- Volume 84, Issue 2, 2003
Volume 84, Issue 2, 2003
- Animal
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- RNA viruses
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Glycosylation inhibitors and neuraminidase enhance human immunodeficiency virus type 1 binding and neutralization by mannose-binding lectin
More LessMannose-binding lectin (MBL), a C-type lectin component of the human innate immune system, binds to the gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). The objective of this study was to assess the effects of inhibitors of endoplasmic reticulum glucosidases and Golgi mannosidase as well as neuraminidase (NA) on the interaction between HIV and MBL. Production of HIV in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM) significantly enhanced binding of HIV to MBL and increased MBL neutralization of an M-tropic HIV primary isolate. In contrast, culturing HIV in the presence of α-glucosidase I and II inhibitors castanospermine and deoxynojirimycin only slightly affected virus binding and neutralization by MBL. Removal of sialic acid from HIV by NA also significantly enhanced virus binding and neutralization by MBL. Treatment of virus grown in the presence of dMM with endoglycosidase F1 substantially reduced binding to MBL, indicating that dMM increased MBL binding by increasing high-mannose carbohydrates on the virus. In contrast, endoglycosidase F1 did not decrease the MBL interaction with NA-treated virus, suggesting that NA exposed novel MBL binding sites. Treatment with dMM increased the immunocapture of HIV by monoclonal antibodies 2F5 and 2G12, indicating that altering the glycosylation of viral glycoproteins increases the accessibility or reactivity of some epitopes. This study shows that specific alterations of the N-linked carbohydrates on HIV gp120/gp41 can enhance MBL-mediated neutralization of virus by strengthening the interaction of HIV-1 with MBL.
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Construction and immunogenicity in a prime–boost regimen of a Semliki Forest virus-vectored experimental HIV clade A vaccine
A novel, experimental subunit human immunodeficiency virus (HIV) vaccine, SFV.HIVA, was constructed. This consists of Semliki Forest virus (SFV), which is a suitable vaccine vector for use in humans, and a passenger gene encoding HIVA, which is an immunogen derived from HIV-1 clade A that is being currently tested in clinical trials of combined DNA- and modified vaccinia virus Ankara (MVA)-vectored vaccines in Oxford (UK) and Nairobi (Kenya). In the mouse, the SFV.HIVA vaccine was highly immunogenic for T cell-mediated immune responses and induced T cell memory that lasted for at least 6 months. SFV.HIVA was also compared to the vaccines currently used in the clinical trials and was shown to be as effective in T cell induction as pTHr.HIVA DNA but less immunogenic than MVA.HIVA. When tested in a prime–boost regimen, SFV.HIVA-induced responses could be boosted by MVA.HIVA. This work is a part of a long-term effort to build a panel of subunit vaccines expressing a common immunogen, which will allow both a direct comparison of various vaccine vectors and combined vaccination regimens in humans and provide more flexibility and/or a potential optimization of vaccinations for individuals based on their pre-existing anti-vector immunity.
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The proline-rich region of the ecotropic Moloney murine leukaemia virus envelope protein tolerates the insertion of the green fluorescent protein and allows the generation of replication-competent virus
More LessSequences encoding the green fluorescent protein (GFP) were inserted into the envelope protein (Env) of ecotropic Moloney murine leukaemia virus, MoMLV. Insertion of these sequences into the proline-rich region (PRR) of Env resulted in a chimeric GFP-Env protein that allowed retrovirus vector transduction of murine cells with titres similar to wild-type Env. However, N-terminal extension with GFP did not result in a functional Env protein. GFP sequences were then inserted into the Env PRR of E-MO virus, a MoMLV that carries epidermal growth factor sequences at the N terminus of its Env protein. The resulting virus, GFP-EMO1, replicates to the same titres as the parental virus. In a chronically infected cell culture, GFP-EMO1 was genetically stable. However, additional insertions of sequences that led to recombination or that may have been incompatible with virus replication were deleted and decreased virus titre. In summary, Env PRR can be used to tag individual virus particles with GFP, which leaves other regions available for modification in studies aimed at altering virus tropism.
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Walleye dermal sarcoma virus Orf C is targeted to the mitochondria
More LessWalleye dermal sarcomas are associated with the presence of a complex retrovirus, walleye dermal sarcoma virus (WDSV). These sarcomas develop and regress seasonally in naturally infected fish. In addition to gag, pol and env, WDSV contains three open reading frames (ORFs), designated orf a, orf b and orf c. orf c is located between the 5′ long terminal repeat and gag. Developing tumours contain low levels of orf a and orf b transcripts, whereas regressing tumours contain high levels of genomic transcripts and virus particles. Orf C protein is encoded by the full-length, genomic transcript and can be detected in tumour extracts with anti-Orf C-specific antisera. To determine the subcellular location of WDSV Orf C, cultured cells were transfected with an expression vector encoding haemagglutinin-tagged Orf C and examined by immunofluorescence. Orf C was observed throughout the cytoplasm and accumulated in cytoplasmic organelles. Dual-antibody staining for Orf C and mitochondrial cytochrome c demonstrated colocalization of Orf C with mitochondria and loss of the normal distribution of mitochondria in the cytoplasm. Cells transiently expressing Orf C exhibited apoptotic morphology and increased levels of surface phosphatidylserine and were unable to retain MitoTracker Orange, a dye that accumulates in active mitochondria. These results imply a functional role for WDSV Orf C in an alteration of mitochondrial function that results in apoptosis contributing to tumour regression.
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Translational regulation of rotavirus gene expression
More LessRotavirus mRNAs are transcribed from 11 genomic dsRNA segments within a subviral particle. The mRNAs are extruded into the cytoplasm where they serve as mRNA for protein synthesis and as templates for packaging and replication into dsRNA. The molecular steps in the replication pathway that regulate the levels of viral gene expression are not well defined. We have investigated potential mechanisms of regulation of rotavirus gene expression by functional evaluation of two differentially expressed viral mRNAs. NSP1 (gene 5) and VP6 (gene 6) are expressed early in infection, and VP6 is expressed in excess over NSP1. We formulated the hypothesis that the amounts of NSP1 and VP6 were regulated by the translational efficiencies of the respective mRNAs. We measured the levels of gene 5 and gene 6 mRNA and showed that they were not significantly different, and protein analysis indicated no difference in stability of NSP1 compared with VP6. Polyribosome analysis showed that the majority of gene 6 mRNA was present on large polysomes. In contrast, sedimentation of more than half of the gene 5 mRNA was subpolysomal. The change in distribution of gene 5 mRNA in polyribosome gradients in response to treatment with low concentrations of cycloheximide suggested that gene 5 is a poor translation initiation template compared with gene 6 mRNA. These data define a regulatory mechanism for the difference in amounts of VP6 and NSP1 and provide evidence for post-transcriptional control of rotavirus gene expression mediated by the translational efficiency of individual viral mRNAs.
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Stable expression of antisense RNAs targeted to the 5′ non-coding region confers heterotypic inhibition to foot-and-mouth disease virus infection
More LessThe antiviral potential of transcripts targeted to the non-coding regions (NCRs) of foot-and-mouth disease virus (FMDV) RNA have been studied during transient and constitutive expression in susceptible BHK-21 cells. Transient expression of antisense transcripts corresponding to the 5′ and 3′NCRs, alone or in combination, confers specific inhibition of homologous (serotype C) virus infection in BHK-21 cells. Constitutive expression of antisense 5′NCR transcripts (5′AS) exerted higher levels of inhibition to homologous and heterologous (serotypes O, A, Asia, SAT 1, SAT 2 and SAT 3) FMDV infection, as estimated by a 10-fold reduction in virus titre in the supernatants from infected clones and by a plaque reduction assay. These inhibitions were also observed, albeit to a lesser extent, in clones stably expressing antisense 3′NCR transcripts. The antiviral response was specific for FMDV, as the picornavirus encephalomyocarditis virus was not inhibited in any of the transformed cell lines. In all cases, a correlation was found between the level of transcript expression and the extent of virus inhibition. The potential to efficiently inhibit FMDV, including isolates representing the seven serotypes, by expressing interfering 5′AS transcripts opens the possibility of developing transgenic animals with a reduced susceptibility to FMDV.
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Interaction of glyceraldehyde-3-phosphate dehydrogenase with secondary and tertiary RNA structural elements of the hepatitis A virus 3′ translated and non-translated regions
More LessProteins interacting with RNA structures at the 3′ non-translated region (3′NTR) of picornaviruses are probably important during viral RNA replication. We have shown previously that a dominant cellular cytoplasmic protein of 38 kDa (p38) interacts with the 3′NTR and upstream regions of the hepatitis A virus (HAV) RNA ( Kusov et al., J Virol 70, 1890–1897, 1996 ). Immunological and biochemical analyses of p38 have indicated that it is identical to GAPDH, which has previously been described as modulating translational regulation of the HAV RNA by interacting with the 5′NTR ( Schultz et al., J Biol Chem 271, 14134–14142, 1996 ). Three separate binding regions for GAPDH in the 3′NTR and in the upstream 3D polymerase-coding region were identified. Structural analysis of these RNA regions by computer modelling and direct enzymatic cleavage suggested the presence of several AU-rich stem–loop structures having the potential for tertiary interactions. Binding of GAPDH to these structures was confirmed by RNA footprint analysis and resulted in the loss of double-stranded RNA regions. A different panel of RNA binding proteins (p28, p41 and p65) was detected in the ribosomal fractions of several cell lines (BSC-1, FRhK-4 and HeLa), whereas RNA binding of the GAPDH that was also present in these fractions was only marginal or absent.
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In vivo activity of Rhopalosiphum padi virus internal ribosome entry sites
More LessThe RNA genome of Rhopalosiphum padi virus (RhPV), like other members of the Dicistroviridae, contains two open reading frames that are preceded by internal ribosome entry sites (IRESs). To compare the activities of the two RhPV IRESs in insect cells, a system was established for the in vivo transcription and translation of plasmid templates containing the IRESs. In this system, the two RhPV IRESs directed initiation of translation from bicistronic plasmids with equal efficiency. Competition was observed between the two IRESs when they were in cis in a bicistronic plasmid. A mutation that disrupted the 3′-proximal pseudoknot of the intergenic (IG) IRES reduced translation initiation in vivo. Similarly, mutations in the RhPV IG IRES disrupted its ability to bind 80S particles in vitro. The two IRESs preferentially labelled proteins of different masses in UV cross-linking experiments, illustrating the different translation initiation mechanisms employed by the two elements.
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Molecular and serological characterization of sporadic acute hepatitis E in a Japanese patient infected with a genotype III hepatitis E virus in 1993
Serum samples collected periodically from a 40-year-old Japanese woman who had not travelled abroad and who had contracted sporadic acute hepatitis E in 1993 were semi-quantitatively tested by enzyme immunoassay for IgM, IgA and IgG antibodies to hepatitis E virus (HEV). Anti-HEV IgM and IgA antibody levels were the highest (1 : 2400 dilution and 1 : 3400 dilution, respectively) on day 9 after the onset of hepatitis and then decreased rapidly in a parallel manner. Anti-HEV IgG antibody levels were the highest (1 : 17000 dilution) on day 145 and then decreased gradually but remained at high titres (1 : 2200 dilution) even 8·7 years after the onset of hepatitis. An HEV isolate, HE-JA10, recovered from the patient's serum at admission was closely related to a genotype III strain isolated in the United States (US1), with 92·2% identity over the full-length genome, and was most closely related to the JMY-Haw isolate of Japanese origin (95·4% identity).
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The extent of homologous recombination in members of the genus Flavivirus
More LessThe family Flaviviridae includes important human pathogens, such as dengue (DEN) virus, yellow fever (YF) virus and hepatitis C virus, many of which have emerged or re-emerged in recent years. Until recently, flavivirus evolution was thought to proceed in a clonal manner, with diversity generated mainly through the accumulation of mutational changes. However, this assumption has now been shown to be invalid, with homologous recombination demonstrated in all three genera of the Flaviviridae. Since recombination has important implications for the study of virus evolution, a survey of recombination in the viruses of the genus Flavivirus was carried out. Using envelope gene sequence data and a combination of graphical and phylogenetic analyses, hitherto unreported recombination in Japanese encephalitis virus and St Louis encephalitis virus was detected, as well as further recombinants in DEN virus. However, no evidence for recombination was found in West Nile or YF viruses, or in the tick-borne flavivirus group. It is proposed that the difference between the mosquito- and tick-borne viruses can be accounted for by their differing modes of transmission, whilst the variation among the mosquito-borne flaviviruses reflects both the ecology of the particular host and vector species and also bias in the sampling process.
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Evolution of hepatitis C virus in blood donors and their respective recipients
This paper describes the study of hepatitis C virus (HCV) evolution in the largest cohort of HCV-infected blood donors (BDs)/blood recipients (BRs) reported to date (25 pairs). A molecular analysis of partial sequences in the E1 (envelope) and NS5-B (polymerase) genes was performed. Phylogenetic reconstruction showed that the evolution of dominant strains was qualitatively and quantitatively different in BDs and BRs. The evolutionary rate was significantly higher in BRs, in which, in addition, most substitutions observed were antonymous. These findings corroborate the hypothesis that a large part of virus evolution – which was evaluated to be equivalent to ∼20 years of chronic evolution – is acquired during the early phase of infection. These findings should be taken into account for the modelling of the long-term evolution of HCV and their possible contribution to improve our understanding of HCV natural history is discussed.
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Insertion of cellular sequence and RNA recombination in the structural protein coding region of cytopathogenic bovine viral diarrhoea virus
More LessThe cytopathogenic bovine viral diarrhoea virus (cp BVDV) strain KS86-1cp was isolated from a calf persistently infected with the noncytopathogenic (ncp) strain KS86-1ncp after it was exposed to cp BVDV strain Nose and developed mucosal disease (MD). Molecular analysis revealed that an insertion of a cellular gene and a duplication of the viral RNA encoding the nucleocapsid protein C and part of Npro had occurred in the C coding region of the Nose and KS86-1cp genomes. The inserted cellular gene was closely related to the cINS sequence. Remarkably, the 5′ upstream region from the insertion of KS86-1cp had high sequence identity to that of Nose, but differed from that of KS86-1ncp. In contrast, the region downstream from the insertion of KS86-1cp showed high identity to KS86-1ncp, but not to Nose. These data reveal that KS86-1cp is a chimeric virus generated by homologous RNA recombination in a calf with MD.
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Upregulation of IL-10 gene expression in porcine peripheral blood mononuclear cells by porcine reproductive and respiratory syndrome virus
More LessSeveral lines of evidence suggest that porcine reproductive and respiratory syndrome virus (PRRSV) may have immunomodulatory effects on the host immune system. To determine the effect of PRRSV on cytokine production, a multiplex PCR was established. This allowed a semi-quantitative analysis of IFN-γ, IL-2, IL-4, IL-10 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression levels from porcine peripheral blood mononuclear cells (PBMCs). These results showed that both live and inactivated PRRSV predominantly upregulated IL-10 gene expression in porcine PBMCs. In addition, when PBMCs from pigs immunized previously with classical swine fever virus (CSFV) vaccine were cultivated with the recall antigen, CSFV, in the presence of PRRSV, significant upregulation of IL-10 gene expression and reduction of IFN-γ gene expression were observed. These findings indicated that the presence of PRRSV in the culture could affect recall antigen response. This study implies that the induction of IL-10 production may be one of the strategies used by PRRSV to modulate host immune responses.
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The complete genomic sequence of hepatitis delta virus genotype IIb prevalent in Okinawa, Japan
More LessThe Miyako Islands, located in the southernmost part of Japan, have been reported to be endemic for hepatitis delta virus (HDV). The majority of HDV patients in this area exhibit a relatively mild course of infection that evolves into a quiescent cirrhotic condition. The entire nucleotide sequence of the Miyako isolate (L215) of HDV obtained from a cirrhotic patient infected with HDV was determined. This isolate, L215, comprises 1682 nt and encodes 213 aa of the hepatitis delta antigen. Phylogenetic analysis showed that L215 is closely related to the Taiwanese genotype IIb HDV isolate. In addition, the predicted folding structure of the antigenomic RNA substrate was different from those of the published genotype II sequences.
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Phylogenetic relationships among members of the genus Phlebovirus (Bunyaviridae) based on partial M segment sequence analyses
Viruses in the Phlebovirus genus of the family Bunyaviridae cause clinical syndromes ranging from a short, self-limiting febrile illness to fatal haemorrhagic fever. The genus currently consists of 68 antigenically distinct virus serotypes, most of which have not been genetically characterized. RT-PCR with four ‘cocktail’ primers was performed to amplify a region of the M segment of the genome of 24 phleboviruses included in the sandfly fever Naples, sandfly fever Sicilian and Punta Toro serocomplexes. Partial M segment sequences were successfully obtained and phylogenetic analysis was performed. The three resultant genotypic lineages were consistent with serological data. The sequence divergences were 27·6 % (nucleotide) and 25·7 % (amino acid) within the Sicilian serocomplex, 33·7 % (nucleotide) and 34·4 % (amino acid) within the Naples serocomplex and 35·6 % (nucleotide) and 37·5 % (amino acid) within the Punta Toro serocomplex. Overall, the diversities among viruses of Sicilian, Naples and Punta Toro serocomplexes were 48·2 % and 57·6 % at the nucleotide and amino acid levels, respectively. This high genetic divergence may explain the difficulties in designing a consensus primer pair for the amplification of all the phleboviruses using RT-PCR. It also suggests that infection with one genotype may not completely immunize against infection with all other genotypes in a given serocomplex. These findings have implications for potential vaccine development and may help explain clinical reports of multiple episodes of sandfly fever in the same individual.
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Effect of fusion protein cleavage site mutations on virulence of Newcastle disease virus: non-virulent cleavage site mutants revert to virulence after one passage in chicken brain
More LessVirulence of Newcastle disease virus (NDV) is mainly determined by the amino acid sequence of the fusion (F0) protein cleavage site. Full-length NDV cDNA clone pNDFL was used to generate infectious NDV with defined mutations in the F0 cleavage site (RRQRR↓L, GRQGR↓F, RRQGR↓F, RGQRR↓F and RKQKR↓F). All the mutants were viable and the mutations were maintained after virus propagation in embryonated eggs. The mutants showed single-cell infections on chicken embryo fibroblasts, which suggested that they were non-virulent. However, virulence tests in 1-day-old chickens resulted in an intracerebral pathogenicity index (ICPI) between 0 and 1·3. Moreover, virulent virus was isolated from chickens that had died in the virulence tests. Subsequent sequence analysis showed that the mutants RRQRR↓L, RRQGR↓F, RGQRR↓F and RKQKR↓F gave rise to the appearance of revertants containing the virulent cleavage site RRQ(K/R)R↓F and an ICPI of 1·4 or higher. This indicated that reversion to virulence was caused by alteration of the amino acid sequence of the F0 cleavage site from a non-virulent to a virulent type. Furthermore, the ICPI of the revertants was higher than that of cDNA-derived strain NDFLtag, which has the same cleavage site, RRQRR↓F (ICPI=1·3). NDFLtagPass, which was isolated from dead chickens after intracerebral inoculation of NDFLtag, also showed an increase in the ICPI from 1·3 to 1·5. This study proves that reversion to virulence occurs within non-virulent NDV populations and that the virulence may increase after one passage in chicken brain.
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A molecular epidemiological study of Australian bat lyssavirus
The genetic diversity of Australian bat lyssavirus (ABL) was investigated by comparing 24 ABL isolate glycoprotein (G) gene nucleotide sequences with those of 37 lyssaviruses representing Lyssavirus genotypes 1–6. Phylogenetic analyses indicated that ABL forms a monophyletic group separate from other lyssaviruses. This group differentiates into two clades: one associated with Pteropus (flying fox) species, the other with the insectivorous bat Saccolaimus flaviventris. Calculation of percentage nucleotide identities between isolates of the two clades revealed up to 18·7 % nucleotide sequence divergence between the two ABL variants. These observations suggest that ABL is a separate lyssavirus species with a similar epidemiology to chiropteran rabies virus (RV), where two distinct ABL variants co-exist in Australia in bat species with dissimilar ecology. Analyses of selection pressures in ABL G gene sequences provided some evidence of weak positive selection within the endodomain at amino acids 499 and 501, although in general the dominant evolutionary process observed was purifying selection. This intimates that, in nature, isolates of ABL, like those of RV, are subject to relatively strong selective constraints, suggesting a stability of host species, cell tropisms and ecological conditions.
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- DNA viruses
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Nuclear matrix localization and SUMO-1 modification of adenovirus type 5 E1b 55K protein are controlled by E4 Orf6 protein
More LessHuman adenovirus serotype 5 encodes three proteins, E1b 55K, E4 Orf3 and E4 Orf6, which interact with each other and with components of the nucleus to regulate mRNA processing and export, viral DNA replication and p53-dependent apoptosis. Previous studies have shown that, during wild-type infection, 55K associates initially with structures termed ND10, which are sites of localization of the promyelocytic leukaemia protein, and then moves, dependent upon its interaction with Orf6, to the establishing virus replication centres. Absence of either Orf3 or Orf6 affects the localization of 55K and so may affect its function. In this study, the influence of Orf3 and Orf6 expression on the association of 55K with the insoluble matrix fraction of the nucleus and with ND10 particularly was examined. Overexpression of Orf6 was sufficient to block the association of 55K with this fraction, irrespective of the presence of Orf3. This effect depended upon the two proteins being able to interact. However, the association of 55K with ND10, which persists throughout infection in the absence of Orf6, required Orf3 to be present, thus distinguishing two subsets of matrix-associated 55K. A modified form of 55K, formation of which was blocked by mutating the known site of SUMO-1 attachment, was more abundant in the absence of Orf6 but unaffected by the absence of Orf3. Thus, this modification is favoured when 55K remains associated with the matrix but does not correlate with its stable association with ND10, many components of which are modified by SUMO-1.
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A ‘first loop’ linear epitope accessible on native hepatitis B surface antigen that persists in the face of ‘second loop’ immune escape
More LessMurine monoclonal antibodies (mAbs) were raised following immunization with native mutant hepatitis B surface antigen (HBsAg) purified from human sera. A set of antibodies binding to a linear epitope carried between residues 121 and 129 of the s region was demonstrated. These antibodies were shown by cross-competition assays to bind to a single epitope whose antigenicity was influenced by the TTP motif lying between residues 125 and 127. This first loop epitope remained accessible on the surface of HBsAg in spite of major second loop mutations abrogating the normal a conformational epitopes. The mAb and its binding region in the first loop are important diagnostically and may represent an importance immunological target, one that is stable in the face of immunologically driven escape.
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Purification and biochemical characterization of the E1 replication initiation protein of the cutaneous human papillomavirus type 1
More LessThe E1 and E2 proteins encoded by papillomaviruses are required for viral DNA replication. Although E1 is the replication initiator protein, previous studies have shown that the full-length E1 protein binds to the origin weakly and with low sequence specificity. The E2 protein facilitates binding of the E1 protein to the origin, triggering the initiation of replication. The E1 protein contains ATPase, helicase and DNA unwinding activities. In vivo studies with mucosal human papillomavirus (HPV) types 11 and 18 have shown that while E1 is absolutely essential for replication, the E1 binding site is dispensable. However, both the E2 protein and E2 binding sites are required for their replication. In contrast to these HPVs, transient replication of HPV type 1, which infects cutaneous tissue, requires only the viral E1 protein and E1 binding site. To understand the basis for these differences, we have overexpressed and purified the HPV-1 E1 and E2 proteins and studied their biochemical properties. The purified E1 protein was shown to have an ATPase activity with a very low K m value, similar to that of the SV40 large T antigen. The E1 protein bound to the HPV-1 origin in the absence of the E2 protein and without the use of any cross-linking agents. Our results suggest that the ability of the HPV-1 E1 protein to initiate DNA replication in vivo in the absence of the E2 protein may be due to its stable interaction with the HPV-1 origin.
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Induction of humoral and cell-mediated immunity to hepatitis B surface antigen by a novel adjuvant activity of Oka varicella vaccine
More LessOka varicella vaccine induces humoral and cell-mediated immunity to varicella-zoster virus (VZV), even in immunocompromised hosts. This vaccine showed novel adjuvant activity against co-inoculated hepatitis B surface antigen (HBsAg). Either a mixed inoculation of HBsAg with heat-inactivated Oka varicella vaccine at one site or a separate inoculation of HBsAg and live vaccine at different sites induced an antibody response but failed to induce delayed type hypersensitivity (DTH) to HBsAg. In contrast, immunization of HBsAg mixed with live vaccine induced DTH and an enhanced antibody response to HBsAg. The adjuvant activity of Oka varicella vaccine was similar in terms of antibody production to that of alum adjuvant. A T helper cell-dominant immunity to VZV and HBsAg continued for 1 year. Oka varicella vaccine combined with a foreign antigen may serve as a novel polyvalent vaccine for the infectious diseases for which cell-mediated immunity is beneficial.
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Down-regulation of MHC class I expression by equine herpesvirus-1
More LessThere is good evidence that cytotoxic T lymphocytes play an important role in the clearance of equine herpesvirus-1 (EHV1) in horses. We have demonstrated that, in common with other alphaherpesviruses, EHV1 infection can lead to dramatic down-regulation of MHC class I expression at the cell surface, a common strategy for pathogen evasion of the host immune response. This down-regulation is specific for MHC class I and does not reflect a general shut-off of host-cell protein synthesis. The use of monoclonal antibodies that recognize different MHC class I epitopes has demonstrated that the effect may be allele- or locus-specific. Use of the viral DNA synthesis inhibitor phosphonoacetic acid, which prevents late viral gene expression, showed that the effect is mediated by an immediate-early or early viral gene, and use of the protein translation inhibitor cycloheximide confirmed that an early gene is primarily responsible. The data indicate that EHV1 infection results in enhanced endocytosis of MHC class I from the cell surface; the only other herpesvirus reported to use this mechanism is human herpesvirus-8. Elucidation of the precise mechanisms used by EHV1 in this process and identification of the genes responsible may lead to improved vaccination strategies.
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Mutagenesis of a bovine herpesvirus type 1 genome cloned as an infectious bacterial artificial chromosome: analysis of glycoprotein E and G double deletion mutants
More LessThe genome of bovine herpesvirus type 1 Schönböken was cloned as a bacterial artificial chromosome (BAC) by inserting mini F plasmid sequences into the glycoprotein (g) E gene. The resulting BAC clone, pBHV-1ΔgE, was transfected into bovine kidney cells and viable gE-negative BHV-1 (BHV-1ΔgE) was recovered. By RecE/T mutagenesis in Escherichia coli, the gG open reading frame was deleted from pBHV-1ΔgE. From the mutated BAC, double negative BHV-1ΔgE-gG was reconstituted and its growth properties were compared to those of rescuant viruses in which the gE gene was restored (BHV-1rev, BHV-1ΔgG). The mutant viruses did not exhibit markedly lowered virus titres. Plaque sizes of BHV-1ΔgE, BHV-1ΔgE-gG and BHV-1ΔgG, however, were reduced by 19 to 55 % compared to parental strain Schönböken or BHV-1rev. Our results suggested that gE and gG function independently from each other in cell-to-cell spread, because an additive effect on plaque formation was observed in the gE/gG double deletion mutant.
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Induction of HLA-G-restricted human cytomegalovirus pp65 (UL83)-specific cytotoxic T lymphocytes in HLA-G transgenic mice
The non-classical major histocompatibility complex class I molecule HLA-G is expressed mainly by extravillous trophoblasts at the materno–foetal interface. HLA-G has been found to bind endogenously processed nonameric peptides but its function as a restriction element for a cytotoxic T cell response to viruses with tropism for trophoblastic cells has never been demonstrated. In this study, candidate viral peptides derived from human cytomegalovirus (HCMV) pp65 (UL83), which stabilized the HLA-G molecule on HLA-G-transfected T2 cells, were identified. The specific anti-pp65 cytotoxic T lymphocyte (CTL) response restricted by HLA-G in triple transgenic mice (HLA-G, human β2m, human CD8α) was then investigated by injection of dendritic cells loaded with synthetic pp65-derived peptides or by infection with canarypox virus expressing pp65. Results showed that CTLs from HLA-G mice have the capacity to kill target cells either infected with recombinant vaccinia viruses expressing pp65 or loaded with specific pp65-derived peptides using HLA-G as an antigen-presenting molecule. It was also demonstrated that these HLA-G-restricted pp65-specific T cells are able to kill the human astrocytoma cell line U373, which was transfected with HLA-G and infected with HCMV. Moreover, using HLA-G tetramers refolded with a synthetic pp65-derived peptide, peptide-specific CD8+ cells restricted by HLA-G have been detected in vivo. These findings provide the first evidence that HLA-G can select anti-HCMV-restricted CTLs in vivo, although the potency of this cytolytic response is limited (20–25 %). The weak HLA-G-restricted anti-HCMV response is probably due to HLA-G-mediated inhibitory signals on the development of an antiviral CTL response.
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Physical interaction of Epstein–Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) with human oestrogen-related receptor 1 (hERR1): hERR1 interacts with a conserved domain of EBNA-LP that is critical for EBV-induced B-cell immortalization
More LessEpstein–Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) consists of W1W2 repeats and a unique C-terminal Y1Y2 domain and plays a critical role in EBV-induced transformation. To identify the cellular proteins associating with EBNA-LP, we performed a yeast two-hybrid screen using EBNA-LP cDNA containing a single W1W2 domain as bait and an EBV-transformed human peripheral blood lymphocyte cDNA library as the source of cellular genes. Our results were as follows. (i) A cDNA in the positive yeast colony was found to encode a cellular protein, human oestrogen-related receptor 1 (hERR1), which is a constitutive transcriptional activator of the various types of oestrogen response elements. (ii) A purified chimeric protein consisting of glutathione S-transferase (GST) fused to hERR1 specifically formed complexes with EBNA-LPs containing one (EBNA-LPR1), two (EBNA-LPR2) or four W1W2 repeats (EBNA-LPR4) transiently expressed in COS-7 cells. Reciprocally, GST fused to EBNA-LPR1 or EBNA-LPR2 pulled down hERR1 transiently expressed in COS-7 cells. (iii) Mutational analyses of EBNA-LP revealed that the Y2 domain of EBNA-LP is responsible for the interaction with hERR1 and two leucines in the Y2 domain (Leu-78 and -82), which are conserved among a subset of primate gammaherpesviruses, are interactive sites for hERR1. So far, it has been reported that the only domain of EBNA-LP critical for EBV-induced transformation is the Y1Y2 domain. Potential roles of hERR1 in EBV-induced transformation are discussed.
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Auto-activation of the transforming viral interferon regulatory factor encoded by Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8)
More LessKaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8-encoded viral interferon regulatory factor (vIRF) transforms NIH3T3 cells, represses interferon signal transduction and regulates the expression of other KSHV genes. Here, we have shown that vIRF is a transcriptional activator and auto-activates its own expression. Ectopic expression of vIRF activated the vIRF promoter in KSHV-negative 293, COS7, HeLa and BJAB cell lines in a dose-dependent fashion in a reporter assay and the expression of vIRF transcripts from endogenous viral genomes in BCBL-1 and BC-1 cells latently infected with KSHV. Deletion analysis identified two cis elements, named Vac1 and Vac2, in the vIRF promoter that were responsive to vIRF activation. vIRF auto-activation via Vac1 but not Vac2 was repressed by Tis, a transcriptional silencer in the vIRF promoter. Neither Vac1 nor Vac2 contain any interferon-stimulated response element (ISRE)-like sequences and are unresponsive to induction with interferon-β and -γ. These results indicate that KSHV uses the mechanism of auto-activation to regulate the expression of a viral transforming protein to efficiently evade host tumour suppressor pathways.
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Absence of a functional defect in CD8+ T cells during primary murine gammaherpesvirus-68 infection of I-Ab−/− mice
More LessThe murine gammaherpesvirus-68 kills I-Ab−/− mice despite the presence of virus-specific CD8+ cytotoxic T lymphocytes (CTL). This has raised the possibility that these CTL are functionally abnormal. Here, no difference was observed between I-Ab−/− mice and I-Ab+/+ controls in virus-specific CTL function, T cell receptor usage, or surface phenotype. Thus CTL immunity was independent of CD4+ T cells in a chronic herpesvirus infection, but was still inadequate to control virus replication.
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Early pathogenesis of Autographa californica multiple nucleopolyhedrovirus and Helicoverpa zea single nucleopolyhedrovirus in Heliothis virescens: a comparison of the ‘M’ and ‘S’ strategies for establishing fatal infection
More LessNucleopolyhedroviruses (NPVs) (Baculoviridae) produce fatal infections in larval lepidopteran insects. NPVs are designated SNPVs or MNPVs based on whether the occlusion-derived virus (ODV) that initiates primary midgut infections contains single (S) or multiple (M) nucleocapsids. The principal consequence of this ODV packaging is that primary target cells infected with the M phenotype receive multiple nucleocapsids, whereas those infected by the S phenotype receive only one. To determine the biological significance of this difference in the initial infection strategy, a comparison of the primary and secondary infection patterns of the recombinants Helicoverpa zea SNPV (HzSNPV-hsp70/lacZ) and Autographa californica MNPV (AcMNPV-hsp70/lacZ) in orally inoculated larvae of Heliothis virescens was carried out. At dosages yielding similar final mortalities (∼85 %), primary midgut infections by HzSNPV-hsp70/lacZ (indicated by lacZ expression) were observed 6 h earlier and in greater numbers than those generated by AcMNPV-hsp70/lacZ. Infection of secondary target cells in the tracheal epidermis, however, occurred at the same time and at the same rate for both NPVs. A 2 h delay was observed between the onset of primary and secondary AcMNPV-hsp70/lacZ infection, supporting the hypothesis that early tracheal infections were initiated by ODV nucleocapsids repackaged as budded virus. In contrast, an 8 h delay was observed with HzSNPV-hsp70/lacZ, suggesting that systemic infections were established only after virus replication in primary targets. Significant numbers of both MNPV- and SNPV-infected primary target cells were sloughed from the midgut beginning as early as 16 h post-infection. Midgut cell sloughing may be an important host-mediated selection pressure influencing the evolution of NPV morphology and gene regulation, shaping, in part, baculovirus infection strategies.
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The catalytic subunit of protein kinase CK2 phosphorylates in vitro the movement protein of Tomato mosaic virus
More LessThe movement protein (MP) of Tomato mosaic virus (ToMV) was reported previously by us to be phosphorylated in vitro by a cellular protein kinase(s) that exhibited several characteristics of casein kinase 2 (CK2). To characterize further this CK2-like cellular kinase, we have cloned cDNAs encoding the CK2 catalytic subunit from tobacco and compared the properties of the recombinant protein with those of the CK2-like cellular kinase. The recombinant CK2 catalytic subunit formed a complex with ToMV MP and phosphorylated it, similar to the CK2-like cellular kinase. Phosphoamino acid analyses of various mutant MPs altered near the C terminus revealed that the recombinant CK2 catalytic subunit phosphorylated serine-261, while the CK2-like cellular kinase phosphorylated both serine-261 and threonine-256. Both kinases were suggested to phosphorylate an additional serine residue(s) in regions other than the C-terminal peptide. The results are consistent with our previous prediction of involvement of CK2 in phosphorylation of ToMV MP.
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