- Volume 78, Issue 4, 1997
Volume 78, Issue 4, 1997
- Articles
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Role of p53 mutation in polyomavirus-induced tumorigenesis
Small DNA tumour viruses, such as simian virus 40 (SV40), papilloma viruses and adenoviruses, encode proteins that form complexes with and inactivate the p53 and retinoblastoma (RB) proteins. This convergent evolution reflectsthe common need of these viruses to inactivate these two important regulators of cell cycle progression and cell survival. Polyomavirus, a close relative of SV40, is different. Its large T protein complexes only with RB, not with p53. We have examined whether this is compensated by the frequent appearance of p53 mutations in polyomavirus-induced tumours. We tested the p53 status of 15 polyomavirus-induced sarcomas. Two sarcomas were p53-negative while six carried mutant p53. Another six sarcomas expressed low levels of wild-type p53. One tumour expressed high levels of wild-type p53 protein as shown by DNA sequencing and immunofluorescence staining. MDM2 amplification was not detected in any of the tumours, but Northern blotting showed that MDM2 was overexpressed in at least two tumours that expressed wild-type p53 and in one tumour that expressed both wild-type and mutant p53. Treatment with the DNA-damaging agent mitomycin C caused p53 protein accumulation followed by induction of MDM2 and WAF1/p21 mRNA in four of the tumours expressing wild-type p53, indicating that p53-mediated transcriptional activation was unaltered in these tumours. However, p53-mediated transactivation of WAF1/p21 was impaired in the wild-type p53-expressing tumours that expressed elevated levels of MDM2. These results demonstrate that p53 mutation and inactivation are frequently but not invariably involved in polyomavirus-induced tumorigenesis.
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Analysis of the interaction between human papillomavirus type 16 E7 and the TATA-binding protein, TBP
More LessThe E7 protein encoded by human papillomavirus type 16 shows transforming and immortalizing activities which are mediated, in part, through the interaction of the viral oncoprotein with the pRB protein family. This interaction is not solely responsible for E7 function, however, and other properties of E7, such as the interaction with basal transcription factors such as TBP, are likely to be of importance. We show here that three regions of the viral protein contribute to the interaction between E7 and TBP; the pRB-binding domain, the casein kinase II phosphorylation region and the C-terminal dimerization domain. Mutations within each region reduced the interaction of E7 with TBP in vitro, and simultaneous alterations within each of these regions completely abrogated binding. Unlike the pRB interaction, the association of E7 with TBP was enhanced following phosphorylation of E7 by casein kinase II, demonstrating a functional significance for phosphorylation of the viral protein.
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Characterization of the DNA-binding activity of the E1 and E2 proteins and the E1/E2 complex of human papillomavirus type 33
More LessThe E1 and E2 proteins of papillomaviruses are essential for the initiation of viral DNA replication. We have purified the E2 protein of human papillomavirus type 33 (HPV-33) by immunoaffinity chromatography. The purified E2 protein bound with high affinity to all four consensus binding sites of HPV-33 (Kd ≌ 2×10 10 M). A putative E2 binding site differing at one position in the second stem of the palindrome was not bound by E2. The E1 protein of HPV-33 purified by affinity chromatography using glutathione S-transferase as tag displayed specific DNA-binding activity in footprint analyses protecting HPV-33 nucleotides 7896 to 7909/1 to 18 from DNasel digestion. Hypersensitive sites at position 6 on the sense and position 1 on the antisense strand were observed in the middle of the protected region. An E1/E2 complex protected the E1 binding site and E2 binding sites from DNasel digestion suggesting that both proteins retain DNA-binding activity in the complex.
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Serological and T-helper cell responses to human papillomavirus type 16 L1 in women with cervical dysplasia or cervical carcinoma and in healthy controls
More LessIn a cross-sectional study we have investigated serological and T-helper (Th) cell responses to human papillomavirus type 16 (HPV-16) L1 in women with HPV-16 related diseases and related them to cervical histology and HPV DNA status. Using a virus-like particle (VLP) based ELISA to detect antibodies to the HPV-16 L1 capsid protein, 45% (33/73) of women with cervical dysplasia, 40% (2/5) of women with cervical cancer, 36% (4/11) of healthy adult female controls and 6% (2/35) of healthy children were found to be seropositive. Amongst women with cervical dysplasia, the highest levels of seropositivity were found in those who were HPV-16 DNA positive (60%, 15/25) or positive for any of the ‘high-risk’ HPV types, 16/18/33 (58%, 18/31), when compared with those with HPVtype ‘X’ (25%, 5/20) or with healthy children (6%, 2/35; P < 0·05 for all comparisons). There was a trend for women with cervical dysplasia to show an increased level of seropositivity with increasing grade of lesion. There was no direct correlation found between seropositivityand Th cell responses in all groups studied. However, a combined analysis of each individual’s Th and B cell responses suggests that a Th1 pattern of response is predominant amongst healthy adult controls (80% of responders) but reduced in women with cervical dysplasia (55% of responders). A trend towards a decrease in Th1 type responses was also noted with increasing grade of dysplastic lesion. These findings provide further evidence for the importance of the Th response in the control of genital HPV infections.
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Tropic determinant for canine parvovirus and feline panleukopenia virus functions through the capsid protein VP2
More LessCanine parvovirus (CPV) can productively infect canine and feline cell lines whereas feline panleukopenia virus (FPV) is restricted to the latter. The major determinants of tropism are two amino acids in the sequence shared by the capsid proteins, VP1 and VP2. We have shown that a rodent parvovirus-derived transducing genome, containing the luciferase reporter, can be packaged by VP1 and VP2 from separate helper sources. Canine A72 cells and feline CFK cells were transduced with recombinant virions generated using VP1 and VP2 combinations from CPV and FPV. Both VP1 and VP2 were necessary for production of transducing virions. Efficient transduction of A72 cells required VP2 of CPV. Therefore, the capsid determinants of tropism for CPV and FPV are in VP2, although a source of VP1 is also necessary to produce infectious particles. The results extend similar observations on the tropic determinants of different strains of minute virus of mice.
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Evidence for the occurrence of two distinct subgroups of peanut stunt cucumovirus strains: molecular characterization of RNA3
More LessStrains of peanut stunt cucumovirus (PSV) were classified into two distinct subgroups, I and II, based on Western and Northern blot analyses using antisera and cloned cDNA probes to strains PSV-ER and PSV-W. These results were corroborated by nucleotide sequence analyses of full-length cDNA clones of RNA3 from representative strains of the two subgroups. Whereas the percentage nucleotide sequence identity between PSV-ER (or PSV-J) and PSV-W RNA3s was determined to be 80%, the corresponding value between strains ER and J was 91%, confirming that strains ER and J belong to the same subgroup (subgroup I) whereas strain W belongs to a separate subgroup (subgroup II). PSV-W and PSV-ER RNA3s are 2173 and 2188 nucleotides long, respectively. Each is dicistronic, encoding a putative movement protein (3a protein) and a coat protein (CP). The intercistronic and 5′ untranslated region (UTR) sequences of PSV strains, unlike those of cucumber mosaic cucumovirus (CMV) strains, are highly conserved and thus not useful for distinguishing the two subgroups. However, the 3′ UTR sequences of PSV strains, like those of CMV strains, can discriminate between the two subgroups since strains within the same subgroup are 95% identical in their 3′ UTRs whereas those in different subgroups are only 74–78% identical. PSV-W and PSV-ER RNA4s were determined to be 994 and 1006 nucleotides long, respectively. PSV 3a and CP genes have higher percentage nucleotide sequence identities to those of tomato aspermy cucumovirus than to those of CMV.
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Formation of multimers of cucumber mosaic virus satellite RNA
More LessDouble-stranded RNA multimers of cucumber mosaic virus (CMV) satellite RNA were detected in CMV-infected plants. RT-PCR showed that plussense and minus-sense monomers and plus-sense multimers of satellite RNA were present. Multimeric minus-sense RNA was not present except in the form of multimeric dsRNA. Sequence analysis of 52 cloned junction regions in head-to-tail repeats of unit-length satellite RNA indicated that about 35% of the junction sequences were precise fusions of monomer units, 56% lacked sequence of the 5′ component, and 10% lacked sequence of both 3′ and 5′ components. No junction contained additional nucleotides. Deletions at the junction regions may have accumulated during CMV multiplication in inoculated plants. These data suggest that replicase is not released from the template during synthesis of multimeric molecules of satellite RNA.
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Analysis of the infectivity of monomeric clones of pepper huasteco virus
The infectivity of several monomeric clones of pepper huasteco virus was investigated. All clones were infectious when inoculated excised from the plasmid DNA. However, only certain clones were infectious when inoculated in the non-excised form. Constructs in which the cloning site lies inside regions or genes involved in replication (e.g. Repbinding site, rep and AC2-AC3 genes) were not infectious, whereas constructs in which the site was located inside the CP or BC1 genes were infectious. A clone that interrupts the BV1 gene was not infectious suggesting an early role of BV1 during the establishment of the infection. Linear viral clones containing different DNA fragments at both extremes were also infectious although with a lower efficiency. Analysis of the progeny suggested a precise excision mechanism since in most cases only wild type virus was recovered. The results suggest that excision could be linked to replication through a very specific recombination process.
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Baculovirus-mediated trans-epithelial transport of proteins in infected caterpillars
More LessBaculovirus-mediated abnormal protein trafficking was studied in infected caterpillars by using heterologous proteins. The gene for human complement C1r was expressed in larvae of Mamestra brassicae by a recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) vector. By following the time-course of recombinant C1r distribution among various tissues, cell types and cell organelles, we concluded that the dominant site of recombinant protein synthesis was the fat body, although some production in the haemocytes and midgut was also observed. Only about 4% of the cells of the infected organs expressed recombinant C1r, which was then secreted into the haemolymph. The tracheal and integumental cuticle was rich in recombinant protein from the fourth day after infection although epidermal cells did not synthesize recombinant C1r. The morphological picture suggested that the accumulation was a consequence of a trans-epithelial transport. This transport process was checked by following the fate of the 49 kDa haemolymph protein and injected ovalbumin in AcMNPV-infected Mamestra brassicae and in Lymantria dispar nuclear polyhedrosis virus- infected Lymantria dispar larvae. Both proteins were able to pass the basal membrane of the epidermis and accumulated in the cuticle, while in control larvae neither was transported. The observed transepithelial transport points to the role of baculo- viruses in directing recombinant, endo- and exogenous proteins to cuticulated tissues. Based on these results we conclude that the permeability of basal membranes undergoes a characteristic change during the course of baculovirus infection.
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Comprehensive physical map of the Cydia pomonella granulovirus genome and sequence analysis of the granulin gene region
More LessA cloned strain of Cydia pomonella granulovirus, CpGV-M1, was obtained using successive rounds of an in vivo limiting dilution method. A detailed physical map of the genome was constructed using 11 restriction enzymes. The region containing the granulin gene and an open reading frame immediately upstream of the granulin gene was sequenced. This region showed a high degree of homology to the equivalent region from Cryptophlebia leucotreta granulovirus with 98% amino acid identity for the granulins and 68% identity for the putative polypeptides encoded by the upstream ORFs. These latter polypeptides contained two zinc finger-like motifs and showed a low degree of homology to ME53 from Autographa californica nucleopoly- hedrovirus (AcMNPV). Evidence is presented for a similar upstream ORF in Artogeia rapae GV also. Hybridization studies showed that the CpGV genome had a similar overall organization to the Artogeia rapae GV genome. Hybridization between CpGV and AcMNPV was limited to fragments spanning about 15% of each genome suggesting that very few genes are highly conserved between GVs and NPVs.
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PrP genotypes and experimental scrapie in orally inoculated Suffolk sheep in the United States
One-hundred and three United States Suffolk sheep were inoculated orally with a scrapie agent preparation and monitored for clinical disease and histopathological lesions characteristic of scrapie. A retrospective study of the polymorphisms at codon 171 of the prion protein (PrP) gene was performed on these sheep. All 63 sheep that developed scrapie during the observation period were homozygous for the glutamine 171 (171-QQ) PrP allele. Twelve 171-QQ sheep failed to develop disease. All 5 sheep homozygous for arginine (171-RR) and all 23 heterozygous (171-QR) sheep remained free of scrapie.
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