- Volume 75, Issue 1, 1994
Volume 75, Issue 1, 1994
- Review Article
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- Bacterial
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Nucleotide sequence determination, characterization and purification of the single-stranded DNA-binding protein and major coat protein of filamentous phage ϕLf of Xanthomonas campestris pv. campestris
More LessϕLf is a filamentous bacteriophage of Xanthomonas campestris pv. campestris, which can integrate its genome into the host chromosome. The nucleotide sequence of an EcoRV-SphI fragment (1018 bp) from the ϕLf replicative form DNA was determined. Four contiguous open reading frames (ORFs), orf98-orf43-orf38-orf42, were revealed. ORFs 98 and 42 were identified as the genes encoding a single-stranded DNA-binding protein (Sbp) and a major coat protein, respectively. Sbp was purified and found to bind with a high affinity to ssDNA prepared from ϕLf phage particles. The major coat protein showed sequence features similar to those of the typical major coat proteins of other filamentous phages. However, it appears to be synthesized as a mature product, similar to the situation with Pf3 but different from that with other filamentous phage major coat proteins which are synthesized as pre-coats and are subject to post-translational processing. The M rs, estimated by SDS-PAGE, of both the Sbp (10·9K) and the major coat protein (4·1K) coincide with the values deduced from the nucleotide sequences. ORFs 43 and 38 are proposed to be the genes encoding two minor coat proteins. The order of these four genes is similar to that found in the Escherichia coli filamentous phages.
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- Animal
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Similar genetic susceptibility in iatrogenic and sporadic Creutzfeldt-Jakob disease
More LessCreutzfeldt-Jakob disease (CJD) is one of the transmissible spongiform encephalopathies (TSEs). In all TSEs host susceptibility is an important factor in the development of clinical disease. The prion protein (PrP) gene appears to confer the main component of this susceptibility. The appearance of spontaneous neurodegeneration in PrP transgenic mice carrying a human mutation has raised the possibility that the origin of sporadic CJD is solely genetic. We studied PrP codon 129 polymorphism in 23 of the 25 CJD cases in France related to human growth hormone (hGH) therapy. They constitute the largest and most homogeneous hGH- related iatrogenic CJD population yet analysed. All these CJD cases were homozygous at codon 129, compared with only 50% in the healthy control group (P< 0·00002). These iatrogenic cases also displayed a genotype frequency distribution similar to that observed in sporadic CJD. These results underline the importance of the PrP gene and especially the homozygous codon 129 genotype in determining the risk of developing CJD after contamination by a TSE agent. They also suggest that highly susceptible individuals may exist and raise the possibility that sporadic CJD may have an environmental origin.
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Complex structure of the nuclear translocation signal of influenza virus polymerase PA subunit
More LessThe protein regions involved in the nuclear translocation of the influenza virus PA polymerase subunit have been identified by deletion analysis of the protein expressed from a recombinant simian virus 40. Two regions seem to play a role in the process: region I (amino acids 124 to 139) and region II (amino acids 186 to 247). A nucleoplasmin-like nuclear translocation signal (NLS) has been identified in region I and an additional NLS appears to be present in region II, although no consensus targeting sequence can be detected. Alteration in any of the regions identified by short deletions completely prevented nuclear transport, whereas elimination of the regions I or II by large amino- or carboxy-terminal deletions did not prevent nuclear targeting of the truncated protein. In addition, a point mutation at position 154 completely eliminated nuclear transport. A β-galactosidase fusion protein containing the 280 amino acid terminal region of the PA protein was partially transported to the nucleus and mutant PA proteins with a cytoplasmic phenotype could not be rescued by superinfection with influenza virus. These results suggest that the PA protein contains a functional nuclear targeting region which is required in influenza virus infection, with two independent NLSs, one in region I and the other in region II.
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A small percentage of influenza virus M1 protein contains zinc but zinc does not influence in vitro M1-RNA interaction
More LessA peptide containing the CCHH motif, the putative zinc-binding sequence of influenza virus M1 protein, was found to bind zinc in a one-to-one complex with the characteristics of a typical zinc-binding peptide. Intact influenza virus also contained zinc and we show that this zinc is bound to the M1 protein in the virus. However, only a small proportion of M1 contained zinc: 4% in virus and 6 to 9% in isolated protein. One strain, B/Yamagata/16/88, consistently contained more zinc: 15 to 20% both in virus and in isolated protein. We also determined the RNA binding and transcription inhibition activities of various M1 proteins and found that the zinc content of M1 had no influence on either activity. We suggest that the zinc in M1 has a structural role in the virion other than nucleic acid binding.
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An amino acid change in the non-structural NS2 protein of an influenza A virus mutant is responsible for the generation of defective interfering (DI) particles by amplifying DI RNAs and suppressing complementary RNA synthesis
More LessThe mutated non-structural NS2 protein of an influenza A virus mutant, Wa-182, has been shown to be responsible for the production of defective interfering (DI) particles lacking the PA gene after a single cycle high-multiplicity infection. Using a subclone of Wa-182, A3/e-3, that inherited the Wa-182 phenotype but contained only a marginal amount of DI RNAs derived from the PA gene, we showed that replication of the PA genome RNA was suppressed primarily at the step of complementary RNA (cRNA) synthesis. On the other hand, the small amounts of DI RNA species present in the stock of A3/e-3 were shown to be replicated efficiently. These findings suggested that the suppression of cRNA synthesis of the PA gene was caused by preferential amplification of the DI RNAs. The suppression of PA gene cRNA synthesis subsequently resulted in suppression of both virion RNA synthesis and secondary transcription of the PA gene. Such aberrant replication of the PA gene was found to be attributable to an amino acid change in the NS2 protein at position 32, from isoleucine to threonine. These results suggest that the NS2 protein plays a role in promoting normal replication of the genomic RNAs by preventing the replication of short-length RNA species.
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Modulation of antiviral immune responses by exogenous cytokines: effects of tumour necrosis factor-α, interleukin-1α, interleukin-2 and interferon-γ on the immunogenicity of an inactivated rabies vaccine
In vivo administration of exogenous cytokines may influence elicited immune responses, and hence may change the efficacy of a vaccine. We investigated the effects of tumour necrosis factor-α (TNF-α), interleukin- 1α (IL-1α), interleukin-2 (IL-2) and interferon-γ (IFN-γ) on the immune response elicited by inactivated rabies virus vaccine in a mouse model. Each of the cytokines increased virus-specific IgG responses after primary and after secondary immunization. A single dose of 1·3 ng TNF-α or 1L-1α, when injected shortly before vaccination, only marginally stimulated resistance to challenge infection (four- and seven-fold, respectively) without enhancing virus neutralizing antibody (VNAb) responses. In contrast, a single injection of 103 units of IFN-α or five daily injections of 1·6 μg IL-2 increased vaccine dilutions protecting 50% of mice (PD50 values) 77- to 50-fold, respectively, with a concomitant enhancement of VNAb. At a 1:10000 dilution of a standard inactivated rabies vaccine preparation both ILN-γ and 1L-2 increased protective immunity without enhancing VNAb responses; in non-vaccinated animals this treatment had no effect on resistance to challenge. Combined administration of IFN-y and 1L-2 syner- gistically enhanced VNAb responses. In contrast to the other cytokines tested, 1FN-γ preferentially stimulated virus-specific IgG2a production. It also augmented the vaccine-induced priming of rabies virus-specific spleno- cyte proliferation. These results document that certain cytokines alone or in combination are potent immunological adjuvants which may direct and modulate immunization-induced antiviral immune responses.
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Molecular evolution and epidemiology of dengue-3 viruses
More LessThe nucleic acid sequences of the pre-membrane/ membrane and envelope protein genes of 23 geographically and temporally distinct dengue (DEN)-3 viruses were determined. This was accomplished by reverse transcriptase-PCR amplification of the structural genes followed by automated DNA sequence analysis. Comparison of nucleic acid sequences revealed that similarity among the viruses was greater than 90 %. The similarity among deduced amino acids was between 95 % and 100 %, and in many cases identical amino acid substitutions occurred among viruses from similar geographical regions. Alignment of nucleic acid sequences followed by parsimony analysis allowed the generation of phylo-genetic trees, demonstrating that geographically independent evolution of DEN-3 viruses had occurred. The DEN-3 viruses were separated into four genetically distinct subtypes. Subtype I consists of viruses from Indonesia, Malaysia, the Philippines and the South Pacific islands; subtype II consists of viruses from Thailand; subtype III consists of viruses from Sri Lanka, India, Africa and Samoa; subtype IV consists of viruses from Puerto Rico and the 1965 Tahiti virus. Phylogenetic analysis has also contributed to our understanding of the molecular epidemiology and worldwide distribution of DEN-3 viruses.
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Clonality, expression and methylation patterns of the Epstein-Barr virus genomes in lethal midline granulomas classified as peripheral angiocentric T cell lymphomas
We analysed the terminal repeats of Epstein-Barr virus (EBV) in DNAs isolated from six lethal midline granuloma (LMG) biopsies. A single fused terminal fragment could be detected in each case, indicating that these angiocentric peripheral T cell lymphomas represent clonal proliferations of cells infected with EBV on a single occasion. Using reverse transcriptase-PCR, we detected EBV nuclear antigen (EBNA) 1 and latent membrane protein (LMP) 1, but not EBNA 2 messages in LMG biopsy RNAs. The splicing pattern of the EBNA 1 message was consistent with the usage of a promoter localized in the BamHI F fragment (F promoter). The BamHI W fragment repeats and LMP-coding sequences were highly methylated in all cases. In contrast, the LMP regulatory sequences were found to be hypomethylated or partially methylated, as in LMP-expressing nasopharyngeal carcinomas.
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Detection of multiple ‘Ebnotypes’ in individual Epstein-Barr virus carriers following lymphocyte transformation by virus derived from peripheral blood and oropharynx
Transformation of a B lymphocyte into a lymphoblastoid cell line (LCL) by Epstein-Barr virus (EBV) results in the expression of EBV nuclear antigens (EBNAs) of which the size spectrum (‘Ebnotype’) is characteristic for the transforming virion. Ebnotyping has been used as an epidemiological tool for studies of EBV infection. We compared the occurrence of a single and of multiple Ebnotypes, as defined by EBNAs 1, 2 and 6, in healthy and diseased EBV carriers. Cases from which two or more LCLs could be established from peripheral blood or oropharyngeal cultures were considered informative. The frequency of multiple Ebnotypes was relatively low in healthy individuals and in patients with infectious mononucleosis or with haematological diseases who were awaiting a bone marrow transplant [blood, 11 of 74 patients (15%); oropharynx, 12 of 49 patients (24%)], whereas it was relatively high in recipients of bone marrow or cardiac allografts and one patient with AIDS [blood, 12 of 34 patients (35%); oropharynx, 11 of 16 patients (69%)]. Three patterns of the simultaneous presence of multiple Ebnotypes were distinguished. The first, most frequent, pattern observed predominantly in oropharyngeal cultures of all groups consisted of minority Ebnotypes differing from the majority type by only a single EBNA protein (usually EBNA 1). The second, less frequent, pattern observed in the healthy carriers and the (candidate) transplant recipients consisted of minority Ebnotypes differing from the majority type by two EBNA proteins (mostly EBNAs 1 and 6). The third pattern, characterized by the simultaneous presence of totally different Ebnotypes, was restricted to the (candidate) transplant recipients and the AIDS patient and was more frequently observed in the blood than in the oropharynx. We suggest that the first two patterns result from heterologous recombinations occurring during viral replication at repeat sequences within the EBNA coding regions, whereas the third pattern reflects multiple infections with exogenous viruses.
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Heterogeneity within the Epstein-Barr virus nuclear antigen 2 gene in different strains of Epstein-Barr virus
More LessDNA isolated from biopsies of endemic Burkitt’s lymphoma (BL) from New Guinea was analysed for the presence of Epstein-Barr virus (EBV) sequences using the polymerase chain reaction. Primers were designed to amplify sequences within the Epstein-Barr virus nuclear antigen (EBNA) 1 and 2 genes. These analyses detected the EBNAl sequence in all the biopsies studied. Additional sets of primers directed against the EBNA2 gene were used in order to categorize the EBV strains as A-type or B-type (39% A-type; 50% B-type; 5% A- and B-type; 5% untypeable). These results indicated that DNA sequence heterogeneity within the EBNA2 gene region may exist in different strains of EBV. The extent of DNA sequence heterogeneity among different strains of EBV was determined by sequencing of a region within the EBNA2 gene in a number of different A-type and B-type strains of EBV originating from Africa or New Guinea. The results demonstrated DNA sequence heterogeneity within the EBNA2 gene in different strains of EBV. This heterogeneity was more extensive among A-type strains than B-type strains of EBV.
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Late phase inhibition of murine cytomegalovirus replication by synergistic action of interferon-gamma and tumour necrosis factor
More LessWe have shown previously that the antiviral function of CD4+ T lymphocytes against murine cytomegalovirus (MCMV) is associated with the release of interferon- (IFN-γ). We now demonstrate that IFN-γ and tumour necrosis factor alpha (TNF-α) display synergism in their antiviral activity. As little as 2 ng/ml of IFN-γ and TNF-α reduced the virus yield by about three orders of magnitude. There was no effect on immediate early (IE) and early (E) gene expression as far as the candidate genes IE1, El and those encoding the major DNA- binding protein and the DNA polymerase were concerned. Late gene transcription, assayed by the candidate genes encoding glycoprotein B and the MCMV homologue of ICP 18·5, was blocked and MCMV DNA replication was found to be reduced but not halted. The most prominent finding of the cytokine effect, seen by electron microscopy, was an alteration of nucleocapsid formation. Altogether, the synergism is multifaceted and acts at more than one stage during viral morphogenesis. Because the cytokines clearly do not act at an early stage of infection we conclude that the mode of cytokine activity differs between alpha- and betaherpesviruses.
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Inhibition by iota-carrageenan of the spread of murine cytomegalovirus from the peritoneal cavity to the blood plasma
More LessThe mechanism of iota-carrageenan (ι-CAR)-induced protection against murine cytomegalovirus (MCMV) infection of mice was analysed. The virus titres in the target organs on the fourth day correlated with that in the plasma at 1 h after intraperitoneal inoculation of MCMV, irrespective of ι-CAR treatment. Pretreatment of mice with ι-CAR induced infiltration of polymorphonuclear neutrophils into the peritoneal cavity and inhibition of viral spread from the peritoneal cavity to the plasma. Although the direct relationship of these two phenomena was unclear, the inhibition by ι-CAR of viral spread resulted in protection against MCMV infection of mice.
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Virulence and pathogenesis of non-virulent and virulent strains of pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus
Pseudorabies virus (PRV) expressing the envelope glycoprotein El (E1) of hog cholera virus (HCV) was used as a model to study the potential risks connected with the use of a live herpesvirus vaccine expressing a foreign gene. The gene encoding E1 was inserted into the glycoprotein X (gX) locus of both a virulent PRV strain and a non-virulent PRV strain in which the virulence genes encoding glycoprotein I (gI) and thymidine kinase (TK) had been inactivated. We investigated whether strain M205 (gI −, TK −,gX − , E1+) had a changed cell or host tropism or virulence compared with strain M206 (gI − ,TK − ,gX − ) in pigs, rabbits, hamsters, rats, mice and rhesus monkeys. The insertion of E1 into this non-virulent PRV strain caused no change in cell or host tropism. However, pigs inoculated with M205 shed less virus over a shorter period than pigs inoculated with M206. Theoretically, virulent PRV strains expressing E1 (gX −, E1+) could arise through transfer of the E1 gene of M205 to a virulent PRV strain. Therefore, we inoculated pigs with strain M12 (gX −, E1 − ) or the control strain M104 (gX − ) and compared the virulence and pathogenesis. M12 and M104 were of approximately equal virulence and the pathogenesis of both strains was similar. We concluded that incorporating E1 of HCV into the gX locus of PRV did not change cell or host tropism, nor did it change the virulence of either nonvirulent or virulent PRV.
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Immunogenicity of high expression adenovirus-hepatitis B virus recombinant vaccines in dogs
High yielding adenovirus (Ad)-hepatitis B recombinant (Ad-Hep B) viruses were prepared by insertion of the hepatitis B surface antigen (HBsAg) gene into the early region 3 (E3 region) of Ad4 or Ad7 vectors containing intact or largely deleted E3 regions. Both E3-deleted and non-deleted recombinants produced about six- to eightfold higher amounts of HBsAg than previously reported recombinants. These recombinant viruses were evaluated for immunogenicity in dogs which sustain abortive lung infections by Ad4 and Ad7. Recombinants containing E3 deletions elicited 10- to 12-fold stronger anti- HBs primary responses than previously evaluated low yield recombinants. Further immunizations with heterotypic Ad-Hep B recombinants induced substantial anti-HBs booster responses as well as anti-‘a’ epitope responses. In contrast, recombinant viruses containing intact E3 regions induced only weak or negligible anti- HBs responses, although such viruses induced strong antibody responses to the Ad vectors. The immunogenicity of high-yielding Ad recombinants correlated with temporal expression of HBsAg and thus the dog represents a valuable model for evaluation of immune responses to heterologous proteins that are expressed early and that do not require efficient DNA replication. Recombinants expressing HBsAg late in the infectious cycle require further testing in the fully permissive chimpanzee model. This study establishes that the E3- deleted high yield Ad4 and Ad7 recombinants represent promising live oral hepatitis B vaccine candidates.
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Expression of a foreign epitope on the surface of the adenovirus hexon
More LessTo present short protein sequences to the host immune system a foreign epitope has been expressed on the surface of the adenovirus virion as part of the hexon. As the trimeric hexon constitutes 240 out of the 252 capsomers of the virus, the foreign epitope is repeated 720 times on the virion surface. An eight amino acid sequence from the major antigenic site in the VP1 capsid protein of poliovirus type 3 was engineered into two regions of the adenovirus type 2 hexon. The two loop regions chosen to accommodate the foreign sequences are exposed on the surface of the virion, show sequence variation between serotypes and are the sites of interaction with neutralizing antibodies. Virus with substitutions in loop I had wild-type growth characteristics, whereas virus with substitutions in loop II grew poorly. Adenoviruses with poliovirus sequences in loop I were recognized and efficiently neutralized by antisera specific for the poliovirus sequence; an antiserum raised against the adenovirus with the poliovirus insert specifically recognized the VP1 capsid protein of poliovirus type 3. It is therefore feasible to alter the surface properties of the adenovirus virion and in doing so to manipulate the immune response to this virus.
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Sequence and structural analysis of murine adenovirus type 1 hexon
More LessThe nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank database and assigned accession number M81889.
The genomic region encoding the major capsid protein (hexon) of murine adenovirus type 1 (MAV-1) has been isolated and sequenced. The sequence predicts a 908 residue MAV-1 hexon protein and is flanked by a portion of the upstream pVI gene and the downstream endoproteinase gene. The order of these genes and their location in the middle of the genome are the same as those found in other adenoviruses sequenced to date. Multiple sequence alignment with the other five known hexon protein sequences reveals an overall residue identity of 51% and residue conservation of 66%. In comparison with human adenovirus type 2 (Ad2), MAV-1 hexon has major deletions between residues 141 to 170, 270 to 284 and 446 to 455. Since these regions in the Ad2 hexon are partially exposed on the outer surface of the virion, they may represent type-specific antigenic determinants. The MAV-1 hexon sequence has been modelled using the known three-dimensional structure of the Ad2 hexon. The variable regions in which the mutations, deletions and insertions occur are located in the l1 and 12 loops of the molecule that form the protruding hexon towers on the external surface of the virion.
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Production of mink enteritis parvovirus empty capsids by expression in a baculovirus vector system: a recombinant vaccine for mink enteritis parvovirus in mink
More LessThe VP-2 gene of mink enteritis parvovirus (MEV) was amplified by the polymerase chain reaction using MEV DNA isolated from the faeces of a naturally infected mink. Subsequently the VP-2 gene was cloned into a baculovirus expression vector. Recombinant baculo-viruses were isolated and the MEV VP-2 gene product was characterized after expression in Sf9 insect cells. The MEV VP-2 product had the same size as that reported for the wild-type MEV VP-2 protein and was recognized by convalescent sera from MEV-infected mink and a panel of monoclonal antibodies reactive to MEV. Furthermore, the VP-2 protein was able to form parvovirus-like particles, which had haemagglutinating properties comparable with the wild-type MEV. The cloned VP-2 gene was sequenced and only five nucleotide differences were found after alignment with the known sequences of the MEV type 1 and type 2 isolates. Surprisingly, the VP-2 gene encoded a valine and a tyrosine at amino acid positions 232 and 234, identical to the situation found in MEV type 1, but at position 300 there was a valine which is a determinant of MEV type 2. Immunization of mink with approximately 40000 haemagglutinating units of recombinant MEV VP-2 induced a measurable antibody response as tested by haemagglutination inhibition. Furthermore, the immunized mink did not excrete virus and did not develop clinical disease upon challenge with a virulent isolate of MEV.
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Immune response to human papillomavirus type 16 E6 gene in a live vaccinia vector
L. Gao, B. Chain, C. Sinclair, L. Crawford, J. Zhou, J. Morris, X. Zhu and H. StaussImmunization of mice with a recombinant vaccinia virus expressing the human papillomavirus type 16 (HPV-16) E6 gene elicits specific antibody, proliferative and cytotoxic T lymphocyte responses. T and B cell epitopes were mapped by using synthetic peptides. This study provides the background to future investigation aimed at developing prophylactic and therapeutic vaccines against HPV-16 infection and cervical cancer.
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Delayed-type hypersensitivity response to human papillomavirus type 16 E6 protein in a mouse model
More LessA mouse model incorporating the epitheliotropic nature of human papillomavirus (HPV) infections has been used to study an immune response to HPV type 16 (HPV-16) E6 protein in vivo. Using a transplantation technique, a novel immortal keratinocyte cell line expressing the E6 protein has been grafted onto syngeneic mice to re-form a differentiated epithelium overlying a granulation tissue bed. By this approach the presentation of viral antigens to the immune system can be modelled in a way analogous to the natural infection. Here we report a delayed-type hypersensitivity (DTH) reaction in grafted mice challenged intradermally with a recombinant vaccinia virus expressing the HPV-16 E6 protein. The specificity of the response was confirmed by the absence of a DTH reaction to challenge with virus expressing either HPV-16 E7 or L1 protein.
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Characterization and biosynthesis of the woodchuck hepatitis virus e antigen
More LessThe biosynthesis of the secretory core gene product of the woodchuck hepatitis virus (WHV) was studied in human cells. We have shown that the WHV e antigen was a N-glycosylated (most likely a diglycosylated) protein, with an apparent M r of 24K. To demonstrate that the WHV precore protein was correctly processed in human cells, we engineered chimeric proteins in which signal peptides or arginine-rich domains of WHV and hepatitis B virus (HBV) precore proteins were exchanged. Our results showed that both the signal peptide and the arginine-rich region of WHV precore protein were cleaved off during the secretion pathway, as previously reported for precore protein of human HBV and duck HBV. These observations demonstrate that the maturation process of the e antigen is conserved in hepadnaviruses. In addition, on the basis of inhibition experiments, we suggest that the cleavage of the carboxy terminus of the WHV precore protein occurred in a post-endoplasmic reticulum compartment, most likely beyond the medial Golgi, and that this cleavage was catalysed by an aspartyl protease.
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Bovine herpesvirus 1 gIV-expressing cells resist virus penetration
More LessThe interaction of bovine herpesvirus 1 (BHV-1) with the BHV-1 glycoprotein IV (glV)-expressing cell line Dl-1 was examined by radiolabelled virus adsorption assays, in situ autoradiography and electron microscopy. Adsorption of radiolabelled BHV-1 to Dl-1 cells was similar to that observed in control cell lines but in situ radiography revealed that virus moved to the nucleus of control but not the gIV-expressing cells. Electron microscopy studies showed that BHV-1 attached to the cell membranes of Dl-1 and control cells at 4 °C but penetration of virus was observed only in control cells when the temperature was shifted to 37 °C. These results provide further evidence that cellular expression of gIV does not prevent viral adsorption, but does prevent the entrance of the virus into the cell.
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Expression of the murine cytomegalovirus glycoprotein H by recombinant vaccinia virus
More LessThe sequence of the gene encoding glycoprotein H (gH) of murine cytomegalovirus (MCMV) strain Smith was determined and compared with the sequence of the gH of MCMV strain K181. Transcriptional analysis showed that gH is encoded by a large mRNA of 5·0 kb, which is synthesized late in infection. A recombinant vaccinia virus expressing the MCMV gH open reading frame was constructed (Vac-gH). Anti-MCMV serum precipitated a protein of 87K from Vac-gH-infected cells. Reactivity with a monoclonal antibody showed the identity of the MCMV gH with a 87K envelope glycoprotein described previously by Loh and Qualtiere. Immunization of mice with the Vac-gH recombinant gave rise to an anti-gH serum, which neutralized MCMV without complement in vitro.
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Expression of the human cytomegalovirus 65K tegument phosphoprotein in insect cells by baculovirus vectors
More LessThe gene encoding the 65K tegument phosphoprotein (pp65) of human cytomegalovirus (HCMV) was cloned into pAc373 to construct a recombinant baculovirus (Acpp65-3) expressing pp65 in insect Sf9 cells. A baculovirus that carried a fragment of the gene, corresponding to the first 442 amino acids of pp65, was also developed, using vector pVL941 (Acpp65-2). Recombinant proteins migrating in SDS-polyacrylamide gels with an M r of either 65K (Acpp65-3) or 56K (Acpp65-2) were detected in cytoplasmic and nuclear extracts of infected Sf9 cells. The 56K and 65K proteins were recognized in immunoblots by monoclonal antibodies (MAbs) 28–77 and 28–19, which are specific for pp65. The insect cell-expressed antigens were also analysed on Western blots using MAbs 4D11, 7D2, 8E3, 7B4 and 8E10, which recognize the HCMV antigen GP66 in immunoblots. The truncated pp65 antigen of Acpp65-2 was reactive with MAbs 4D11, 7D2, 8E10 and 7B4. The protein expressed by Acpp65-3 reacted only with MAb 4D11. The data proved that the epitopes recognized by MAbs 4D11, 7D2, 8E3 and 7B4 mapped in the region of pp65, comprising amino acids 1 to 442, and also that GP66 and pp65 represent the same HCMV antigen. Immunoblot analysis of human sera from individuals seropositive for HCMV showed that the recombinant pp65 products were as antigenic as the native 65K phosphoprotein produced in HCMV-infected human embryonic fibroblasts.
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Human immunodeficiency virus type 1 interaction with the membrane of CD4+ cells induces the synthesis and nuclear translocation of 70K heat shock protein
In the last few years a growing body of experimental evidence has indicated that the interaction of human immunodeficiency virus type 1 (HIV-1) surface glycoprotein (gpl20) with the membrane of CD4+ cells may deliver negative signals, eventually leading to programmed cell death (apoptosis) of either mature CD4+ lymphocytes or CD34+ haematopoietic progenitor cells, in the absence of cell infection with HIV-1. However, information on the possible activation of the classical signal transduction pathway through gpl20 engagement of cell surface CD4 is contradictory. Heat shock proteins (hsp) or ‘ stress ’ proteins’ are involved in protecting cells from the deleterious effects of heat and other stresses and perform various cell roles. In mammalian cells there is evidence that hsp70 is involved in the transport of proteins to lysosomes, mitochondria and the nucleus. The results obtained in our study demonstrate that early (3 h) after the exposure of permissive CD4+ cells to HIV- 1 (or to purified recombinant gpl20) a peak of increased synthesis and nuclear translocation of a 70K hsp (and possibly other proteins) is observed. These data indicate that gpl20 possesses the capacity to trigger a cascade of events through a transmembrane signalling activity.
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Morphological differentiation of human SH-SY5Y neuroblastoma cells inhibits human immunodeficiency virus type 1 infection
More LessWe have studied human immunodeficiency virus type 1 (HIV-1) infection in human SH-SY5Y neuroblastoma cells at various stages of morphological differentiation. Two days’ treatment of the cells with retinoic acid (RA) or dibutyryl cAMP (db-cAMP) resulted in the appearance of elongated neurites and enhanced production of 160K to 200K neurofilament proteins as shown by indirect immunofluorescence. DNA synthesis was reduced only in RA-treated cells as detected by 5-bromo- 2-deoxyuridine incorporation. The cells were infected with two T-lymphotropic virus strains (IIIB and NDK) and two fresh isolates (39001 and 46001) from bron- choalveolar lavage samples of AIDS patients. The latter two isolates were unable to form syncytia in infected CD4-positive T-lymphoblastoid C8166 cells which was in contrast to our T-lymphotropic virus strains. Interphase in situ hybridization showed that 14 to 16 % of SH- SY5Y cells become positive for HIV-1 DNA. Regardless of the virus strain, morphological differentiation of the cells with RA or db-cAMP inhibited infection by 50 % at a single cell in situ resolution. Nested PCR confirmed the presence of proviral DNA in the infected cells. These results show that human neuroblastoma cells, tumour cells of neuroectodermal origin, can be infected by different HIV-1 isolates and that the infection is inhibited by neurotypic cell differentiation.
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Expression, characterization and purification of simian immunodeficiency virus soluble, oligomerized gp160 from mammalian cells
More LessThe envelope glycoprotein, gpl60, of human (HIV) and simian (SIV) immunodeficiency viruses mediates virus- host cell binding followed by fusion of the viral and plasma membranes. The envelope proteins are known to exist as non-covalently associated oligomers on the virus surface. The production of permanent mammalian cell lines that constitutively secrete relatively high levels of soluble forms of SIV gpl60 is described and we show that these proteins are secreted predominantly as tetramers with lower levels of dimer forms. Oligomeric forms were purified to greater than 90 % purity using a simple gel filtration method. The purified proteins bind CD4 suggesting that they remain in their native conformation. The purified oligomeric proteins provide the basis for more relevant structural, functional and immunological studies than recombinant gp120 as they more closely resemble the envelope protein oligomer.
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Amino acid sequence similarity of the VP7 protein of human rotavirus HCR3 to that of canine and feline rotaviruses
More LessThe sequence of the VP7 gene of human rotavirus strain HCR3 was determined and its predicted amino acid sequence was compared with that of other rotavirus strains. The VP7 gene is 1062 nucleotides long and contains a single open reading frame of 981 nucleotides capable of encoding a protein of 326 amino acids. The VP7 amino acid sequence similarity of strain HCR3 to those of various human and animal G3 serotypes ranged from 88·7 to 99·4%, and from 60·4 to 88·3 % to strains representing each of the other 13 G serotypes. Alignment of four variable regions [VR4, VR5(A), VR7(B) and VR8(C)] of HCR3 with those of G3 strains of different host species showed that HCR3 possesses a sequence almost identical to that of canine rotaviruses and feline rotavirus strain CAT97 in all four regions. A considerable divergence in regions VR4, VR7(B) and VR8(C) was found with strains of human, mouse, monkey, horse and rabbit rotaviruses. This observation together with results of our previous study on VP4 indicated that human rotavirus HCR3 is genetically more closely related to animal rotaviruses than to other human rotaviruses.
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Cloning of the Nilaparvata lugens reovirus genome: conserved terminal nucleotide sequences and nucleotide sequence of genome segment S10
More LessThe segmented double-stranded RNA genome of Nila- parvata lugens reovirus (NLRV) was cloned, and the nucleotide sequence of genome segment S10 and terminal nucleotide sequences of the rest of the segments were determined. Genome segment S10 of NLRV consisted of 1430 nucleotides with a single open reading frame extending for 1293 nucleotides from nucleotide 46. It encoded a polypeptide of 431 amino acids with an M rof 49·4K, which was a non-structural protein. The plus-strand RNA of all genome segments had the same conserved trinucleotide 5′AGU- and hexanucleotide- GUUGUC 3′in the 5′ - and 3′ -terminal regions, respectively. These conserved terminal sequences resembled those found in the segments of the members of the genus Fijivirus.
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Analysis of the structural protein gene sequence shows Kyasanur Forest disease virus as a distinct member in the tick-borne encephalitis virus serocomplex
More LessKyasanur Forest disease (KFD) virus is a highly pathogenic member of the family Flaviviridae producing a haemorrhagic disease in infected human beings. Despite this high pathogenicity and potential epidemiological importance, there have been relatively few detailed antigenic or molecular studies on KFD virus. The nucleotide sequences of the genes encoding the structural proteins of the virus have now been determined. From these data we conclude that KFD virus is a distinct member in the tick-borne flavivirus complex with characteristic protease cleavage sites, fusion peptide, signal sequences and hydrophobic transmembrane domains. Comparison of the deduced amino acid sequences of KFD virus showed close relationships with other tick-borne flaviviruses. Among the structural proteins, the E protein showed maximum similarity (77·4% to 81·3 %) to tick-borne flaviviruses. Alignment of the amino acid sequence with those of other known tick-borne flaviviruses revealed many conserved regions confirming its identity as a member of the tick-borne encephalitis group, although the genetic marker EHLPTA showed a T→K substitution in KFD virus. The proposed genetic marker at amino acid positions 232 to 234 (AQE) was unique for KFD virus. A dendrogram derived from the amino acid alignment showed a phylogenetic relationship similar to those obtained on the basis of serological studies. The question of the sudden emergence of KFD virus in India and the possibilities of developing recombinant virus vaccines are discussed.
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Cloning of the nucleocapsid protein gene of peste-des-petits-ruminants virus: relationship to other morbilliviruses
More LessTwo independent cDNA clones, identified as representing the mRNA of the nucleocapsid protein gene of peste-des-petits-ruminants virus, were sequenced. The longest insert was 1662 nucleotides, not counting the poly(A) tail, and it was estimated that about 21 nucleotides were missing from the complete gene sequence. The sequence contained one long open reading frame encoding a protein of 525 amino acids with a predicted relative molecular mass of 58008.
Comparisons of the nucleic acid and protein sequences of all the morbillivirus nucleoproteins so far determined indicated two major subgroups in the morbillivirus genus of the Paramyxoviridae: one group included canine and phocine distemper viruses, and the other rinderpest, measles and peste-des-petits-ruminants viruses. Peste- des-petits-ruminants virus was found to be slightly more related to canine and phocine distemper viruses than were measles and rinderpest viruses.
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Unusual amino-terminal sequence repeat characterizes the capsid protein of dasheen mosaic potyvirus
More LessThe 3′-terminal region of a Florida isolate of dasheen mosaic potyvirus (DMV-LA) genome including the coat protein (CP) gene was cloned and sequenced. Protease digestion was predicted to occur between the glutamine and alanine residues at positions 79 and 80 of the 408 residue long polypeptide to produce a CP of 329 amino acids with an estimated M r of 36229. Following the putative protease recognition site is a DAG sequence, which is conserved among aphid-transmitted potyviral CPs. There is an unusual and unique stretch of 52 amino acids after the DAG that is repetitive and rich in threonine and asparagine. A sequence of 10 residues (GNNTNTNTN ST) was repeated three times in tandem within this stretch and was followed by six proline residues. Several potential glycosylation sites were found clustered within this region. Expression in Escherichia coli and Western blotting of the CP confirmed its size and serological identity. Sequence comparisons and phylogenetic reconstructions indicated that DMV is a distinct potyvirus within the passionfruit woodiness virus subgroup cluster.
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Nucleotide sequence of carnation ringspot dianthovirus RNA-1
More LessThe nucleotide sequence of carnation ringspot virus (CRSV) RNA-1, the type member of the dianthovirus genus, has been determined. The 3756 nucleotide genomic RNA-1 contains three large open reading frames (ORFs), capable of encoding 27K, 54K and 38K polypeptides. In addition, a small ORF encoding a 10K polypeptide at the 3′ terminus of the RNA has been identified. The gene organization of CRSV RNA-1 is similar to those of red clover necrotic mosaic (RCNMV) and sweet clover necrotic mosaic (SCNMV) diantho- viruses with the exception that CRSV RNA-1 contains the additional 3′-terminal ORF. The 27K and 54K proteins possess significant sequence similarity to corresponding polypeptides of the other dianthoviruses. The 54K protein also contains the conserved RNA- dependent RNA polymerase motif. The identification of a shifty heptanucleotide preceding the p27 ORF termination codon and a predicted secondary structure following the terminator suggest that a translational frameshifting event allows translation to continue past the p27 ORF into the p54 ORF, which is in the − 1 frame, generating an 88K fusion protein. Amino acid sequence alignment of the 38K protein with the corresponding RCNMV and SCNMV polypeptides indicate that this is the viral capsid protein.
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