- Volume 77, Issue 2, 1996
Volume 77, Issue 2, 1996
- Animal
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- RNA viruses
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Amino acids involved in distinguishing between monotypes of rotavirus G serotypes 2 and 4
More LessNeutralizing monoclonal antibodies (N-MAbs) to serotype G2 and G4 rotaviruses were used to study intraserotypic variation by selection and characterization of N-MAb-resistant antigenic variants and reaction of N-MAbs with prototype rotavirus strains. Two G2-specific N-MAbs reacted with G2 rotaviruses S2, DS-1, RV-5 and RV-6 but not with 1076. Sequence analysis of the gene encoding VP7 of 1076 virus showed that the differences in amino acid sequence between 1076 virus and the other G2 strains at position 147, 213 and 217 correlated with the loss of N-MAb reactivity. Rotavirus variant mutation mapping data suggested that the amino acid difference at position 213 was likely to be of greatest importance. Rotavirus 1076 was defined as monotype b within G2 strains, whereas S2, DS-1, RV-5 and RV-6 belong to monotype a. The molecular basis for G4 subtypes/monotypes was also studied. The monotype G4b N-MAb 3A3 selected an antigenic variant with an amino acid mutation at position 96, whereas variants of the G4a-reactive N-MAb ST-3:1 showed a mutation at position 94, which produced a new, utilized glycosylation site. Neutralization by N-MAb ST-3:1 was also affected by amino acid changes at position 96. Reactions with these N-MAbs show that serotype G2 viruses can be divided into monotypes and confirm the observation that serotype G4 rotaviruses can be subdivided into subtypes/monotypes a and b. The G2 monotypes relate to differences at particular amino acids within antigenic region C and possibly region B, whereas antigenic region A is most important for G4 monotype differentiation.
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Identification of hepatitis A virus non-structural protein 2B and its release by the major virus protease 3C
More LessThe RNA genome of hepatitis A virus (HAV) encodes a giant polyprotein that is putatively cleaved proteolytically into four structural and seven non-structural proteins. So far, most of the proposed non-structural proteins and their respective cleavage sites have not been identified. A vaccinia virus recombinant (vRGORF) containing the complete HAV ORF under the control of the bacteriophage T7 promoter was used to express HAV in recombinant animal cells (BT7-H) that constitutively expressed T7 DNA-dependent RNA polymerase. A HAV-specific 27.5 kDa expression product was identified as peptide 2B. The 27.5 kDa 2B antigen was also found in HAV-infected MRC-5 cells. The N-terminal amino acid residues of the new peptide 2B are Ala-Lys-Ile-Ser-Leu-Phe and polyprotein cleavage between 2A and 2B occurred at amino acids 836–837 (Gln-Ala). Furthermore, heterologous expression in the same system of regions P1–P2 and of the protease 3C (3Cpro) gene, showed that P1–P2 polyprotein is not cleaved autocatalytically but by 3Cpro. Hence, 3Cpro is effective in cleaving the polyprotein 2A–2B junction.
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Antibody and host cell recognition of foot-and-mouth disease virus (serotype C) cleaved at the Arg-Gly-Asp (RGD) motif: a structural interpretation
More LessFoot-and-mouth disease virus (FMDV) of serotype C (isolate C-S8c1) was cleaved in situ by trypsin at the Arg-Gly-Asp (RGD) motif, which is involved both in attachment of FMDV to cells and in recognition of a major antigenic site (site A) by antibodies. Though 99.4% of the RGD moieties were cleaved, the virus remained infectious. A synthetic peptide which represented the sequence of the VP1 G-H loop of C-S8c1, including the RGD motif, greatly inhibited FMDV attachment to cells. The same peptide inhibited, very effectively and to the same extent (50% inhibition at about 1 µm), the infectivity of both intact and trypsin-treated virus. Replacement of Asp with Glu at the RGD motif abolished the inhibitory effects of the peptide. Thus, the RGD motif is involved in the infectivity of both intact and RGD-cleaved serotype C FMDV. Trypsin treatment did not affect the reactivity of the virus with some monoclonal antibodies (MAbs) directed to site A whose epitopes involve mainly residues contiguous to the cleaved bond, but diminished the reactivity with site A MAbs whose epitopes include the RGD sequence and flanking residues. However, high concentrations of any site A MAb tested neutralized close to 100% of the infectious trypsin-treated virus. We propose that, in spite of covalent cleavage, the high number of intramolecular non-covalent interactions observed within the G-H loop of FMDV C-S8c1 (complexed to antibody) may hold the RGD in a nearly correct conformation and allow — albeit with reduced affinity — antibody and cell receptor recognition of RGD-cleaved FMDV.
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Recognition of the initiation codon for protein synthesis in foot-and-mouth disease virus RNA
More LessFoot-and-mouth disease virus (FMDV) RNA utilizes two in-frame initiation codons to produce two precursor proteins with identical carboxy termini. The 5′ untranslated region (5′UTR) directs the ribosome to internal sequences without the need for a cap structure as used in host mRNAs. The FMDV 5′UTR was cloned upstream of the reporter gene chloramphenicol acetyltransferase (CAT) in order to study the selection of initiation site and to facilitate quantification of the translation products. After in vitro transcription with T7 RNA polymerase and translation in rabbit reticulocyte lysate, the two CAT products, resulting from initiation from the two initiation codons, were quantified. The downstream initiator AUG (AUGLb) was selected more efficiently in the wild-type 5′UTR. In truncated RNA, the upstream initiation site (AUGLab) was more efficiently utilized than in the wild-type 5′UTR. Protein synthesis initiation factors were added to translation assays to determine whether these factors influenced initiation site selection. Addition of eIF-2 and of eIF-2B changed the selection process for both types of RNA. These factors induced a 2.5-fold higher usage of the upstream AUGLab for wild-type and 5′UTR-truncated RNA. A change in mRNA concentration also induced a change in the usage of initiation codons; however, the effect of eIF-2 was measured over a broad range of mRNA concentrations. In conclusion, eIF-2 mediates the recognition of the initiation codon during both cap-dependent and internal ribosome entry site-dependent initiation.
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Limited genetic changes in the Sabin 1 strain of poliovirus occurring in the central nervous system of monkeys
Replication of attenuated poliovirus strains results in their partial deattenuation. Recently we identified mutations accumulating in the Sabin 1 poliovirus in cell cultures. Here we report genetic changes occurring in this virus during replication in the central nervous system (CNS) of monkeys. Viruses isolated from different parts of the CNS of rhesus monkeys (inoculated into the spinal cord) were screened for sequence heterogeneities and newly identified mutations were independently confirmed and quantified using mutant analysis by PCR and restriction enzyme cleavage (MAPREC). All consistently accumulating mutations identified in this study were located in untranslated regions: GU → AU or GU → GC substitution at a complementary pair formed by nucleotides 480 and 525, U → C substitution at nucleotide 612, and GU → AU or GU → GC substitution of a base pair formed by the nucleotides 7427/7441 immediately preceding the poly(A) tract. All these mutations except one (7427) were previously identified in cell culture passages or stool isolates from vaccinees. Sequencing of 11 CNS isolates also identified a few random silent mutations that accumulated as neutral ‘passengers’, passively coselected with genuinely selectable mutations present on the same RNA molecule. One isolate also contained the wild-type base at nucleotide 2741 (Ala88 → Thr in VP1). Our results demonstrate a remarkable genetic stability of the Sabin 1 poliovirus in the CNS of monkeys, suggesting that deattenuation is determined by a very limited number of mutations. These mutations can be assayed by MAPREC to monitor the consistency of oral poliovirus vaccine (OPV) production.
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Role of immune responses in protection and pathogenesis during Semliki Forest virus encephalitis
More LessThe course of Semliki Forest virus (SFV) A7(74) infection in immunocompetent BALB/c, athymic nu/nu and severe combined immunodeficient (SCID) mice was compared. BALB/c mice remained healthy and exhibited transient viraemia and infectious virus in the brain from days 2 to 7. Antibodies were detectable by day 5. In comparison, SCID mice displayed a high incidence of paralysis and died: the average day of death was day 23. From infection until death, virus was present in blood and brain. No antibodies were detectable. Athymic mice were intermediate with a transient viraemia and a persistent (> 210 days) sub-clinical central nervous system (CNS) infection. These mice produced anti-viral IgM but not IgG. The pattern of infection in BALB/c or nu/nu mice could be recreated in infected SCID mice by transfer of immune serum from BALB/c or nu/nu mice, with the important exception that although BALB/c immune serum could abolish infectivity titres in the CNS, scattered cells positive for viral RNA remained. Transfer of serum decreased mortality and delayed the onset of paralysis. Transfer to infected SCID mice of a non-neutralizing IgG anti-E2 monoclonal antibody did not affect the viraemia but could also reduce brain virus titres. Irrespective of specific immune responses, virus replication in CNS cells was restricted, was generally non-cytopathic and in the absence of specific immune responses could persist. From day 14 lesions of inflammatory, primary demyelination were observed throughout the CNS of BALB/c mice. In contrast, despite prolonged brain virus titres, no demyelinating lesions were observed in infected nu/nu or SCID mice. Lesions could be initiated in the latter by transfer of spleen cells but not antibody. In summary, the focal restricted infection in the CNS of adult mice infected with SFV A7(74) is independent of specific immune responses. IgM antibodies clear the viraemia. IgG antibodies including non-neutralizing antibodies reduce and clear infectious virus but cells positive for viral RNA remain. These may normally be cleared by T cell responses which are damaging and give rise to lesions of demyelination.
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Hepatitis C virus variants from Jakarta, Indonesia classifiable into novel genotypes in the second (2e and 2f), tenth (10a) and eleventh (11a) genetic groups
Hepatitis C virus (HCV) isolates from 126 hepatitis patients in Jakarta, Indonesia were genotyped by PCR with genotype-specific primers deduced from the HCV core gene. Fifty-five isolates (44%) were classified as genotype II/1b, 15 (12%) as 1c, 33 (26%) as III/2a, and 1 (1%) as V/3a, while the remaining 22 (17%) were not classifiable into any of the five common genotypes (I/1a, II/1b, III/2a, IV/2b and V/3a) or 1c. Sequences of a part of the NS5b region [1093 bp (nucleotides 8279–9371)] of the 22 isolates of unclassifiable genotype were subjected to pair-wise comparison and phylogenetic analysis along with those of 62 isolates of 25 genotypes in nine genetic groups. Seven of the isolates were classified into 2e and two into 2f, representing novel genotypes in genetic group 2, while ten and three were classified into two new genetic groups, 10 and 11, respectively, and their genotypes were provisionally designated 10a and 11a. The isolates of genotype 10a (JK049) and 11a (JK046) were sequenced in full. Comparison of 24 HCV genomes including those of JK049 and JK046, over the entire genome and subgenomic regions, supported the classification of HCV into 11 genetic groups.
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Pathway of rubella virus infectious entry into Vero cells
The mechanism and the kinetics of rubella virus (RV) penetration into Vero cells were studied. By using pronase or acid treatment to inactivate virus which had adsorbed to cell membrane but had not been internalized, it was found that a period of 7 h was required in order for all of the adsorbed virus to enter the host cells. Lysosomotropic agents (monensin, methylamine, ammonium chloride and chloroquine) were used to study the mechanism by which RV penetrates host cells. Virus replication was inhibited if treatment of cells with these compounds was performed for at least 9 h after infection. However, if extracellular adsorbed virions were eliminated by acid treatment following removal of the lysosomotropic compounds, RV replication was completely inhibited by treatment with these drugs for any time period after adsorption. This indicated that the prolonged period of treatment with these compounds necessary to inhibit virus replication is due to the slow rate of RV internalization. None of the compounds had any effect on infection initiated by transfection of RV RNA, confirming that these drugs were exerting their inhibitory activity at penetration. The inhibition of RV replication by lysosomotropic compounds indicates that RV penetrates host cells by the endosomal pathway.
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An adenovirus recombinant expressing the spike glycoprotein of porcine respiratory coronavirus is immunogenic in swine
More LessThe full-length spike (S) gene of porcine respiratory coronavirus (PRCV) was inserted into the genome of human adenovirus type 5 downstream of the early transcription region 3 promoter. The recombinant virus replicated in cultures of the swine testicle ST cell line and directed the synthesis of S antigen with a maximum yield of approximately 26 µg per 106 cells. The antigen was cell-associated except in the late phase of the infection, when a small amount (3.5 µg per 106 cells) was released. The cell-associated antigen consisted of polypeptides of molecular mass 160 kDa and 175 kDa, comigrating with the authentic precursor S′ and the mature S protein of PRCV, respectively. The extracellular recombinant antigen corresponded to the 175 kDa mature protein. Some recombinant S protein was exposed on the cell surface and was recognized by neutralization-mediating anti-S monoclonal antibodies. Piglets, inoculated oronasally with the recombinant adenovirus vector developed PRCV-neutralizing serum antibodies and were partially protected against PRCV challenge, demonstrating the potential of live adenovirus as vaccine vector.
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The JHM strain of mouse hepatitis virus induces a spike protein-specific Db-restricted cytotoxic T cell response
More LessCytotoxic T lymphocyte (CTL) activity specific for mouse hepatitis virus (MHV) JHM strain (JHMV or MHV-4) was examined using in vitro stimulated spleen cells derived from immunized C57BL/6 (H-2b) mice. Target cells infected with JHMV were specifically recognized; however, analysis of target cells expressing the virus structural proteins via recombinant vaccinia viruses showed no recognition of the viral nucleocapsid (N), membrane (M), small membrane (sM) or haemagglutinin-esterase (HE) proteins. Only target cells expressing the virus spike (S) protein were recognized. Furthermore, the majority of CTL activity was restricted to target cells expressing the MHC class I Db molecules. Analysis of truncations and deletions of the S protein expressed by recombinant vaccinia viruses and peptide coated targets identified a single antigenic epitope, aa 510–518, conforming to the Db binding motif. These amino acids are contained within a domain deleted from a number of strains of mouse hepatitis virus, suggesting a role for immune pressure. To determine the potential for CTL specific for an epitope(s) within a non-structural protein, 24 CTL lines were established and characterized. No evidence for the induction of non-specific CTL activity or virus-specific CTL restricted to an epitope in a non-structural protein was obtained. These data indicate that the predominant CTL activity in JHMV-infected C57BL/6 mice is Db restricted and specific for a single epitope contained within aa 510–518 of the S protein.
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Identification of the sequences responsible for nuclear targeting of the V protein of human parainfluenza virus type 2
In human parainfluenza virus type 2 (hPIV-2)-infected cells, anti-phosphoprotein (P)-specific monoclonal antibody (MAb) densely stained the perinuclear regions of infected cells throughout infection, indicating that the P protein was localized exclusively in the cell cytoplasm. By contrast, antigens recognized by MAbs directed against the P-V-common domain of hPIV-2 were located predominantly in the cytoplasm, but in some hPIV-2-infected cells they were also found in the nuclei, suggesting that a fraction of hPIV-2V protein is localized there. hPIV-2 V protein expressed from a cDNA clone was localized in the nuclei of transfected cells. By using indirect immunofluorescence analyses, we examined the intracellular localization of various sequentially deleted V proteins, to determine the nuclear localization signals (NLS) of the V protein. Two noncontiguous regions in the V protein were required for nuclear localization and retention, since deletion of these regions [region I (aa 1–46) and region II (aa 175–196)] resulted in cytoplasmic localization. Both regions resulted in nuclear localization independently. A nucleoplasmin-like NLS was identified in region II but no consensus targeting sequence could be found in region I. When NP protein was co-expressed with V protein or the N-terminal fragment (aa 1–46) of V protein, a fraction of the NP protein was translocated into cell nuclei.
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Immunodominant epitopes defined by a yeast-expressed library of random fragments of the rabies virus glycoprotein map outside major antigenic sites
More LessNineteen yeast colonies secreting rabies virus glycoprotein (G) peptides immunoreactive with polyclonal anti-rabies virus sera were selected from a random expression library. The peptides, around 80 amino acids long, spanned amino acids 54–494 of the G protein. These peptides, together with two constructions including, respectively, immunodominant sites II and III, were analysed for their immunoreactivity with 40 anti-G protein monoclonal antibodies (MAbs) composed of 12 MAbs that reacted with SDS-treated protein in Western blot under reducing conditions (WB+) and 28 representative MAbs that did not react after denaturation (WB−). This last category represents 98% of anti-rabies virus G MAbs. None of the WB− MAbs bound peptides. Of the 12 WB+ MAbs, one bound two peptides situated before the transmembrane domain of the protein and six bound peptides overlapping a region situated between amino acids 223 and 276. These six MAbs define a new antigenic region that would be considered ‘immunodominant’ if the peptide strategy had been used to study the antigenicity of the protein; however, this region is only recognized by about 1% of our MAbs. Three of these WB+ MAbs had significant neutralizing activity; two were used for the selection of antigenic mutants (MAR mutants). Some mutants had a substitution within the region delimited by the peptides, confirming the pertinence of both the peptide and escape mutant approaches. However, a few mutants had a substitution outside the peptide-delimited region, suggesting that remote mutation(s) could affect epitope accessibility in the native protein.
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The genomic structure of a new simian T-lymphotropic virus, STLV-PH969, differs from that of human T-lymphotropic virus types I and II
More LessA new simian T-lymphotropic virus, STLV-PH969, was recently isolated from a wild-born Hamadryas baboon. Previous analysis had revealed that it differs sufficiently from the other HTLV/STLVs to be considered a new type, provisionally designated primate T-lymphotropic virus-L. Here we analyse a 3850 bp cDNA fragment spanning the 3′ part of the STLV-PH969 genome. The fragment encodes three major proteins: Env, Tax and Rex. Sequence comparison and phylogenetic analysis indicate that in general STLV-PH969 tends to be more closely related to HTLV-II than to HTLV-I, although separate gene regions might have evolved under different constraints. Detailed comparison of the Env, Tax and Rex proteins among the HTLV-I, -II and STLV-PH969 prototypes reveals that the amino acid sequence of each protein shows a preferential conservation of functionally important domains. RNA-PCR on cytoplasmic messengers demonstrated splicing between a splice donor site immediately downstream of the env start codon, and two splice acceptor sites identified in the pX region. The predominant spliced messenger encodes both Tax and Rex. The other messenger potentially encodes a new viral protein from the proximal part of the pX region that is similar in amino acid composition to p12I and p10xI of HTLV-I and HTLV-II respectively. This genomic organization of the proximal pX region of STLV-PH969 is different from that found in HTLV-I and HTLV-II. Therefore, the distinct classification of this virus can be justified, not only in terms of sequence divergence but also in terms of its different genomic structure.
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The three human T-lymphotropic virus type I subtypes arose from three geographically distinct simian reservoirs
To investigate the origin of human T-lymphotropic virus I (HTLV-I), strains of diverse geographical origin were analysed. We sequenced the LTR and env genes of HTLV-I strains from Brazil, Central African Republic, Taiwan and Zaire, and the simian T-lymphotropic virus type I (STLV-I) strain PHSu1 from a baboon from the Sukhumi primate centre. We performed phylogenetic analyses using neighbour-joining, parsimony and maximum likelihood methods. Three separate HTLV-I clusters were identified interspersed between STLV-I clusters. The Brazilian and the Taiwanese strains were within the first well-supported cluster containing all cosmopolitan HTLV-I strains flanked by west African STLV-I strains. The HTLV-I strains from Central African Republic and Zaire fell into a central African cluster close to the chimpanzee STLV-I isolates. The third well-supported cluster included all Melanesian HTLV-I strains and had Indonesian STLV-I strains as closest neighbours. Therefore, currently known HTLV-I strains represent three HTLV-I subtypes that most probably have originated from three geographically distinct interspecies transmission events. The highly divergent PHSu1, isolated from Papio hamadryas, was closely related to PCY-991, isolated from Papio cynocephalus, both from the Sukhumi primate centre. Both clustered together with Asian wild-caught rhesus macaque STLV-I strains suggesting recent interspecies transmission of virus from rhesus macaques to colony-bred African baboons at the Sukhumi primate centre. In the rooted env trees obtained using the STLV strain PH969 as an outgroup, the Asian strains branched off before the African strains, implying an Asian origin for HTLV/STLV type I based on presently available strains.
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Transfer of human T cell lymphotropic virus type I to human term trophoblast cells in vitro
More LessWe studied the susceptibility of term placental trophoblast cells to in vitro infection with human T cell leukaemia/lymphotropic virus type I in order to provide further insight into the role of syncytiotrophoblast in transplacental passage of the virus. Pure villous trophoblast cultures were exposed to cell-free virus and the extent of infection was analysed by semiquantitative PCR assay to detect integrated proviral DNA. Four different primer pairs targeting the gag, pol, env and pX regions invariably revealed that virus sequences were present in amounts 102–103 times less than in the reference cell line MT-2. Virus expression was studied at both the transcriptional and translational levels. Whereas doubly spliced mRNAs coding for the Tax and Rex regulatory proteins could be detected by RT-PCR no virus-specific proteins were found in the cells by immunoperoxidase staining. The present data lend support to the notion that the placental trophoblast may represent a barrier effectively protecting the fetal compartment from exposure to the virus.
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Characterization of the human endogenous retrovirus K proteinase
The proteinase of the human endogenous retrovirus K (HERV-K) shows similarity to retrovirus aspartic proteinases. It is translated from a transcript composed of gag and prt. The proteinase was expressed either as full-length native protein or as truncated protein in Escherichia coli. Functional protein was demonstrated by its autocatalytic cleavage into an 18 kDa fragment recognized by a polyclonal antiserum. This autocatalytic cleavage was specifically inhibited by a human immunodeficiency virus type 1 proteinase inhibitor. The HERV-K proteinase expressed in E. coli was capable of cleaving HERV-K Gag translated in vitro. Major protein fragments of 39 and 30 kDa, and minor protein fragments of 26, 22 and 21 kDa were obtained. Similar fragments are also observed in the human teratocarcinoma cell line Teral. Our data suggest that the HERV-K proteinase is functionally equivalent to other retrovirus proteinases and thus probably functions in the processing of Gag precursor protein.
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- DNA viruses
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The hepatitis B virus X protein is a potent AMP kinase
More LessThe hepatitis B virus X-protein (HBx) has been expressed in Escherichia coli both as an unfused protein and with an N-terminal hexaHis-containing fusion sequence. Both forms of HBx, after purification, displayed a potent AMP kinase activity, in which HBx phosphorylates AMP to ADP, using ATP as the exclusive phosphate donor. We also found that HBx has previously unreported GTPase and GTP-ADP nucleoside diphosphate kinase activities.
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Unusual activation of the integrated preS1 promoter of woodchuck hepatitis virus in a liver tumour
More LessWe have analysed abnormal virus RNAs produced from integrated woodchuck hepatitis virus (WHV) sequences in two woodchuck liver tumours. Analysis of cDNA clones revealed that these transcripts consisted of rearranged, virus-specific RNAs encoding the WHV surface antigens. In one tumour, transcription was driven by the major preS2/S promoter and terminated at a cryptic poly(A) signal in the 5′ end of the P gene, giving rise to a truncated version of the normal viral S message. In contrast, the integrated preS2/S promoter remained silent in the second tumour. The start sites of two abundant WHV transcripts encoding the large and middle surface proteins were localized about 100 bp upstream and 300 bp downstream of the preS1 translation initiation codon, corresponding to minor start sites of the normal surface protein mRNAs in chronically infected liver. Thus, the preS1 promoter, a weak promoter in episomal replicative forms of the virus, was activated in the integrated state in this tumour. Our results indicate that alternative usage of the preS1 or the preS2/S promoter in the integrated state may yield differential production of the three virus surface proteins in woodchuck liver tumours.
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Antigenicity of bovine papillomavirus type 1 (BPV-1) L1 virus-like particles compared with that of intact BPV-1 virions
More LessVirus-like-particles (VLPs) of various papillomavirus (PV) types have been produced by expressing recombinant L1 proteins in eukaryotic cells. Although VLPs have the same ultrastructural appearance as native virions and their immunogenicity appears to be similar, their antigenicity has not been carefully evaluated. For this reason, the antigenicity of intact bovine PV type 1 (BPV-1) virions was compared with that of BPV-1 recombinant L1 VLPs by ELISA using a well-characterized panel of polyclonal and monoclonal antibodies generated against intact and denatured BPV-1 particles. The structural integrity of the authentic virions and recombinant VLPs was verified by electron microscopy. The specificity of antibodies raised against intact BPV-1 virions and their reactivity with VLPs revealed that the immunodominant, type-specific, conformational epitopes of intact virions were reproduced on VLPs. However, many monoclonal antibodies that define cross-reactive, non-conformational (linear) epitopes cryptic to the authentic BPV-1 virion tested positively when reacted with intact VLPs. One monoclonal antibody, which recognizes a BPV-1 and deer PV surface conformational epitope, did not react with VLPs. Therefore, although VLPs can be used to immunize animals against infection, the external exposure of broadly cross-reactive epitopes of intact L1 VLPs suggests that the use of L1 VLPs in antigenicity studies such as serological screening should be done with caution.
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Sp1 is critical for basal and E2-transactivated transcription from the bovine papillomavirus type 1 P89 promoter
More LessThe bovine papillomavirus type 1 (BPV-1) long control region (LCR) contains at least three consensus binding sites for the transcription factor Sp1 at nucleotides (nt) 7800, 7833 and 7854. A high basal-level P89 expression vector consisting of an origin-deleted LCR fused to the chloramphenicol acetyltransferase (CAT) gene was utilized to determine the role of these Sp1 sites in the regulation of transcription from the BPV-1 P89 promoter. The three Sp1 sites were capable of binding Sp1 in vitro. Mutation of these sites in the background of the origin-deleted LCR-CAT or a wild-type LCR-CAT construct resulted in decreased basal expression from P89. In addition, mutation of the Sp1 sites in the wild-type background caused a reduction in E2-transactivation potential. These data illustrate the importance of these Sp1 sites in regulating both basal and E2-transactivated P89 expression.
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Suppression of amber nonsense mutations of herpes simplex virus type 1 in a tissue culture system
We have investigated the ability of monkey kidney cell lines (SupD3 and SupD12) inducibly expressing an amber suppressor tRNAser to suppress amber nonsense mutations in three genes of herpes simplex virus type 1 (HSV-1). HSV-1 mutant TK4, which contains a nonsense mutation in the non-essential viral thymidine kinase (TK) gene, synthesized a full-length TK polypeptide at about 30% of the wild-type (wt) level in induced SupD3 cells but not in the parental non-suppressor (Sup0) cells. Using complementing cells, we constructed HSV-1 mutants carrying nonsense mutations in an essential gene, UL8, encoding a protein essential for viral DNA replication (ambUL8) or in a partially dispensable gene, UL12, encoding alkaline nuclease (ambUL12). The growth of the mutants in Vero or Sup0 cells was either totally (ambUL8) or severely (ambUL12) impaired, whereas in cells expressing suppressor tRNA the mutants produced infectious virus. However, the yields were much lower than obtained with wt HSV-1. In Vero or Sup0 cells the mutants ambUL8 and ambUL12 failed to synthesize full-length UL8 and UL12 protein products, respectively. Western immuno-blotting showed that the virus ambUL12 produced full-length UL12 protein in SupD12 cells which yielded a level of 25.9% of the alkaline nuclease activity of the wt HSV-1 control. Our results show that the levels of suppression of the nonsense mutations in ambUL8 and ambUL12 are insufficient to allow their continuing propagation in the available Sup+ cells. Possible reasons are discussed.
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Induction of varicella-zoster virus-neutralizing antibodies in mice by co-infection with recombinant vaccinia viruses expressing the gH or gL gene
More LessRecombinant vaccinia viruses (VV) expressing the varicella-zoster virus (VZV) glycoprotein H (gH) or glycoprotein L (gL) were constructed. The 94 kDa gH intermediate glycoprotein was synthesized in cell cultures infected with the VV-gH recombinant, but only coinfection with both recombinants resulted in the synthesis of the fully processed 118 kDa gH molecule. The VV-expressed gH and gL formed a complex that displayed the conformational neutralization epitope detectable by means of human VZV gH-specific monoclonal antibody V3. Formation of this epitope was inhibited by tunicamycin but not by monensin. Simultaneous intraperitoneal inoculation of mice with high doses of both VV-gH and VV-gL viruses resulted in the development of VZV-neutralizing, complement-independent antibodies; these antibodies were not detected in mice infected solely with either the VV-gH or the VV-gL recombinant.
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Lack of MHC class I complex expression has no effect on spread and control of cytomegalovirus infection in vivo
It has been claimed that MHC class I proteins serve as receptors for murine cytomegalovirus (MCMV) and that this interaction is the most important mechanism for virus entry in most cells. This claim is based on the observation that the MHC haplotype contributes to the susceptibility to cytomegalovirus (CMV) infection in vivo. Results from in vitro studies support the concept that stable expression of correctly folded MHC class I molecules contributes to infection, since the individual properties of MHC class I alleles, the availability of β 2-microglobulin (β 2m) and also the degree of peptide charging of the MHC class I heavy chain β 2m heterodimers determined the infection phenotype of cell lines. To assess the biological relevance of proper MHC class I expression we investigated CMV infection in β 2m-deficient mice which fail to express ternary MHC class I complexes and lack peripheral CD8+ T lymphocytes. We found that organ virus titres and virus clearance kinetics were not altered in β 2m mutant mice. In addition, there was no indication of diminished virus propagation in β 2m−/− embryonic fibroblasts. β 2m−/− mice suffered from the lack of CD8+ T lymphocytes that was partially compensated for by the function of CD4+ T lymphocytes. An organ-specific anti-virus function of natural killer (NK) cells was observed, independent from the β 2m deletion. The immune control unique for salivary gland infection was maintained. From the data presented here, we confirm the role of MHC class I molecules in the immune surveillance of CMV infection but question the biological impact of correct MHC class I complexes for productive infection.
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Epstein—Barr virus nuclear antigen 2 (EBNA2)-oestrogen receptor fusion proteins complement the EBNA2-deficient Epstein—Barr virus strain P3HR1 in transformation of primary B cells but suppress growth of human B cell lymphoma lines
To develop a transformation system with a conditional Epstein—Barr virus nuclear antigen 2 (EBNA2) gene, we fused the hormone binding domain of the oestrogen receptor to the N or C terminus of EBNA2. In promoter transactivation as well as primary B cell transformation assays these chimeric EBNA2 proteins are able to substitute for wild-type EBNA2 in the presence of oestrogen. Here we provide evidence that this transformation is the result of double infection of a cell with two virions, the P3HR1 virus genome and a mini-EBV plasmid carrying the chimeric EBNA2 gene. Unexpectedly, expression of the same EBNA2-oestrogen receptor fusion protein in established human B cell lymphoma lines resulted in growth retardation or growth arrest upon the addition of oestrogen. By titrating the oestrogen concentration in these stably transfected cells, the growth retarding and the transactivating function of the chimeric proteins could not be dissociated. We propose that growth inhibition of established B cell lymphoma lines is a novel function of EBNA2 which has not been detected in the absence of an inducible system. It remains open whether the growth retarding property of the EBNA2-oestrogen receptor fusion protein in B cell lymphoma lines is due to unphysiologically high expression of the chimeric protein or to interference with a cellular programme driving proliferation in these cell lines.
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- Insect
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Physical map of Anagrapha falcifera multinucleocapsid nuclear polyhedrosis virus
More LessA physical map of Anagrapha falcifera multinucleocapsid nuclear polyhedrosis virus (AfMNPV) DNA was constructed for restriction endonucleases EcoRI, HindIII, PstI and XhoI. The genome size was estimated to be 130 kbp. Ordering of the restriction fragments was accomplished by cross-blot hybridization, double digestion and DNA—DNA hybridization. The polyhedrin gene and homologous repeat (hr) regions were located by hybridization to the Autographa californica MNPV (AcMNPV) polyhedrin gene and hr4, respectively. Restriction pattern comparison and Southern blot analysis suggest that AfMNPV is closely related to AcMNPV.
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- Plant
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Do light-induced pH changes within the chloroplast drive turnip yellow mosaic virus assembly?
More LessTurnip yellow mosaic virus (TYMV) induces gross morphological and biochemical changes in the chloroplasts of infected cells. Viral RNA is synthesized in vesicles formed by invagination of the outer chloroplast bilayer. Virion assembly occurs at the neck of these vesicles and requires illumination. Data collected over the last three decades are consistent with the hypothesis that light-induced generation of a low pH drives TYMV assembly within the intermembrane space of chloroplasts. In a low-pH environment, poly(C) regions within the genomic RNA of TYMV may interact to form tertiary structures, and the recognition of these structures by TYMV coat protein initiates virion assembly.
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