhibited by heparin to a similar extent. The pattern of inhibition by heparin of the gpV mutant was similar to that reported for a herpes simplex virus mutant that does not contain gC, but different from that described for a pseudorabies virus mutant devoid of gill. These results indicate that VZV gpV is not required for viral replication in vitro.
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Glycoprotein gE-negative Pseudorabies Virus has a Reduced Capability to Infect Second- and third-order Neurons of the Olfactory and Trigeminal Routes in the Porcine Central Nervous System
We investigated the spread of glycoprotein gE (gE)- negative pseudorabies virus (PRV) and its rescued ‘wild- type’ strain into and within the central nervous system (CNS) of 3- and 10-week-old pigs. This is the first study that demonstrates PRV invasion of the porcine CNS via the synaptically linked neurons of the olfactory and trigeminal routes and that demonstrates the role of gE in this invasion. After intranasal inoculation with high doses of virus, gE-negative PRV replicated less efficiently in peripheral tissues. The titres of the gE-negative virus in the oropharyngeal mucosa, olfactory epithelium, draining lymph nodes and trigeminal ganglion were approximately 100-fold lower in 3-week-old pigs and 10fold lower in 10-week-old pigs than titres of the ‘wild- type’ virus. In contrast to the ‘wild-type’ virus, titres of the gE-negative virus were very low or undetectable in the olfactory bulb, brain stem and other tissues of the CNS. Viral antigen of rescued ‘wild-type’ PRV and of gE-negative PRV was detected immunohistochemically in the olfactory epithelium and in neurons of the trigeminal ganglion, and also in the olfactory and trigeminal axons leading towards the CNS. But, in contrast to ‘wild-type’ virus, no viral antigen of the gEnegative virus was detected in second- or third-order neurons in the olfactory bulb or in the brain stem. We conclude that gE-negative PRV can infect first-order neurons of the olfactory and trigeminal routes and is able to spread via their axons towards the CNS. Yet, gEnegative PRV has a greatly reduced capacity to infect second- or third-order neurons. Finally, we report lateral spread of ‘wild-type ’ PRV in the trigeminal ganglion, i.e. nonsynaptic transport from neuron to neuron. Possible mechanisms that could explain the reduced levels of the gE-negative virus in the CNS are discussed.
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The Gene Downstream of the gC Homologue in Feline Herpes Virus Type 1 is Involved in the Expression of Virulence
Feline herpesvirus type 1 (FHV-1) mutants were constructed, carrying a β-galactosidase marker gene integrated into the region downstream of the gene encoding the homologue of glycoprotein C (gC) of herpes simplex virus type 1. In cell culture, no differences in replication were observed between mutants and the parent FHV-1 strain. However, in experimentally infected cats, mutants caused fewer clinical signs after oronasal administration although they replicated to the same extent as the parental strain. Sequence analysis in the region of the UL segment surrounding the insertion site revealed an open reading frame (ORF 2) encoding a putative polypeptide of 2IK. RNA analysis indicated a corresponding transcript of 0·8 kb that was detected late after infection of cells in culture. This particular UL locus downstream of the gC gene has not been thoroughly investigated in any of the herpesviruses. The putative gene product showed only limited evolutionary conservation since similarity could be found only with the assumed homologue of equine herpesvirus type 1. Further characterization of this newly identified FHV-1 gene involved in virulence may provide insight into the development of disease owing to herpesvirus infection.
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Identification and Characterization of the Protein Product of Gene 71 in Equine Herpesvirus 1
More LessEquine herpesvirus 1 (EHV-1) strain Ab4 gene 71 is predicted to encode a primary product with a M r of 80·1K. We have previously constructed a deletion/lacZ insertion mutant, ED71, and demonstrated that gene 71 is dispensable for growth of virus in cell culture. We have now constructed a gene 71 revertant, Re71. To identify and characterize the product of gene 71, we produced a specific antiserum, anti-71, against a β-galactosidase fusion protein containing the carboxy terminus of the gene 71 polypeptide. Using the anti-71 serum, mutant ED71 and the revertant Re71, we have demonstrated that gene 71 encodes a 192K polypeptide. Experiments with glycosylation inhibitors revealed that the protein product of gene 71 is N-glycosylated and heavily O-glycosylated. When the 192K polypeptide is synthesized in the presence of monensin, the M r of the polypeptide is reduced to 80K, the predicted unmodified M r of the gene 71 polypeptide. The gene 71 product is found in virions and L particles in a fully processed form that runs as a diffuse band in electrophoresis, with a M r in excess of 200K. Immunofluorescence and virion surface labelling experiments showed that the polypeptide product of gene 71 is located on cellular membranes and the virion envelope. A time course of infection confirmed that gene 71 is regulated as a leaky late gene in infected cells. Finally, using wild-type EHV-1 Ab4, mutant ED71, revertant Re71 and two antibodies (P19 against EHV-1 glycoprotein gp300, and anti-71) we conclusively demonstrated that gene 71 encodes gp300. This contradicts published results with P19 alone, which indicated gp300 was the product of EHV-1 gene 28.
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Role of the Carboxy Terminus of Herpes Simplex Virus Type 1 DNA Polymerase in its Interaction With UL42
More LessSeveral recent reports implicate sequences at or near the C terminus of the catalytic subunit (POL) of herpes simplex virus type 1 (HSV-1) DNA polymerase in its interaction with the accessory protein UL42. We have investigated further the involvement of this region by three different approaches: anti-idiotype antibodies, a competition ELISA and inhibition of the interaction by peptides. Antibodies raised in rabbits to peptides corresponding to regions of POL all reacted in Western blots with POL. Surprisingly, the sera raised against C-terminal peptides (amino acids 1221 to 1235 and 1224 to 1235) also reacted with UL42. The UL42 reactivity was shown to be due to the presence of anti-idiotype antibodies, providing direct evidence for complementarity of the structure of the extreme C terminus of POL to a region of UL42. To measure the contribution of the C terminus of POL to UL42 binding we developed a competition ELISA using POL, a truncated polymerase lacking the carboxyl-terminal 27 amino acids (POLdl) and UL42. UL42 binding to immobilized POL was inhibited approximately four times more effectively by competition, in solution, with POL than with POLdl, indicating that the C-terminal 27 amino acids of POL are responsible for at least 75 % of the binding energy. A peptide corresponding to these 27 amino acids (residues 1209 to 1235) inhibited both the POL-UL42 interaction and the stimulation of POL by UL42 and did so more effectively than peptides corresponding to amino acids just away from the C terminus (residues 1195 to 1223 and 1177 to 1195).
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Nucleotide Sequence of the Polyhedrin Gene Region of Helicoverpa zea Single Nucleocapsid Nuclear Polyhedrosis Virus: Placement of the Virus in Lepidopteran Nuclear Polyhedrosis Virus Group II
More LessThe polyhedrin gene (polh) of Helicoverpa zea single nucleocapsid nuclear polyhedrosis virus (HzSNPV) was identified and shown by sequence analysis of the iscoRI I genomic fragment to encode a 246 amino acid polypeptide that has greater than 80 % sequence identity to known polyhedrins. It is preceded by an AT-rich region containing the conserved late promoter motif TAAG, which was identified as a transcription start point. Downstream ofpolh there were several similarities in genome arrangement to other nuclear polyhedrosis viruses (NPVs). These include open reading frame (ORF) 8, immediately downstream of polh, encoding a 412 amino acid protein with multiple tandem proline residues, which is homologous to ORF8 (ORF1629) of Autographa califomica multiple nucleocapsid NPV. Phylogenetic analysis of the polh gene region shows that HzSNPV is a member of the previously described lepidopteran NPV group II and that it is most closely related to polh of the NPVs of Malacosoma nuestria, Spodoptera littoralis, Orgyia pseudotsugata (single nucleo- capsid-type virus) and Buzura supressaria.
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DNA Sequence and Transcriptional Analysis of the Simian Varicella Virus Glycoprotein B Gene
More LessThe varicella-zoster virus (VZV) glycoprotein B (gB) is a major viral antigen which elicits immunity and neutralizing antibodies. In this study, the genomic map position and DNA sequence of a simian varicella virus (SW) homologue of the VZV gB gene was identified and the transcript analysed. A 32P-labelled VZV gB DNA probe hybridized to a subclone of the SW BamHl B restriction endonuclease fragment indicating the fine map position of SW DNA sequences homologous to the VZV gB gene. The SW gB DNA sequence was determined and analysis revealed a 2751 base pair open reading frame (ORF) with 71·1% identity to the VZV gB gene and 53·8% identity to the herpes simplex type 1 gB gene. The SW gB ORF encodes a 916 amino acid polypeptide with a predicted molecular mass of 104K. The deduced SW and VZV gB polypeptides share 78·9 % amino acid identity and predicted TV-linked glycosylation sites, cleavage sites and transmembrane regions. 32P-labelled SW gB DNA and RNA probes hybridized to a 3·5 kilobase SW polyadenylated transcript. Primer extension experiments identified transcript start sites for the SW and VZV gB genes and permitted a comparison of the sequences upstream of the SW and VZV gB ORFs. The SW and VZV gB promoter elements are similar in content and align closely. The VZV gB transcript start site suggests a gB polypeptide initiation site which is inconsistent with the previously reported ATG start codon.
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Delineation of a Sequence Required for Nuclear Localization of the Protein Encoded by Varicella-zoster Virus Gene 61
More LessAll characterized alphaherpesviruses encode a protein whose N-terminal region contains a novel zinc-binding motif, the C3HC4 domain. Homology between the different proteins is in general limited to key residues in this domain. In order to identify a separate landmark site in the C3HC4 protein encoded by varicella-zoster virus gene 61, namely the region required for nuclear localization, we have analysed a range of mutants in transient expression and immunofluorescence experiments. A basic region (RGAKRR) at residues 387 to 392 was found to be required for nuclear localization, and residues 390 and 391 were critical.
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Identification, Sequencing and Expression of the Glycoprotein L Gene of Murine Cytomegalovirus
DNA sequence analysis of the genome of the Smith strain of murine cytomegalovirus (MCMV) revealed an open reading frame (ORF) with amino acid sequence identity to glycoprotein L (gL) of other herpesviruses. The ORF is 822 bp in size and has the capacity to encode a protein of 274 amino acids. It has significant identity with the gL genes of human CMV and human herpesvirus 6. The coding sequence of the gL gene of MCMV strain K181 was also determined, and expressed in Escherichia coli as a fusion protein with glutathione S- transferase using the pGEX expression system. Two antibody-binding regions were identified on the basis of the reactivity of a series of truncated gL constructs with anti-MCMV antibodies. One was mapped to residues 1 to 38 and the other between residues 230 and 274. Polyclonal antibodies specific to gL were raised against the lull-length gL fusion protein. The antisera were shown to react with a 46K protein present in purified virions by Western blotting. Treatment of purified virions with endoglycosidase-H or -F resulted in reductions in M r of the 46K species to 42K and 3IK, respectively. The antisera did not exhibit any neutralizing activity in a plaque reduction assay.
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Expression of the Epstein–Barr Virus Envelope Fusion Glycoprotein gp85 Gene by a Recombinant Baculovirus
More LessThe gp85 envelope glycoprotein of Epstein-Barr virus (EBV) has a role in the molecular mechanism of infection, enabling fusion between the viral and host cell envelopes, a role in common with the homologous gH glycoproteins in other herpesviruses. A glutathione S- transferase bacterial fusion protein (GST85N-S) was generated, containing 178 amino acids from the C terminus of gp85 and including a known gp85 linear epitope. A panel of EBV-positive human antisera contained no antibodies to linear epitopes presented on the purified GST85N-S protein, indicating that primary protein structure in this region of gp85 is not a B cell target. This bacterial fusion protein was used to raise a rabbit monospecific polyclonal antiserum capable of detecting gp85 in a Western blot. The majority of recombinant baculovirus-expressed gp85 obtained from cell extracts prepared with SDS appeared on Western blots as heterogeneous high M r protein aggregates and consistently included 84K, 81K and 70K bands. Recombinant gp85 aggregation was increased by boiling the sample prior to gel electrophoresis. The 84K and 81K proteins were completely sensitive to endoglycosidase H treatment, indicating that these glycosylated species did not undergo further post-translational processing. Immunofluorescence studies revealed that recombinant gp85 was not transported to the insect cell surface. It reacted only with antibodies recognizing denatured gp85 and not with antibody to native gp85. Therefore expression of the gene encoding gp85, BXLF2, alone in the baculovirus expression system is insufficient for the synthesis of a correctly transported, processed, folded and antigenically native form of recombinant gp85.
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Molecular Determinants of the V3 Loop of Human Immunodeficiency Virus Type 1 Glycoprotein gp120 Responsible for Controlling Cell Tropism
More LessWe and others have identified the major determinant of cell tropism in human immunodeficiency virus type 1 (HIV-1) as the V3 loop of glycoprotein gpl20. We have conducted a detailed study of two molecularly cloned isolates of HIV-1, HIVJR-CSF and HIVNL4-3, that differ in their tropism for immortalized CD4+ cell lines, by constructing a series of site-directed mutations within the V3 loop of HIVJR-CSF based on the sequence of HIVNL4-3. The phenotypes of these mutants fall into two classes, those which are viable and those which are not. A spontaneous mutant with significantly altered growth properties was also recovered and found to have an additional single amino acid change in the V3 loop sequence. The carboxy-terminal β-strand part of the V3 loop is the major determinant of cell tropism. However, the results presented here indicate that the functional role of the V3 loop sequences can only be interpreted properly in the context of the original gpl20 backbone from which they were derived. These findings show that over-simplistic interpretation of sequence data derived from unknown mixtures of HIV variants in infected persons may be highly misleading.
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Inhibition of Human T Cell Leukaemia Virus Type I Long Terminal Repeat Expression by DNA Methylation: Implications for Latency
More LessHuman T cell leukaemia virus type I (HTLV-I) provirus DNA was found to be methylated in patients with adult T cell leukaemia. We have therefore examined the possibility that DNA methylation might contribute to HTLV-I latency. In vitro methylation of HTLV-I long terminal repeat (LTR)-chloramphenicol acetyltransfer- ase or LTR-Luciferase constructs at eight HpaII sites, a subset of the eukaryotic methylation site CpG, resulted in a three- to fourfold inhibition of transcription in transfected cells. Inhibition of transcription by methylation of all CpG methylation sites using Sxsl methylase was much more pronounced (50- to 80-fold). As partial methylation of the LTR showed, methylation of the promoter region was responsible for most of the effect. Whereas cellular stimulation by a combination of phorbol 12-myristate 13-acetate and Tax was able to reverse the HpaII methylation effect, the inhibition by SsjI methylation was not suppressible under these conditions. The results are in line with a possible function of DNA methylation in HTLV-I latency.
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Reovirus λ1 Protein: Affinity for Double-stranded Nucleic Acids by a Small Amino-terminal Region of the Protein Independent From the Zinc Finger Motif
More LessThe reovirus λ1 protein, a major component of the inner capsid, has been shown to exhibit an affinity for dsRNA in a ‘Northwestern’ filter-binding assay. In the present study it was demonstrated that the protein can bind dsDNA as well as dsRNA. A bacterial expression system was used to study the protein region able to bind to nucleic acids. The amino-terminal 187 amino acids of λ1 were fused to the bacterial maltose-binding protein and shown to be sufficient for binding to nucleic acids. The putative zinc finger present on λ1 is not encompassed in this fragment of the protein. Site-directed mutagenesis also indicated that this zinc finger motif is unrelated to binding. In contrast, mutations introduced in a previously suggested nucleotide-binding motif almost completely prevented the binding. These data indicate that the amino-terminal end of λ1, encompassing its nucleotide-binding motif, is involved in the affinity of this protein for nucleic acids.
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Rotavirus Causes Selective Vimentin Reorganization in Monkey Kidney CV-1 Cells
More LessThe effect of rotavirus infection on cytoskeletal organization was examined in cultured African green monkey kidney (CV-1) cells. Rhesus rotavirus caused significant and selective changes in the organization of the vimentin filament network without having any effect on microtubules or actin. Double-immunofluorescence studies showed that at 6 h post-infection, and in the absence of cytopathic effect, the normal arrays of vimentin fibres radiating from multiple sites around the nucleus were lost. Vimentin fibres became irregularly distributed in the cytoplasm and were totally disrupted in the later stages of infection. Vimentin reorganization occurred independent of extracellular Ca2+ levels.
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Association of Serine in Position 1124 of Hantaan Virus Glycoprotein With Virulence in Mice
More LessHantaan virus (HV) of the genus Hantavirus causes a fatal disease in suckling mice following intraperitoneal or intracerebral infection. HV cl-1, which was obtained from the 76–118 strain of HV by growth in Vero E6 cells, exhibited high mortality rates in mice whereas mice infected with HV cl-2 survived without any clinical signs. To determine the molecular basis for the marked difference in virulence, we compared the nucleotide sequences of the large (L), medium (M) and small (S) segments of HV cl-1 genome with those of HV cl-2 and found that there was only one predicted amino acid substitution. This amino acid substitution was in position 1124 of the glycoprotein encoded by the M genome segment, in which serine in HV cl-1 was replaced by glycine in HV cl-2. Although there were several nucleotide and amino acid differences between the parental 76–118 strain and HV cl-1, the serine in position 1124 of the glycoprotein was common to the pathogenic parent and the pathogenic mutant. These results suggest that this substitution may be responsible for the virulence of this hantavirus.
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Full-length Sequence of the Genome of Hepatitis C Virus Type 3a: Comparative Study With Different Genotypes
Hepatitis C virus (HCV) type K3a (type 3a), which represents a minor genotype in Europe, the U.S.A. and Asia, appears to be significantly distributed throughout Australia and Brazil. We amplified the HCV-K3a/650 genome by reverse transcription polymerase chain reaction in ten overlapping fragments and determined the nucleotide sequences. The total sequence was 9454 bases in length and contained an open reading frame of 3021 amino acids, which is 10 or 11 amino acids longer than in HCV type 1 and 12 amino acids shorter than the sequence of type 2. These differences were due to the different lengths of both the putative envelope protein E2 and the NS5A regions, whose nucleotide lengths differ between types 1 and 2 also. Phylogenetic analysis of the putative core region and a portion of NS5B encoding the Gly-Asp-Asp motif indicated that HCV- K3a closely matched the corresponding type 3a group. The deletion and addition of amino acids in both E2 and NS5A may be associated with their pathobiological features.
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Variation in Biological Properties of Cauliflower Mosaic Virus Clones
More LessInfectous clones were prepared from virion DNA of three cauliflower mosaic virus (CaMV) isolates, 11/3, Xinjiang (XJ), and Aust, to investigate pathogenic variation in virus populations. Of 10 infectious clones obtained for isolate 11/3, four pathotypes were identified, each producing symptoms in turnip that differed from those of the 11/3 wild-type. Virus from two clonal groups of 11/3 was transmissible by aphids whereas that from two others was not. Of the five infectious clones obtained from isolate XJ, two groups were identified, one of which differed symptomatically from the wild- type. Only one infectious clone was obtained from isolate Aust and this had properties similar to the wild- type. Restriction enzyme polymorphisms were found in some clonal groups and these correlated with symptoms. Other groups with different pathogenic properties could not be distinguished apart by restriction site polymorphisms. Further variation was observed in the nucleotide sequences of gene II (coding for aphid transmission factor) from these viruses as compared with other CaMV isolates. In the aphid non-transmissible clones of isolate 11/3, one had a Gly to Arg mutation in gene II similar to that of other non-deleted non-transmissible CaMV isolates. The second had a 322 bp deletion at the site of a small direct repeat similar to that of isolate CM4-184 although occurring in a different position. The gene II deletion of isolate 11/3 produced a frame-shift that separated genes II and III by 60 bp. Most CaMV clones studied remained biologically stable producing similar symptoms during subsequent passages. However, one clone (11/3–7) produced two new biotypes during its first passage suggesting that it was relatively unstable. Our results show that wild-type populations of CaMV contain a range of infectious genome variants with contrasting biological properties and differing stability. We suggest that a variety of significant viral phenotypic changes can occur during each infection cycle resulting from relatively small genome changes.
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