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Volume 75,
Issue 4,
1994
Volume 75, Issue 4, 1994
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Transcriptional analysis and genome expression of chicken anaemia virus
More LessStrand-specific riboprobes representative of either strand of the chicken anaemia virus (CAV) replicative form (RF) DNA indicated that only one strand of the RF was transcribed to produce a major 2·0 kb transcript and that the encapsidated DNA strand was of negative sense. Primer extension analysis located a single transcriptional start site at nucleotide position 360 of the CAV sequence. Amplification, cloning and sequencing of the 3′ end of the major transcript revealed the polyadenylation site at nucleotide position 21. Northern blot analysis using a series of genomic probes indicated that the 2·0 kb transcript was devoid of splicing and identified a non-transcribed region of the genome. This non-transcribed region was shown to possess promoter activity, enhancing the expression of the human growth hormone reporter gene in a transient gene expression assay. These observations suggest a simple strategy of genome expression involving a functional polycistronic message.
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Sequence variation in the capsid protein genes of human papillomavirus type 16
More LessWe have cloned and sequenced the L1 and L2 genes from human papillomavirus type 16 (HPV16) DNA- containing cervical cytology samples collected from the U.K. and Trinidad. Samples containing high copy numbers of HPV16 DNA were selected as being likely to contain fully functional virus DNA molecules in an episomal state, rather than in an integrated and possibly altered state. In comparison with the previously published sequence of HPVI6 isolated from an invasive cancer a variety of differences were detected in both L1 and L2. The pattern of changes appears to be different in samples from the two geographic regions. One of the differences (resulting in D at position 202 of the LI protein) reported recently to be functionally important for virus particle assembly was found to occur in all the samples examined. Variations in LI found within known immunoreactive regions or hydrophobic domains should be taken into account in design of prophylactic vaccines for HPV16 based on virus-like particles. All variations within L2 protein were found in hydrophilic domains in the carboxy-terminal half of L2. These positions were highly variable among other types of papillomavirus and are located outside the known L2 immunoreactive region.
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Effect of frameshift mutation in the pre-C region of hepatitis B virus on the X and C genes
More LessWe have previously cloned a mutant hepatitis B virus (HBV) genome which had one thymidine addition in the pre-C region resulting in a frameshift mutation in the pre-C region and fusion of the X and C genes. We constructed plasmids containing serially deleted and/or back-mutated (authentic) pre-C regions to study the effect of the frameshift mutation. COS cells transfected with plasmids containing the frameshifted pre-C region produced a 21K C protein (P21c) but not a 22K partially processed pre-C protein (P22). On the other hand, COS cells transfected with plasmids containing the back-mutated pre-C region produced P22. This result was also observed in HepG2-K8 cells producing the mutant HBV particles. Therefore, the pre-C region of HBV is likely to be non-essential for virus replication. COS cells transfected with the plasmid containing a fused X-C open reading frame (ORF) produced a 40K X-C fusion protein. This X-C fusion protein exerted transcriptional trans-activation. These results suggest that the mutant HBV has a C gene with a defective pre-C region and a fused X-C ORF, and hence cannot synthesize 16K HBeAg (P16e).
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Determination of the 5′ end of the lactate dehydrogenase-elevating virus genome by two independent approaches
More LessWe have determined the 5′ end of the lactate dehydrogenase-elevating virus (LDV) genome (strain LDV-P) using two independent approaches. In one approach, methylmercuric hydroxide-denatured genomic RNA was reverse-transcribed using as primer an oligonucleotide complementary to the 5′ end of open reading frame (ORF) la. The first-strand cDNA was ligated with T4 RNA ligase to an oligonucleotide of which the 3′ end was blocked. The ligated product was amplified by PCR, cloned and sequenced. In the second approach, untreated or decapped genomic RNA was ligated between the 3′ and 5′ ends, reverse-transcribed across the ligation junction and the product was amplified by PCR, cloned and sequenced. Both approaches yielded the same results, indicating that the 5′ leader of LDV-P is 156 nucleotides long, inclusive of the 5′ UAUAACC 3′ sequence involved in the linkage of the 5′ leader to the bodies of the seven subgenomic mRNAs of LDV. The 5′ leader of LDV is about 50 nucleotides shorter than those of the related viruses, equine arteritis virus and Lelystad virus, but at least twice as long as the leaders of the coronaviruses. The finding that untreated LDV RNA was ligated 5′ to 3′ end as efficiently as RNA treated with decapping enzyme suggests that genomic LDV RNA may not possess a 5′ cap but terminates with 5′ phosphoryl-A.
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Hepatitis C virus variants from Nepal with novel genotypes and their classification into the third major group
Five isolates of hepatitis C virus (HCV) RNA from patients with chronic liver disease in Nepal were not classifiable into the known genotypes I/1a, Il/lb, III/2a, IV/2b or V/3a using PCR with type-specific primers deduced from the HCV core gene. Their nucleotide sequences were determined for the 5′ -terminal 1·5 ldlobases and 3′ -terminal 1·2 kilobases, covering 30% of the entire genome, and compared with each other and with reported sequences of HCV isolates of various genotypes. They were more similar to a reported HCV isolate (NZL1) of genotype V/3a (in 81·6 to 84T % of their nucleotides and 85·7 to 88·7% of the deduced amino acid sequence) compared with the genotypes I/1a to IV/2b (in 69·3 to 74·7% and 72·3 to 77·4%, respectively). Hence they were considered to be variants of the third major group (group 3). The five HCV isolates shared 81·3 to 85·2% of nucleotide sequence and 85·4 to 89·3 % of deduced amino acid sequence. Thus they were substantially different from each other. One of them was classified as genotype VI/3b due to an 88·2 % similarity in nucleotide sequence to that of the reported HCV isolates of this genotype, whereas the remaining four were classified into provisional genotypes 3c, 3d, 3e and 3f. These HCV variants have evolved and remained in Nepal, and have not been observed in the other areas of the world.
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Common distribution of antigenic determinants and complementation activity on matrix proteins of two vesicular stomatitis virus serotypes
More LessTo compare the antigenic and functional domains of the matrix (M) proteins of vesicular stomatitis virus (VSV) serotypes Indiana (VSV-Ind) and New Jersey (VSV-NJ), deletion mutants and chimeras were cloned in pBSM13 and expressed as in-frame lacZ fusion proteins in Escherichia coli. Non-cross-reactive monoclonal antibodies directed to the two antigenically distinct M proteins were tested by Western blot analysis to map three epitopes of VSV-Ind M protein and four epitopes of VSV-NJ M protein. Epitope 1 of the VSV-Ind M protein and epitope II of the VSV-NJ M protein both mapped to the highly basic N-terminal 34 amino acids of each homotypic M protein. Epitopes 2 and 3 of the VSV-Ind M protein and epitopes III and IV of the VSV-NJ M protein mapped to a region spanning amino acids 35 to 74. Epitope I of the VSV-NJ M protein mapped to a region between amino acid 75 and the C terminus. The similarity in location of the serotypically unique antigenic determinants of the two M proteins suggested that they may have a common functional domain. This hypothesis was substantiated by the finding that the two M proteins and various chimeras expressed in CV-1 cells by a recombinant vaccinia virus system were able to rescue M gene temperature-sensitive mutants of both VSV serotypes.
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Role of virion M2 protein in influenza virus uncoating: specific reduction in the rate of membrane fusion between virus and liposomes by amantadine
More LessThe anti-influenza virus drug amantadine was shown to reduce the rate of fusion of liposomes with influenza A viruses whose replication is inhibited by this drug. The fusion with amantadine-resistant viruses was unaffected. Experiments with reassortant and mutant viruses showed that this effect was linked to the M2 protein and not to the haemagglutinin of the virus. The proton ionophore monensin, on the other hand, substantially increased the rate of fusion of the viruses tested. These results indicate that the kinetics of virus-liposome fusion can be modulated by the virus M2 protein, the target of amantadine action, and it is postulated that the M2 ion channel functions by transporting protons into the virion interior and facilitating virus uncoating.
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Nucleotide sequence of the carlavirus associated with blueberry scorch and similar diseases
We have synthesized and mapped a library of cDNA clones representing the RNA genome of a strain of blueberry scorch carlavirus (BBScY) associated with a disease known locally, in New Jersey, U.S.A., as Sheep Pen Hill disease. The nucleotide sequence of that strain was determined to be 8514 residues, excluding the poly(A) tail. In addition, cDNA clones representing the 3′ terminus of another strain of the virus from the same field were synthesized, mapped and sequenced. The overall identity between sequences of these two strains was approximately 90% spanning the 1634 residue overlap, confirming their identity as distinct strains and not simply different isolates of a single strain. Finally, the coat protein gene of a distinct strain of the virus, isolated from plants with blueberry scorch disease in the Puyallup Valley in Washington State, U.S.A., was cloned from total cDNA by PCR. Sequence analysis revealed that the strain from Washington was more divergent from the two New Jersey strains than they were from each other. Comparisons of these sequences with other carlavirus sequences indicated that BBScV is more closely related to lily symptomless virus and potato virus S than to potato virus M, Helenium virus S, carnation latent virus or poplar mosaic virus. BBScV and potato virus M shared approximately 54 % nucleotide sequence identity overall.
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Simultaneous regulation of tomato golden mosaic virus coat protein and AL1 gene expression: expression of the AL4 gene may contribute to suppression of the AL1 gene
More LessThe tomato golden mosaic virus (TGMV) coat protein and AL1 genes are located in opposite directions on either side of an intergenic region. To enable the effects of the AL1, AL2 and AL3 gene products on expression of the coat protein and AL1 genes to be studied simultaneously, a plasmid was constructed, containing the intergenic region linked on one side to a 5′ -terminal portion of the AL1 gene fused to a β -glucuronidase (GUS) reporter gene (to replace most of the AL1 gene) and on the other side to a neomycin phosphotransferase (NEO) reporter gene (to replace the coat protein gene). This GUS-NEO plasmid was mixed with plant expression plasmids containing the AL1, AL2 or AL3 coding regions, the DNA was transformed into Nicotiana benthamiana protoplasts and GUS activities and NEO protein levels were measured. Control transformations were carried out with the GUS-NEO plasmid mixed with the AL1, AL2 or AL3 plasmids in which mutations were introduced to prevent translation of the open reading frames (ORFs). The results showed that transactivation of the coat protein gene by the AL2 gene product and suppression of the AL1 gene by the expression of AL1 DNA (both reported previously) can occur simultaneously. It was also shown that expression of AL4, a small ORF contained within AL1 DNA but in a different reading frame, as well as expression of ORF AL1, can cause significant suppression of AL1 gene expression. Neither the AL1 nor the AL3 gene products affected the expression of the coat protein gene.
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A stable 463 nucleotide variant of citrus exocortis viroid produced by terminal repeats
More LessAn unusual variant of citrus exocortis viroid (CEV) was detected when an inoculum source from Gynura auran- tiaca D.C. was used to infect a hybrid tomato (Lycopersicon esculentum Mill, × L. peruvianum). The 92 nucleotide larger variant, CEV D-92, which displayed the characteristic circular and linear viroid structural forms, contained two repeated sequences spanning the V and T2 domains. A dramatic moderation of symptom expression in Gynura accompanied the incorporation of these repeated sequences. A comparison of the sequence and structure of CEV D-92 with coconut cadang-cadang viroid revealed similarities in the regions generating the naturally occurring terminal repeats suggesting a possible preferred site for RNA recombination between viroids.
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