- Volume 75, Issue 5, 1994
Volume 75, Issue 5, 1994
- Review Article
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- Animal
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Corticosteroid Immunosuppression and Monoclonal Antibody-mediated CD5+ T Lymphocyte Depletion in Normal and Equine Infectious Anaemia Virus-carrier Horses
More LessThe immune control of chronic equine infectious anaemia (EIA) lentiviral infection was investigated by specifically depleting CD5+ T lymphocytes in vivo with monoclonal antibody (MAb) or by immunosuppression with corticosteroids. MAb was given at 25 to 50 mg/day intravenously for 11 days. Murine IgG1 anti-equine CD2 MAb (n = 2 horses) or IgG1 (n = 2) and IgG2a control MAb (n = 2 normal; 2 EIA-infected) did not deplete CD2+ T lymphocytes in horses. Horses given murine IgG2a anti-CD5 MAb HB19A (n = 4 normal; 5 EIA-infected) had depletion of peripheral blood CD5+ T lymphocytes during treatment. These horses, however, maintained a residual population of CD2+ T lymphocytes [15 ( ± 3)% of pretreatment numbers] that did not express CD5 but expressed either CD4 or CD8. These antigenically modulated CD5− T lymphocytes responded normally in vivo to intradermal inoculation with phyto-haemagglutinin and in vitro to allogeneic leukocyte stimulation in one-way mixed lymphocyte reactions. EIA virus-infected horses (n = 5) did not develop recrudescent viraemia or disease following in vivo CD5+ T lymphocyte depletion. Immunosuppression of EIA virus-infected horses with corticosteroids (1 mg/kg body weight/day, intravenously for 9 days) resulted in detectable recrudescent EIA viraemia in 6/11 horses (55%) and recrudescent disease in 9/11 horses (82%). Normal horses (n = 3) treated with corticosteroids developed no clinical disease. These results demonstrate that the use of murine IgG2a MAbs to appropriate equine lymphocyte antigens will facilitate in vivo investigation of the role of T lymphocyte subpopulations in the control of EIA or other important equine diseases.
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The Construction and Characterization of Poliovirus Antigen Chimeras Presenting Defined Regions of the Human T Lymphocyte Marker CD4
More LessDefined regions of the CDR2-like region of the T cell antigen CD4 that are implicated in the binding of the surface glycoprotein (gpl20) of human immunodeficiency virus type 1 (HIV-1) to CD4+ T lymphocytes have been engineered in place of antigenic site 1 of Sabin type 1 poliovirus. The antigenic properties of the recovered chimeric virus particles were investigated using monoclonal antibodies (MAbs) and polyclonal serum to CD4. None of the MAbs tested neutralized the chimeras, presumably because they are directed against conformational determinants on the VI domain of CD4. In contrast, the three antigen chimeras were neutralized by polyclonal serum to CD4, which suggested that the CD4-derived sequences were presented in a relevant conformation. A panel of six MAbs were raised against one of the chimeras, and the epitopes were mapped by the selection of neutralization-resistant mutants and cross-neutralization studies. Five of the six MAbs reacted with soluble CD4 (sCD4) in ELISA, and one (MAb 1686) bound to CD4 expressed at the surface of HeLa cells. The high affinity interaction between gpl20 and sCD4 was not blocked by MAb 1686, and the poliovirus-CD4 chimeras did not interact with gpl20. These results demonstrate that poliovirus can be used as an epitope expression vector for the presentation of sequences in an immunodominant location on the virus particle which adopt a native or near-native conformation, and supports the findings of previous studies involving the presentation of epitopes derived from pathogens.
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Neurological Abnormalities Associated with Feline Immunodeficiency Virus Infection
Specific pathogen-free cats were infected with the Maryland strain of FIV (FIV-MD) for the purpose of assessing the effects of FIV infection on the central nervous system (CNS). Two separate studies were performed, involving a total of 13 infected cats and six age-matched, sham-inoculated controls. All animals infected with FIV-MD seroconverted by 8 weeks postinfection and virus was recovered from peripheral blood mononuclear cells of all infected cats. All of the infected animals had lower absolute CD4+ cell counts and decreased CD4+/CD8+ ratios. Virus was recovered from the cerebrospinal fluid (CSF) of certain infected individuals, and antiviral antibody and pleocytosis were evident in the CSF of the majority of infected cats. Additionally, virus was recovered from tissue explants from the cerebellum, midbrain and brainstem of one sacrificed FIV+ cat. Specific neurological changes included anisocoria, delayed righting reflex and delayed pupillary reflex, as well as delayed visual and auditory evoked potentials, and marked alterations in sleep patterns similar to those reported for human immunodeficiency virus (HlV)-positive individuals. Histological evaluation revealed the presence of perivascular cuffing and glial nodules in FIV-infected cats. These results indicate that FIV causes an acute neurological disease that closely resembles the early neurological effects of HIV infection in humans and should serve well as an animal model for lentivirus-induced CNS disease.
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PrP Genotype and Agent Effects in Scrapie: Change In Allelic Interaction With Different Isolates of Agent in Sheep, a Natural Host of Scrapie
More LessMan and sheep are the two species in which spongiform encephalopathies occur naturally, and in which there are recognized genetic components that predispose an individual person or sheep to clinical disease. In both species mutations/polymorphisms in the PrP gene have been linked to the incidence of natural disease, but only in sheep is it possible to investigate by deliberate exposure to infection whether these polymorphisms are directly correlated with survival time. Cheviot sheep of different PrP genotypes were challenged with one of two isolates of scrapie or an isolate of bovine spongiform encephalopathy and the survival time and incidence of disease were monitored. Genotype analysis showed that dimorphisms in codons 136 and 171 of the ovine PrP gene correlated with control of disease incidence and modulation of incubation time. Crucially, the functional effects of these domains of PrP were shown to alternate depending on the isolate of infecting agent.
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Deletion Mutation in the Signal Anchor Domain Activates Cleavage of the Influenza Virus Neuraminidase, a Type II transmembrane Protein
More LessInfluenza vims neuraminidase (NA) is a type II integral membrane protein with a long hydrophobic domain [29 amino acids (aa)] at the N terminus that functions as an uncleaved signal for translocation into the endoplasmic reticulum and anchors the protein in the membrane. The function of the transmembrane domain in intracellular transport was investigated by deletion mutagenesis. Expression of the mutated NA in eukaryotic cells and by in vitro translation in the presence of membranes showed that the deletion of eight amino acids (aa 28 to 35) from the carboxy end of the signal anchor domain resulted in cleavage, probably by the signal peptidase and secretion of NA into the culture medium. The mutant NA (N28–35) was present inside the cell predominantly as dimers, secreted as dimers, and was enzymatically inactive. When translated in vitro in the presence of dog pancreatic microsomal membranes, the N28–35 protein underwent cleavage and did not remain anchored to membranes. Two other deletion mutants in the transmembrane domain, N7–17 and N17–23, were partially cleaved and secreted, whereas two mutants, one (N19–27) lacking nine aa in the central region and the other (N1-14) lacking the first 14 aa from the N terminus remained uncleaved and exhibited a phenotype similar to the wild-type NA. We conclude that the longer transmembrane domain (29 aa) may play an important role in determining that type II signal is not cleaved during translocation; however, in addition, adjacent amino acid sequences also provide determinants important in signal cleavage.
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Post-translational Folding of the Influenza C Virus Glycoprotein HEF: Defective Processing in Cells Expressing the Cloned Gene
The post-translational processing of the influenza C virus glycoprotein HEF was analysed. In cells infected with influenza C virus, HEF protein is synthesized as a glycosylated 80K polypeptide. A post-translational conformational rearrangement involving the formation of intramolecular disulphide bonds results in a decrease in its electrophoretic mobility. Therefore, SDS-PAGE under non-reducing conditions suggests an M r of about 100K, whereas under reducing conditions an 80K protein is observed which is in accordance with the sequence data. The 100K form was detected 10 min after synthesis of HEF, and transport to the cell surface took about 60 min. This result indicates that the conformational change presumably occurs in the endoplasmic reticulum. A difference in post-translational processing was observed when the HEF gene was expressed in the absence of other influenza C virus genes. In cells infected with recombinant simian virus 40, the 80K precursor was synthesized, but this protein was neither converted to the 100K form nor transported to the cell surface. Deletion of the short cytoplasmic tail of HEF (Arg-Thr-Lys) or replacement of the two basic amino acids by hydrophobic (Ile) or acidic residues (Glu) resulted in HEF protein which was partially converted to the 100K form. Influenza C virus glycoprotein obtained after transient expression of the HEF gene using the vaccinia virus system was completely converted to the 100K form. However, in neither expression system was HEF transported to the cell surface. The possibility is discussed that the interaction of HEF with another viral protein is required for the post-translational folding and transport of this glycoprotein. The M protein of influenza C virus is suggested as a candidate for the chaperone which might interact with the cytoplasmic tail of HEF.
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The Sendai Virus Matrix Protein Appears to be Recruited in the Cytoplasm by the Viral Nucleocapsid to function in Viral Assembly and Budding
More LessThe matrix (M) protein is viewed as the regulator of paramyxovirus particle assembly and budding. Accordingly it was observed to be mutated, and/or decreased in amount, in cases where virus particle production was significantly reduced. Here, a non-productive [non-defective and defective interfering (DI)] Sendai virus infection of COS cells is presented where virus particle production is abolished in the presence of a normal amount of intracellular M protein. In this infection the haemagglutinin—neuraminidase envelope glycoprotein is shown to be dispensable for virion production, and the fusion (F0) envelope glycoprotein behaves as in a productive infection. The M protein is shown to accumulate in perinuclear patches within the cytoplasm. In contrast, localization in the plasma membrane is observed in productive infections. However in both productive and non-productive infections a significant fraction of M protein is found in association with cellular membranes. The M protein-membrane association is shown to take place in the absence of any other viral component, and the M protein-membrane complex exhibits properties similar to those observed for the integral membrane protein F0. However these properties are distinct from those of the phosphoprotein, which is thought to associate with membranes in a non-specific manner. Concomitant with the cytoplasmic accumulation of M protein and the reduction of virus particle production in this non-productive infection, DI nucleocapsids are shown not to associate with cellular membrane fractions. This is a property which coincides with their poor envelopment in virus particles. Taken together, these data indicate the need for M protein to be recruited at the perinuclear membranes by the nucleocapsids to participate in viral assembly and budding. This view is consistent with a process of viral assembly taking place on internal cytoplasmic membranes rather than at the plasma membrane.
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Role of N-linked Oligosaccharide Chains in the Processing and Antigenicity of Measles Virus Haemagglutinin Protein
More LessThe effects of N-linked oligosaccharides on the haemagglutinin (H) protein of measles virus (MV) were assessed with respect to the processing and antigenicity of the molecule. The functional glycosylation sites on the H protein were determined by eliminating each of the five potential positions, Asn-168, Asn-187, Asn-200, Asn-215 and Asn-238, for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant H proteins in BHK-21 cells by a recombinant vaccinia virus encoding T7 polymerase indicated that the first four sites were used in the H glycoprotein for the addition of N-linked oligosaccharide chains. Heterogeneity of oligosaccharide processing was demonstrated. One of the four glycosylation sites had a different carbohydrate structure from those of the other three glycosylation sites and this varied glycosylation was responsible for the appearance of two forms of the H protein. The functional glycosylation sites were systematically removed in various combinations from the H protein to form a panel of mutants in which the role of carbohydrate chains, singly or in different combinations, could be evaluated. Investigations of these glycosylation mutants indicated that (i) two of the four individual carbohydrate side-chains have a large influence on the antigenicity of the molecule; (ii) individual carbohydrate side-chains have little effect on the folding and oligomerization of the molecule, and are not sufficient or necessary alone to facilitate the transport of the molecule to the plasma membrane; (iii) at least two carbohydrate side-chains are required for the H protein to move along the exocytic pathway to the plasma membrane and various combinations of oligosaccharide side-chains, irrespective of the carbohydrate localizations, influence equally the processing of the molecule.
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Identification of Genotypes of Hepatitis C Virus by Sequence Comparisons in the Core, E1 and NS-5 Regions
More LessIsolates of hepatitis C virus (HCV) show considerable nucleotide sequence variability throughout the genome. Comparisons of complete genome sequences have been used as the basis of classification of HCV into a number of genotypes that show 67 to 77% sequence similarity. In order to investigate whether sequence relationships between genotypes are equivalent in different regions of the genome, we have carried out formal sequence analysis of variants in the 5′ non-coding region (5′NCR) and in the genes encoding the core protein, an envelope protein (E1) and a non-structural protein (NS-5). In the E1 region, variants grouped into a series of six major genotypes and a series of subtypes that could be matched to the phylogenetic groupings previously observed for the NS-5 region. Furthermore, core and E1 sequences showed three non-overlapping ranges of sequence similarity corresponding to those between different genotypes, subtypes and isolates previously described in NS-5. Each major genotype could also be reliably identified by sequence comparisons in the well conserved 5′NCR, although many subtypes, such as 1a/1b, 2a/2c and some of those of type 4, could not be reliably distinguished from each other in this region. These data indicate that subgenomic regions such as E1 and NS-5 contain sufficient phylogenetic information for the identification of each of the 11 or 12 known types and subtypes of HCV. No evidence was found for variants of HCV that had sequences of one genotype in the 5′NCR but of a different one in the E1 or NS-5 region. This suggests that recombination between different HCV types is rare or non-existent and does not currently pose a problem in the use of subgenomic regions in classification.
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Heterogeneity of Hepatitis C Virus Genotypes in France
The genotypes of French hepatitis C virus (HCV) isolates were investigated by amplification of a domain from the non-structural region 3 (NS3) using nested PCR, followed by hybridization with two genotype- specific probes, FI (HCV type I-specific) and F2 (HCV type II-specific). Among 119 HCV RNA-positive sera, 91 % of samples were NS3 PCR positive. Most samples (83·2 %) hybridized with one or the other probe only, whereas a few samples (4·2 %) hybridized with both F1 and F2 probes (HB). A small percentage (3·4%) of samples appeared unable to hybridize with either probe (HN). For some of these samples (HB1, HB2, HN1, HN2, HN3, HN4), part of the NS3, core and envelope regions were sequenced and the corresponding deduced consensus sequences were compared with those of prototype isolates of the four HCV genotypes (types I to IV). A phylogenetic tree was constructed to illustrate the relationship between these isolates. The results obtained showed that (i) HN4 appears to be more closely related to type III than to type IV HCV genotypes, which suggests that in France there may exist additional although minor genotypes besides the two major types, FI and F2. (ii) HB1, HB2, HN1, HN2 and probably HN3 belong to the type II HCV genotype. The association between sequence diversity and putative biological difference for isolates within the same genotype remains to be elucidated.
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Neonatal Infection of Mice With Lactate Dehydrogenase-elevating Virus Results in Suppression of Humoral Antiviral Immune Response but does not Alter the Course of Viraemia or the Polyclonal Activation of B Cells and Immune Complex Formation
Neonatal infection of FVB mice with lactate dehydrogenase-elevating virus (LDV) prevented the normal formation of anti-LDV antibodies observed in mice infected at 5 days of age or older. Even 22 weeks post-infection, the concentration of circulating anti-LDV antibodies in neonatally infected mice was insignificant. However, the time course and level of persistent viraemia were the same in neonatally infected mice lacking anti-LDV antibodies as in mice infected at 5 or 15 days of age which developed normal antiviral immune responses. The results support the view that LDV replication in mice is unaffected by antiviral immune responses and instead is primarily dependent on the rate of regeneration of LDV-permissive macrophages. This view is further supported by the following findings. Treatment of mice with cyclophosphamide or dexamethasone, which are known to increase plasma LDV levels, increased the proportion of LDV-permissive macrophages in the peritoneum. Injection of mice with interleukin-3, which is known to stimulate macrophage development, increased plasma LDV levels in persistently infected mice 10- to 100-fold. During the first month of age when mice possess a higher proportion of LDV-permissive macrophages than older mice and peritoneal macrophages exhibit self-sustained growth, the persistent plasma LDV titres were also 10- to 100-fold higher than in older mice. The polyclonal activation of B cells induced by LDV that results in a permanent elevation of IgG2a or IgG2b in the circulation, and the formation of 180K to 300K immune complexes containing IgG2a or IgG2b were also the same in neonatally infected mice and mice infected 5 or 15 days after birth. Thus, the polyclonal activation of B cells occurs in the absence of an antiviral humoral immune response and the immune complexes do not contain anti-LDV antibodies. The immune complexes probably consist of autoantibodies formed in the course of the polyclonal activation of B cells and their cellular antigens.
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Characterization of a Herpes Simplex Virus type 1 Deletion Variant (1703) Which Under-produces Vmw63 During Immediate Early Conditions of Infection
More LessThe herpes simplex virus type 1 deletion variant 1703 apparently fails to synthesize the essential IE2 gene product Vmw63 despite the deletion leaving the gene intact. Sequence analysis revealed that the deletion removes a region to the right of IE2 comprising the 3′ end of IE1, UL56 and the 3′ part of UL55, stopping 555 bp downstream of the IE2 polyadenylation signal. Further DNA sequencing has shown that there is no secondary mutation in the IE2 gene. Western blot analysis demonstrated that Vmw63 is made at reduced levels compared to that produced by the wild-type virus during immediate early conditions of infection. SI nuclease protection mapping has revealed that this reduction is also apparent at the level of mRNA synthesis. A direct link between the deletion and the change in mRNA synthesis was provided by the insertion of a deletion-spanning fragment from 1703 into a 17+ genome, which resulted in the recombinant having a 1703-like phenotype. Evidence that down-regulation of IE2 mRNA during immediate early conditions of infection could be due to antisense RNA initiating from the IE1 promoter was obtained by the insertion of a novel transcriptional termination signal between IE1 and IE2 in the variant and the subsequent detection of wild-type levels of IE2 mRNA and protein.
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Localization of the Herpes Simplex Virus Type 1 Major Capsid Protein VP5 to the Cell Nucleus Requires the Abundant Scaffolding Protein VP22a
More LessThe intracellular distributions of three herpes simplex virus type 1 (HSV-1) capsid proteins, VP23, VP5 and VP22a, were examined using vaccinia virus and plasmid expression systems. During infection of cells with HSV-1 wild-type virus, all three proteins were predominantly located in the nucleus, which is the site of capsid assembly. However, when expressed in the absence of any other HSV-1 proteins, although VP22a was found exclusively in the nucleus as expected, VP5 and VP23 were distributed throughout the cell. Thus nuclear localization is not an intrinsic property of these proteins but must be mediated by one or more HSV-1-induced proteins. Co-expression experiments demonstrated that VP5 was efficiently transported to the nucleus in the presence of VP22a, but the distribution of VP23 was unaffected by the presence of either or both of the other two proteins.
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Assembly of Herpes Simplex virus Type 1 Capsids Using a Panel of Recombinant Baculoviruses
More LessImmature or B capsids of herpes simplex virus type 1 (HSV-1) are composed of seven proteins encoded by six viral genes. The proteins encoded by UL18 (VP23), UL19 (VP5), UL35 (VP26) and UL38 (VP19C) are components of the outer capsid shell whereas those specified by UL26 (VP21 and VP24) and UL26.5 (VP22a), are involved in scaffold formation. We have used a panel of recombinant baculoviruses, each expressing one of the capsid protein genes, to examine the requirements for capsid assembly. Coexpression of the six genes in insect cells resulted in the formation of capsids that were indistinguishable in appearance and protein composition from those made during HSV-1 infection of mammalian cells. This demonstrates that the proteins encoded by the known capsid genes contain all the structural information necessary for capsid assembly and that other virus-encoded proteins are not required for this process. Omission of single recombinant baculo-viruses from this system allowed the role of individual HSV-1 proteins in capsid assembly to be determined. Thus, capsid assembly did not take place in the absence of VP23, VP5 or VP19C, whereas lack of VP26 had no discernible effect on capsid formation. Capsids assembled in the absence of the UL26 gene products had a large-cored phenotype resembling that previously described for the HSV-1 mutant ts1201 which has a lesion in this gene. Some apparently intact capsid shells were also made in the absence of the major scaffolding protein, VP22a, whereas the omission of both UL26 and UL26.5 resulted in the appearance of large numbers of partial and deformed capsid shells.
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Orgyia Pseudotsugata Baculovirus p10 and Polyhedron Envelope Protein Genes: Analysis of their Relative Expression Levels and Role in Polyhedron Structure
More LessTo investigate the regulation of p10 and polyhedron envelope protein (PEP) gene expression and their role in polyhedron development, Orgyia pseudotsugata multi-nucleocapsid nuclear polyhedrosis viruses lacking these genes were constructed. Recombinant viruses were produced, in which the p10 gene, the PEP gene or both genes were disrupted with the β-glucuronidase (GUS) or β-galactosidase (lacZ) genes. GUS activity under the control of the PEP protein promoter was observed later in infection and its maximal expression was less than 10% the level for p10 promoter-GUS constructs. Tissues from O. pseudotsugata larvae infected with these recombinants were examined by electron microscopy. Cells from insects infected with the p10− viruses lacked p10-associated fibrillar structures, but fragments of polyhedron envelope-like structures were observed on the surface of some polyhedra. Immunogold labelling of cells infected with the p10−GUS+ virus with an antibody directed against PEP showed that the PEP was concentrated at the surface of polyhedra. Although polyhedra produced by p10 and PEP gene deletion mutants demonstrated what appeared to be a polyhedron envelope by transmission electron microscopy, scanning electron microscopy showed that they had irregular, pitted surfaces that were different from wild-type polyhedra. These data suggested that both p10 and PEP are important for the proper formation of the periphery of polyhedra.
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Stomach Cancer in Transgenic Mice Expressing Human Papillomavirus Type 16 Early Region Genes From a Keratin Promoter
More LessCertain human papillomaviruses (HPVs) have been implicated as important contributory factors in the development of cervical carcinoma and other epithelial malignancies. In order to investigate the role of papillomavirus early gene expression in epithelial oncogenesis in vivo, we produced transgenic mice expressing HPV-16 early region genes from the promoter of the bovine keratin 6 gene. Spliced transcripts were detected in the tongue, forestomach, glandular stomach, female reproductive tract and tail skin of these mice. This expression was initially asymptomatic. However, at increasing frequency after approx. 100 days, solitary glands within the gastric mucosa became colonized with small dysplastic cells. Later this abnormal cell population spread within the glandular mucosa, invaded the submucosa and outer muscular wall of the stomach, and commonly metastasized to local lymph nodes and the liver. The appearance and staining characteristics of the tumours suggested their classification as malignant carcinoids, originating from the neuroendocrine entero- chromaffin-like cells. Expression of HPY mRNAs was increased in the tumours, though it remained comparable to that in forestomach and tongue. The mean age at tumour presentation was 246 days in males and 352 days in females, all transgenic mice in eight independent lines were similarly susceptible. This study confirms the oncogenicity of HPV-16 early region genes, and establishes a model system in which to investigate mechanisms of malignant progression and possible therapeutic strategies for HPV-associated tumours.
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Differentiation-independent Constitutive Expression of the Human Papillomavirus Type 16 E6 and E7 Oncogenes in the CaSki Cervical Tumour Cell Line
More LessCaSki cells are a human papillomavirus type 16 (HPV-16)-positive cell line that serves as a model for the study of advanced cervical carcinoma. Calcium is an important regulator of normal ectocervical epithelial cell differentiation. HPV E6 and E7 gene products are thought to be important in the process of cervical cell immortalization and hence important in the development of cervical cancer. In the present study we examine the relationship between CaSki cell differentiation and expression of the papillomavirus oncogenes. Shifting CaSki cells from medium containing low (0·06 mm) to high (1·4 mm) calcium results in an increase in cell-cell contact and increased differentiation as measured by an increase in the level of mRNA encoding cytokeratin K13, involucrin and type 1 transglutaminase, which are markers of differentiation in the cervical epithelium. In contrast, E6/E7 transcripts are produced in a differentiation-independent constitutive manner. These results and those from our previous experiments with HPV-16-immortalized but non-tumorigenic cell lines suggest that the constitutive, differentiation-independent expression of E6/E7 levels is a property of both tumorigenic and non-tumorigenic HPV-16-positive cancer cells.
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Development of a Broad Spectrum PCR Assay for Papillomaviruses and its Application in Screening Lung Cancer Biopsies
More LessA PCR assay was developed to detect known and as yet unidentified papillomaviruses (PVs). For this purpose we analysed the conserved amino acid sequences in the L1 and E1 open reading frames of 45 human and nine animal PVs. Candidate regions for the design of a primer were identified as those having the least number of amino acid and nucleotide sequence variants among the different PVs. These regions in the L1 ORF have been described previously. We modified the sequences of the backward and the forward primers, as well as the sequence of the oligonucleotide used as the degenerate probe, in order to cover a broader spectrum of PVs. The sensitivity of the assay for the human and animal PVs tested after hybridization with a 32P-labelled degenerate oligonucleotide probe was one genome copy per cell for integrated PV DNA and 10 genome copies per cell for plasmid PV DNA. The only exceptions were human papillomavirus (HPV) type 4, HPV60 and HPV65, for which a lower sensitivity was obtained. This group could be detected only by using additional primers. The assay was used to analyse 85 lung cancer biopsies representing different histological types. Using this system no PV DNA sequences were detected in the biopsies when compared with human placental DNA (a negative control) and PV DNA-positive standards.
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Sequence Rearrangements in the Upstream Regulatory Region of Human Papillomavirus Type 6: are these Involved in Malignant Transition?
Human papillomavirus type 6 (HPV-6) was isolated from a tongue papilloma which subsequently progressed to an invasive carcinoma. Three biopsies were taken from the same patient at different intervals during the tumour development. The HPV-6 genome in all three biopsies contained a GT-rich 94 bp insertion at nucleotide 7350 in the upstream regulatory region (URR). In comparison to previously published HPV-6 DNA isolates, this insertion seems to be the most prevalent and constant modification, not present in the prototype HPV-6b, and allows an improved alignment with the sequence of the HPV-11 genome. The possible biological significance of these GT-rich clusterings at the beginning of the URR, present not only in these HPV-6 isolates but observed in all other ‘genital’ HPVs also, is discussed.
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