- Volume 73, Issue 4, 1992
Volume 73, Issue 4, 1992
- Review Article
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- Animal
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Characterization of primary human fibroblasts transformed by human papilloma virus type 16 and herpes simplex virus type 2 DNA sequences
More LessHuman papilloma virus type 16 (HPV-16) and herpes simplex virus type 2 (HSV-2) are human viruses implicated in the development of cancer, in particular cervical cancer. The ability of HSV-2 and HPV-16 to transform early passage human cells was examined in this report. For these studies, gingival fibroblasts were utilized. One gingival cell strain was derived from a normal individual (N-16). The second cell strain was derived from hyperplastic gingival tissue of an epileptic individual (R-30) treated with phenytoin, an antiseizure drug. A common side effect of phenytoin is the induction of gingival overgrowth. R-30 cells contained a stable chromosomal translocation between chromosomes 8 and 18 and expressed higher steady state levels of c-myc. HPV-16 DNA efficiently immortalized R-30 cells but not N-16 cells. R-30 cells cotransfected with HPV-16, and HSV-2 viral DNAs were more aneuploid than R-30 cells transfected with HPV-16 DNA alone. Additionally, R-30 cells cotransfected with both viral DNAs grew better in soft agar than R-30 cells transfected with HPV-16 DNA alone. HSV-2 DNA was detected in transformed cells by polymerase chain reaction. These results suggested R-30 cells were immortalized more efficiently by HPV-16 and further imply that HPV-16 and HSV-2 DNA fragments can cooperate during multistep transformation.
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Glycoprotein 60 of equine herpesvirus type 1 is a homologue of herpes simplex virus glycoprotein D and plays a major role in penetration of cells
Monoclonal antibodies (MAbs) specific for equine herpesvirus type 1 (EHV-1) glycoprotein 60 (gp60) and gp17/18 (F3132 and 5H6 respectively) were found to react with the same protein, which was identified as a homologue of herpes simplex virus type 1 gD. MAb F3132 strongly neutralized virus infectivity and inhibited the penetration of the virus into the cell. The effects on penetration were shared with three other MAbs against this protein (P68, F3116 and F3129), but no effect on virus penetration was found with any other anti-EHV-1 MAb tested. The level of glycosylation of gp60 was analysed using glycanase enzymes and glycosylation inhibitors, and consisted of mainly N-linked carbohydrate. The M r of non-N-glycosylated gp60 was 50K.
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Immunity in strain 2 guinea-pigs inoculated with vaccinia virus recombinants expressing varicella-zoster virus glycoproteins I, IV, V or the protein product of the immediate early gene 62
The immunogenicity of specific varicella-zoster virus (VZV) proteins, with emphasis upon cell-mediated immune responses, was evaluated by immunizing strain 2 guinea-pigs with vaccinia virus recombinants that express gpI (vac-gpI), gpIV (vac-gpIV) and gpV (vac-gpV) or the IE-62 protein (vac-IE-62). Vac-gpI elicited the highest initial mean T cell proliferation response [stimulation index (S.I.) 3.8 ± 0.9 s.e.m.] whereas inoculation with vac-gpV produced the lowest primary T cell response (S.I. 2.5 ± 1.1 s.e.m.). T cell proliferation was detected for a shorter period after immunization with vac-gpV compared to vac-gpI, vac-gpIV or vac-IE-62. A comparison of the immunogenicity of vac-gpI and vac-IE-62 with the same proteins prepared by immunoaffinity purification showed that immunization with these proteins in either form elicited virus-specific IgG antibodies and T cell recognition. The presence or absence of IgG antibodies to the IE-62 protein was used to assess protection against challenge with guinea-pig cell-adapted infectious VZV in animals that had been inoculated with vac-gpI, vac-gpIV or vac-gpV. Immunization with vac-gpI and vac-gpIV restricted VZV replication but all animals given vac-gpV developed antibodies to IE-62 after challenge with infectious VZV. Priming of the T lymphocyte response was observed in all animals immunized with VZV-vaccinia virus recombinants after subsequent exposure to infectious VZV. These experiments with VZV vac-gpI, vac-gpIV and vac-gpV in guinea-pigs suggest variability in the capacity of herpesviral glycoproteins to elicit cell-mediated immunity in vivo. Induction of virus-specific immunity using IE-62 means that this major tegument protein of VZV could be a useful component for vaccine development.
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The different interactions of a gIII mutant of pseudorabies virus with several different cell types
More LessGlycoprotein gIII of pseudorabies virus (PrV) is multifunctional. It plays a role in the stable adsorption of the virus to its host cells by interacting with a cellular heparin-like substance. It also affects both release of mature virus from infected cell types and virulence. Thus, although non-essential for growth in vitro, gIII plays a central role in the biology of the virus. The primary attachment of a mutant, PrV2, which has an in-frame internal deletion and expresses a shortened version of gIII, and of wild-type (wt) virus, to MDBK cells has been shown to occur similarly. To ascertain whether different domains of gIII control of the expression of the different biological functions of the gIII protein, we have compared several aspects of virus-host cell interactions of PrV2, of a gIII-null virus, and of wt virus. Our results showed that the deletion of the internal segment of the gIII glycoprotein affects adsorption and virus release differently, i.e. that these two functions of gIII appear to be in dependent of each other. Furthermore, we observed that although the primary adsorption of PrV2 and wt virus to MDBK cells is similar, PrV2 behaved like a gIII-null mutant with respect to virulence. The apparent contradiction between these two findings was resolved when it was found that although PrV2 binds as well as does wt to some cell types, it binds poorly to other cell types. The functional importance of different domains of gIII in virus adsorption thus differs, depending on the cell type with which the virus interacts.
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Cloning and sequencing of Puumala virus Sotkamo strain S and M RNA segments: evidence for strain variation in hantaviruses and expression of the nucleocapsid protein
The prototype Puumala virus (PV) Sotkamo strain small (S) and medium (M) RNA genome segments were amplified by the polymerase chain reaction (PCR), cloned and sequenced. The S segment is 1830 nucleotides long with an open reading frame coding for 433 amino acids. The identity to the PV Hällnäs strain was 83% at the nucleotide and 96% at the amino acid level. The M segment in the Sotkamo strain is 3616 nucleotides long and contains one open reading frame of 1148 amino acids with 83% nucleotide and 94% amino acid identity to the Hällnäs strain. Most amino acid changes were conservative and the five predicted glycosylation sites are identical. The amino acid identity to the prototype hantavirus, Hantaan virus, was 62 and 54% for S and M segments, respectively. The coding region of the S segment was further amplified by PCR, ligated to pEX vectors and expressed in Escherichia coli as a β-galactosidase fusion protein and was seen to be specifically detected by nephropathia epidemica sera in immunoblotting.
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Secretion of fowl plague virus haemagglutinin from insect cells requires elimination of both hydrophobic domains
More LessIn the present study we have investigated the role of the hydrophobic domains of the fowl plague virus (FPV) haemagglutinin (HA) on its intracellular transport and maturation in insect cells. To this end processing of full-length HA (A+) has been compared to that of two truncated forms lacking either the cytoplasmic domain and the transmembrane domain (A−) or lacking the entire HA2 subunit, i.e. the transmembrane domain and the fusion peptide (HA- 2). All glycosylation sites present on A− and HA- 2 were glycosylated, indicating that both truncated forms were completely translocated in the endoplasmic reticulum. Unlike A+, A− and HA- 2 did not form trimers as indicated by cross-linking, gradient centrifugation and studies employing conformation-specific antibodies. Whereas HA- 2 was efficiently secreted, A− was retained in the cells in an apparently membrane-bound form. The data show that the carboxy-terminal transmembrane region is essential for the formation and stability of the trimers of the FPV HA. These observations also indicate that, under certain conditions, the fusion peptide of the FPV HA can serve as a membrane anchor.
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Oligomerization and post-translational processing of glycoprotein G of human respiratory syncytial virus: altered O-glycosylation in the presence of brefeldin A
More LessThe post-translational maturation of the attachment G glycoprotein of human respiratory syncytial virus (RSV) was investigated. The G protein formed homooligomers which sedimented in sucrose gradients at the same rate as the fusion F protein tetramer. Oligomerization of the G protein was insensitive to carbonylcyanide m-chlorophenylhydrazine, showing that this step occurs in the endoplasmic reticulum prior to O-glycosylation which initiated in the trans-Golgi compartment. The sedimentation of the G protein oligomer was essentially unchanged by the subsequent addition of O-linked sugars. This indicated that their contribution to the M r of the G protein is less than that estimated by electrophoretic mobility. It also suggested that O-glycosylation is not an important determinant of G protein oligomerization and, by implication, of polypeptide folding. The G protein is palmitylated. In short labelling pulses, the G protein accumulated as two species of 48K and 50K which contained only N-linked sugars, whose difference in M r was due solely to an N-linked sugar, which both assembled into oligomers, but which differed in the rate of subsequent O-glycosylation. The G protein was not detectably O-glycosylated in the presence of monensin, confirming previous work. In the presence of brefeldin A (BFA), it accumulated as a partially O-glycosylated species (BFA-G) of 68K to 78K. But further analysis by chase incubations following BFA-washout, by lectin-binding, and by glycosidase treatment suggested that BFA-G was not a fully authentic processing intermediate. In particular, some of the O-linked side-chains of the BFA-G protein were found to be sialylated. Rather than being a normal step in processing, this sialylation probably was due to altered distribution or activity of sialyltransferases during BFA treatment and may have resulted in the premature termination of elongation of some of the O-linked side-chains. Thus, these studies (i) indicate that O-glycosylation of the G protein begins in the trans-Golgi compartment and (ii) suggest that O-glycosylation is completed in as a subsequent compartment, but this latter suggestion is complicated by the evidence that the BFA-G protein is not a fully authentic intermediate. Finally, an additional species of 48K to 60K was detected under standard conditions of infection and was found to differ in several ways from the 48K and 50K precursors described above: it was detected only following long labelling periods, it was found in monomers rather than oligomers, and it contained a high content of O-linked sugars. Also, antibodies raised against a peptide from the G ectodomain reacted with the 48K, 50K and mature G proteins but did not react with the 48K to 60K species. This indicated that the latter contained differences in conformation or O-glycosylation which masked that part of the ectodomain. The 48K to 60K species was not detected in virions or secreted from infected cells. Thus, it appeared to be a dead-end by-product rather than a true processing intermediate.
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Assignment of mutant tsN19 (complementation group E) of respiratory syncytial virus to the P protein gene
More LessThe mutation responsible for the temperature-sensitive (ts) phenotype of mutant tsN19 (complementation group E) of respiratory syncytial virus has been located to the P protein gene. Viral protein synthesis was completely restricted at 39 °C, and the tsN19 P protein did not react with an anti-P monoclonal antibody (MAb) (3–5) at 33 °C. Reversion of temperature sensitivity restored reactivity with MAb 3–5. Nucleotide sequence determination and in vitro expression of cDNA clones of P mRNA derived from wild-type, tsN19 and non-ts revertant-infected cells, revealed that temperature sensitivity and loss of reactivity with MAb 3–5 were consequences of a Gly → Ser amino acid change at position 172. A low M r polypeptide, which represented the C-terminal 93 amino acids of the P protein, was produced by internal initiation in the P open reading frame during in vitro translation, and a similar product was detected transiently in vivo.
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Antigenic diversity of human parainfluenza virus type 1 isolates and their immunological relationship with Sendai virus revealed by using monoclonal antibodies
Fifty-six monoclonal antibodies (MAbs) directed against human parainfluenza virus type 1 (hPIV-1) were prepared in order to identify the structural proteins of hPIV-1, to examine the immunological relationship between hPIV-1 and Sendai virus (SV), and to determine the antigenic diversity of clinical isolates of hPIV-1. In addition, 41 MAbs characterized previously and directed against SV were used for immunological comparison of SV and hPIV-1 isolates. Of the MAbs against hPIV-1, two reacted with phospho (P) protein, 11 with nucleocapsid protein (NP), 24 with haemagglutinin-neuraminidase (HN) protein and 19 with fusion (F) protein. With the aid of MAbs against hPIV-1 and those against SV showing cross-reactivity with hPIV-1, the structural proteins of hPIV-1 were identified; p83, p56, p34, gp74 and gp60 of hPIV-1 were identified as the P, NP, M, HN and F proteins, respectively. The MAbs against the P protein and NP of hPIV-1 showed limited cross-reactivity with SV, whereas they had high reactivity with clinical isolates of hPIV-1. Interestingly, one MAb against the NP of hPIV-1 lacked reactivity with clinical isolates which were isolated in the 1970s and 1980s. The MAbs against the HN of hPIV-1 also exhibited quite limited reactivity with SV and the clinical isolates; two groups of HN-specific MAbs showed almost no reactivity with the clinical isolates from the 1970s and 1980s, similarly to the NP-specific MAb. However, anti-HN MAbs belonging to the two groups showing specific activities (neuraminidase inhibition and haemolysis inhibition) reacted with almost all clinical isolates. On the other hand, although anti-F protein MAbs had limited reactivity with SV, they showed reactivity with almost all hPIV-1 isolates. The MAbs against the P, NP, M, HN and F proteins of SV also showed limited cross-reactivity with the clinical hPIV-1 isolates, and this reactivity was independent of the time and place of isolation, except for that of the F protein. These results confirm that although hPIV-1 is related to SV, it is antigenically distinct from it.
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Sequence analysis of the genes encoding the nucleocapsid protein and phosphoprotein (P) of phocid distemper virus, and editing of the P gene transcript
The nucleotide and deduced amino acid sequences of two genes of phocid distemper virus (PDV) were determined by cDNA cloning and sequencing. The long open reading frame of the gene encoding the nucleocapsid (N) protein is presented. As with other morbilliviruses, the phosphoprotein (P) gene of PDV was found to be located after the 5′ end of the N gene and before the 3′ end of the matrix protein gene. The P gene was shown to have the capacity to encode three distinct proteins, P, V and C, in analogy to other morbilliviruses. The results presented provide evidence for editing of the PDV P mRNA transcript by insertion of G residues. When the nucleotide and deduced amino acid sequences of the N, P, V and C genes were aligned with corresponding sequences of other established members of the morbillivirus genus, compelling homology was found between PDV and canine distemper virus (CDV), whereas there was markedly less similarity between PDV and measles virus or rinderpest virus. On the basis of the alignments presented, the estimated amino acid sequence similarity between the N and P genes of PDV and CDV was 84% and 76%, respectively. These differences at the genomic level indicate that the viruses are two separate entities.
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Basis of neurovirulence of avirulent rabies virus variant Av01 with stereotaxic brain inoculation in mice
More LessAv01 is a variant of the challenge virus standard strain of fixed rabies virus that was selected with a neutralizing anti-glycoprotein monoclonal antibody, and has a single amino acid change in the glycoprotein. It is avirulent after both intracerebral and peripheral routes of inoculation in adult mice. In this study, Av01 was found to be neurovirulent with stereotaxic brain inoculation in either the striatum or cerebellum of adult mice. Mice that had been inoculated simultaneously with Av01 by the intracerebral and intrastriatal routes recovered. More infectious virus was present in the brains of mice inoculated intrastriatally than intracerebrally, and more neurons contained rabies virus antigen. However, the topographical distribution of infected neurons was similar with both routes. Serum neutralizing antibodies against rabies virus were produced later and in smaller quantities after intrastriatal inoculation. Av01 is probably neurovirulent after stereotaxic brain inoculation because this route produces both a direct site of viral entry into the central nervous system and a low level of immune stimulation.
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Bovine coronavirus uses N-acetyl-9-O-acetylneuraminic acid as a receptor determinant to initiate the infection of cultured cells
More LessThe importance of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) as a receptor determinant for bovine coronavirus (BCV) on cultured cells was analysed. Pretreatment of MDCK I (Madin Darby canine kidney) cells with neuraminidase or acetylesterase rendered the cells resistant to infection by BCV. The receptors on a human (CaCo-2) and a porcine (LLC-PK1) epithelial cell line were also found to be sensitive to neuraminidase treatment. The susceptibility to infection by BCV was restored after resialylation of asialo-MDCK I cells with Neu5,9Ac2. Transfer of sialic acid lacking a 9-O-acetyl group was ineffective in this respect. These results demonstrate that 9-O-acetylated sialic acid is used as a receptor determinant by BCV to infect cultured cells. The possibility is discussed that the initiation of a BCV infection involves the recognition of different types of receptors, a first receptor for primary attachment and a second receptor to mediate the fusion between the viral envelope and the cellular membrane.
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Localization of group-specific epitopes on the major capsid protein of group A rotavirus
More LessChemical cleavage of the VP6 protein of bovine rotavirus showed that VP6-specific monoclonal antibodies (MAbs) reacted with the amino acid sequence between glycine 48 and asparagine 107. Furthermore, three synthetic peptides (amino acids 48 to 64, 60 to 75 and 91 to 108) containing part of this sequence and 22 consecutive overlapping heptapeptides corresponding to the region between amino acids 48 and 75 were analysed for their immunoreactivity using group-specific MAbs. The MAbs recognized peptides 48–64 and/or 60–75, and a set of overlapping heptapeptides located between residues 53 (asparagine) and 67 (glycine), which have two short sequences in common: IRNW (residues 56 to 59), recognized by MAb RV-133, and (NW)NFD (residues 58/60 to 62), recognized by MAbs RV-50, -1026 and -443. These results indicate that the sequence between amino acid residues 48 and 75 is present in one of the immuno-dominant sites of VP6.
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Characterization of the genes encoding two of the major capsid proteins of epizootic haemorrhagic disease virus indicates a close genetic relationship to bluetongue virus
More LessThe sequences of the genes of two of the major capsid proteins of epizootic haemorrhagic disease virus serotype 1 (EHDV-1, Orbivirus genus, Reoviridae) have been determined by analyses of cDNA clones representing the L2 and S7 RNA segments. The EHDV-1 S7 RNA segment, which encodes the VP7 core protein, is 1162 nucleotides in length and has the capacity to encode 349 amino acids (M r 38243). The EHDV-1 L2 RNA segment, which encodes the outer capsid VP2 protein (M r 113249) is 2968 nucleotides in length and has an open reading frame of 971 codons. The potential secondary structure of the EHDV-1 S7 mRNA species, in particular that of the terminal regions, is comparable to those of the corresponding segments of bluetongue virus (BTV) and African horse sickness virus (AHSV); the EHDV-1 L2 mRNA species has a secondary structure similar to that of the L2 mRNA of BTV. The EHDV-1 VP2 and VP7 proteins, as well as those of the other two major structural proteins of EHDV published previously (the inner core VP3 protein and the second outer capsid, VP5), are closely related to the corresponding proteins of BTV. The EHDV and BTV VP7 sequences are more distantly related to the sequence of the AHSV VP7 protein published recently.
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Expression of the major core antigen VP7 of African horsesickness virus by a recombinant baculovirus and its use as a group-specific diagnostic reagent
More LessThe major core protein, VP7, of African horsesickness virus serotype 4 (AHSV-4), the aetiological agent of a recent outbreak of the disease in southern Europe, was expressed in insect cells infected with a recombinant baculovirus containing a cloned copy of the relevant AHSV gene (S7). Analyses of its biochemical and antigenic properties confirmed the authenticity of the protein expressed. The high-level expression of VP7 under the control of the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus induced disc-shaped crystals in infected insect cells. This enabled us to purify the protein by a one-step ultracentrifugation procedure and to utilize it for the detection of antibodies raised in horses to various serotypes of AHSV. A serological relationship between AHSV and two other orbiviruses, bluetongue virus and epizootic haemorrhagic disease virus, was also demonstrated.
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Lack of human immunodeficiency virus type 1 (HIV-1) replication and accumulation of viral DNA in HIV-1-infected T cells blocked in cell replication
More LessHuman immunodeficiency virus type 1 (HIV-1) infection of the CD4+ SupT and CEM cell lines, blocked in cell replication by the polymerase α inhibitor aphidicolin (APC), was studied. The APC-treated cells showed a lack of viral production, but the presence of single cell killing. High levels of unintegrated viral DNA forms were found in the infected APC-treated cells as compared with untreated cells. Moreover, an increased rate of viral replication occurred in the remaining viable cells following removal of APC. The results indicate that HIV-1 entry and reverse transcription can take place in cells blocked in the S phase of the cell cycle. Replication of infectious progeny virions appears to require de novo cell division. Finally, accumulation of viral DNA in cells during APC treatment can result in cytopathological effects and subsequent enhancement of virus production.
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Recombinant interleukin 4 stimulates human immunodeficiency virus production by infected monocytes and macrophages
Recombinant interleukin 4 (IL-4) stimulated extracellular (EC) and intracellular (IC) production of human immunodeficiency virus (HIV) from infected human blood-derived monocytes and macrophages when incubated with the cells after but not before virus inoculation. Significant stimulation was observed in 20 of 27 experiments with monocytes (inoculated with HIV immediately after adherence) and 10 of 13 experiments with macrophages (inoculated after 5 days adherence) using a total of 30 normal donors of monocytes and macrophages, and 11 recent isolates of monocytotropic HIV strains (after one passage in mononuclear cells). Marked increases in EC and IC HIV antigen were observed in some experiments, which were comparable with the maximal stimulatory effects of other cytokines such as IL-2. IL-4 also had similar effects on infectious HIV concentration as measured by reverse transcriptase and TCID50 assays. Antibody to IL-4 prevented the stimulatory effect of the cytokine. The proportion of monocytes and macrophages infected by HIV, as determined by in situ hybridization, also increased after incubation with IL-4 for 7 days. The most marked effects were observed with HIV-infected macrophages, for which the proportion of unstimulated infected cells was lower (35 to 45% increasing to 66 to 70% with IL-4 treatment). There was also an increased proportion of cells with high granule concentrations, suggesting that IL-4 increases the intracellular concentration of viral nucleic acids. This was supported by semi-quantitative hybridization experiments showing that total HIV RNA increased in IL-4-stimulated monocytes 48 to 96 h after HIV inoculation. A marked increase in aggregates was observed on day 7 in HIV-infected monocytes treated with IL-4, compared to that in HIV-infected cells alone or IL-4-treated uninfected monocytes. These findings suggest that IL-4 stimulates HIV replication in the early phases of infection and may also facilitate virus transmission by aggregate formation.
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Murine monoclonal antibodies directed against the transmembrane protein gp41 of human immunodeficiency virus type 1 enhance its infectivity
More LessMonoclonal antibodies (MAbs) were raised against the transmembrane protein (TM) gp41 of human immunodeficiency virus type 1 (HIV-1, strain HTLV-IIIB). The reactivity of three TM-specific MAbs was investigated in several tests, ELISA, immunostaining of Western blots, immunofluorescence and an alkaline phosphatase-anti-alkaline phosphatase assay. Epitope mapping was done by using overlapping gp41 peptides produced as Escherichia coli fusion proteins and synthetic peptides. In an in vitro assay, all three MAbs showed enhancing effects on HIV-1 infection after single or repeated treatment with the purified MAbs at concentrations of 6 to 25 µg/ml. The enhancing domain is located between amino acids 724 and 752 of the env protein sequence. Homologous peptides based on this sequence were used for analysis of sera from 100 individuals at different stages of HIV infection to evaluate the relevance of antibodies against this region to the prognosis of disease. No antibodies reactive with this region were found in ELISA, indicating that this domain is not immunogenic in humans.
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De novo reverse transcription is a crucial event in cell-to-cell transmission of human immunodeficiency virus
More LessThe proposal that replication of human immunodeficiency virus type 1 (HIV-1), mediated by cell-to-cell transmission of the virus, might bypass de novo reverse transcription was tested by using one-step cell-to-cell and cell-free virus infection systems. Two well characterized reverse transcriptase (RT) inhibitors, azidothymidine at 20 µm and phosphonoformic acid at 100 µg/ml, blocked HIV replication completely following both cell-free virus and cell-to-cell transmission infection, as determined from the kinetics of unintegrated viral DNA synthesis and supernatant RT production after virus infection. Our results confirm that de novo reverse transcription is a crucial and mandatory event in HIV-1 replication following cell-to-cell transmission of the virus.
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Detection of adeno-associated virus type 2 in human peripheral blood cells
More LessThe non-pathogenic human parvovirus, adeno-associated virus (AAV) is helper virus-dependent. However, it integrates into the cellular genome in the absence of its helper viruses. Therefore it could become a useful vector for gene therapy. Previous studies and our own results have shown that 40 to 80% of adults are seropositive for AAV and that seroconversion occurs during the first few years of life, but little is known about the route of natural infection with the virus. We used the polymerase chain reaction to detect the AAV-2 genome and identify AAV sequences within peripheral blood leukocytes (PBLs). We could detect AAV in PBLs of two of 55 healthy blood donors, and two of 16 haemophilic patients. AAV DNA replication and viral protein production in PBLs propagated in tissue culture were also examined. AAV DNA replicated very efficiently in the presence of helper adenovirus, but capsid proteins were produced at a lower level and the yield of infectious virus was very low. Our findings prove that in vivo infection of PBLs occurs, and that PBLs could mediate the spread of AAV infection to different body tissues.
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Peripheral replication and latency reactivation kinetics of the non-neurovirulent herpes simplex virus type 1 variant 1716
More LessThe terminal portion of the herpes simplex virus (HSV) genome long repeat region has been shown to contain a neurovirulence gene. Both HSV-1 and HSV-2 mutants deleted in this gene fail to cause central nervous system (CNS) disease in mice. The HSV-1 strain 17 variant 1716, which has a 759 bp deletion encompassing the gene, grows normally in tissue culture but fails to grow following intracerebral inoculation of mice. This paper demonstrates that 1716 is capable of peripheral replication in the footpads of mice. However, no acute replication of virus is detectable in dorsal root ganglia up to 10 days after footpad inoculation. These results imply that the replication defect in 1716 is not host-specific, but is tissue- and/or cell type-specific. Latency reactivation kinetics demonstrate that 1716 is capable of establishing a latent infection, but the kinetics of reactivation are significantly impaired compared to wild-type virus and are dose-dependent. Lack of acute ganglionic replication combined with impaired reactivation kinetics support the conclusion that a proportion of 1716 genomes initiate a lytic infection which then aborts, and a proportion enter the latent state. The results with 1716 imply that its inability to replicate in CNS and peripheral nervous system neurons is specific, and that the block in replication is beyond the stage of adsorption and entry. A prerequisite for any live attenuated HSV vaccine is an inability to initiate CNS involvement following peripheral inoculation. In this respect, 1716 has prototype vaccine potential with the proviso that a direct extrapolation is being made from mouse to man.
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Status of the ICP34.5 gene in herpes simplex virus type 1 strain 17
More LessIn the published DNA sequence of herpes simplex virus type 1 (HSV-1) strain 17 the coding region for the neurovirulence factor ICP34.5 is disrupted by a 2 bp insertion relative to the corresponding sequence in HSV-1 strain F; this difference would render 60% of the ICP34.5 coding sequence out of frame in strain 17. Re-investigation of the strain 17 sequence showed that the plasmid clone used as the major sequencing substrate for the region was atypical in that other clones did not possess the 2 bp insertion. It was concluded that the coding sequence for ICP34.5 is intact in HSV-1 strain 17 and that the HSV-1 DNA sequence should be revised at this locus.
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Relationship between HOX2 homeobox gene expression and the human cytomegalovirus immediate early genes
More LessThe human embryonal carcinoma cell line NT2/D1 is known to be non-permissive for human cytomegalovirus (HCMV) but becomes permissive after being induced to differentiate by retinoic acid (RA). Because homeobox genes have been reported to be specifically activated in the RA-differentiated NT2/D1 cells, we investigated the possible correlation between expression of homeobox (HOX) 2 genes and expression of the immediate early (IE) genes of HCMV both in NT2/D1 cells and in HCMV permissive human embryonic lung (HEL) cells. HCMV infection did not induce activation of the HOX2A, HOX2E and HOX2I genes in undifferentiated NT2/D1 cells nor affect their activation in the RA-differentiated NT2/D1 cells. By in situ hybridization using a HOX2A RNA probe, HOX2A transcript-positive cells appeared as clusters in RA-differentiated NT2/D1 cells. Viral antigen-positive cells detected by immunofluorescence using an antibody specific for the IE-1 antigen of HCMV appeared as clusters among the population of cells in which the HOX2A transcript was detected. The HOX2A gene only was expressed in HEL cells, however none of the HOX2 genes was expressed in non-permissive HeLa, Raji or mouse embryonic cells. These results suggest that activation of the HOX2A may be necessary for the expression of IE genes. HCMV infection markedly increased the expression of the HOX2E gene in HEL cells in the presence, but not in the absence, of cycloheximide. Ultraviolet-inactivated HCMV also displayed this effect. On the other hand, HCMV infection suppressed expression of the HOX2A gene to some degree at the early and late phases of infection in HEL cells. Activation of the HOX2E gene by HCMV might possibly have a role in virus-induced abnormal embryogenesis.
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The amino terminus of human cytomegalovirus glycoprotein B contains epitopes that vary among strains
More LessWe mapped three antigenic domains of continuous epitopes on human cytomegalovirus (CMV) glycoprotein B (gB) by reacting a panel of independently derived monoclonal antibodies with deletion mutants expressed transiently in COS-1 cells. One of these antigenic domains, DC2, maps in the last 75 amino acids of the carboxy terminus. These epitopes are conserved in strains Towne and AD169, as well as in 19 clinical CMV isolates. ELISAs of DC2-reactive antibodies with a set of overlapping synthetic oligopeptides from the carboxy terminus showed that the epitopes of antibodies CH405-1 and CH421-5 map between amino acids 833 and 852 and that the epitope of antibody CH28-2 maps between amino acids 878 and 898. These linear epitopes were grouped into domain DC3. The third antigenic domain, DC1v, maps at the amino-terminal end of CMV strain AD169 gB but is not contained in strain Towne or in 17 of 19 clinical isolates. Epitopes in this domain are likely to map between amino acids 28 and 67, an area where differences occur in the nucleotide sequence of the gB genes from AD169 and Towne. Analysis of CMV-infected cells by flow cytometry with antibodies to the amino- and carboxy-terminal domains revealed that the amino terminus of gB is extracellular and that the carboxy terminus is not exposed on the cell surface.
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Vaccinia virus encodes four putative DNA and/or RNA helicases distantly related to each other
More LessComputer-assisted analysis of the amino acid sequences of vaccinia virus proteins containing the purine NTP-binding pattern revealed the seven motifs typical of the DNA (RNA) helicase superfamily II in the proteins I8R and A18R. Together with the previously described putative helicases D6R and D11L, the number of putative helicases of this superfamily encoded by the genome of vaccinia virus now reaches four. Aside from the helicase motifs, the sequences of I8R and A18R showed no strong similarity to each other, nor to D6R and D11L. Statistically significant similarity was demonstrated between I8R and the putative RNA helicases involved in pre-mRNA splicing in yeast, PRP2, PRP16 and PRP22, whereas A18R appeared to be related to the putative helicases encoded by the human DNA repair gene ERCC3 and the D10 gene of bacteriophage T5. These findings suggest that I8R may be an RNA helicase. Based on the known properties of the virion NTPases of vaccinia virus, it is possible that the I8R protein may be identical to the previously characterized virion NTPase II. A18R is likely to possess DNA and/or RNA helicase activity. Circumstantial evidence suggests that this activity might be involved in melting duplex structures in late mRNAs. The possibility of independent acquisition of the putative helicases I8R, A18R and a common progenitor to D6R and D11L by an ancestral poxvirus is discussed.
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Low pH deforms the influenza virus envelope
More LessInfluenza virus membrane fusion is induced by low pH, which triggers an irreversible conformational change in the viral haemagglutinin (HA). The result of this change is the extrusion of the HA fusion peptide, after which it may act in the fusion of virus and endosomal membranes. Here we describe electron microscopic observations on low pH-treated virus after negative staining or cryo-electron microscopy of virus in the frozen hydrated state. The results indicate a destabilization of the virus membrane at low pH that can be reversed by returning the pH to neutral.
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Bovine respiratory syncytial virus nucleocapsid protein: mRNA sequence analysis and expression from recombinant vaccinia virus vectors
More LessThe nucleotide sequence of the mRNA encoding the nucleocapsid (N) protein of bovine respiratory syncytial (BRS) virus, strain 391-2, was determined. Recombinant vectors containing a cDNA of the complete N gene were constructed, and expression of the N protein in eukaryotic cells was demonstrated using two different vector systems. The BRS virus N mRNA was 1197 nucleotides in length, exclusive of poly(A), and had a single major open reading frame that encoded a polypeptide of 391 amino acids with a calculated M r of 42.6K. The nucleotide and amino acid sequences of the BRS virus N gene were compared to those of human respiratory syncytial (HRS) virus strains A2 and 18537, and to BRS virus strain A51908. The level of nucleic acid identity between the N mRNA of BRS virus 391-2 and both HRS virus subtypes was 80 to 81%, whereas the identity between the two BRS virus strains was 97%. A 93 to 94% level of identity was observed between the deduced amino acid sequences of the N protein of BRS virus 391-2 and the corresponding sequences of the two HRS virus strains. The two BRS virus N proteins differed in amino acid sequence at only three positions. Recombinant BRS virus N protein was expressed using two different vector systems: in cells from a plasmid using the vaccinia virus/T7 polymerase expression system or from a recombinant vaccinia virus. N proteins synthesized by the two vector systems migrated with an electrophoretic mobility identical to that of authentic BRS virus N protein, and were precipitated by anti-BRS virus antibodies.
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Sequence analysis of the large (L) protein of simian virus 5
The complete nucleotide sequence of the large (L) protein gene of simian virus 5 (SV5) was determined from cDNA of the genomic RNA and mRNA, and found to be 6804 bases in length, exclusive of a poly(A) tract. The sequence contained an open reading frame of 6765 nucleotides encoding 2255 amino acids. Results of dot matrix comparisons of the L protein of SV5 with those of human parainfluenza type 3 virus and Sendai virus indicated that there are five conserved domains, and that each domain contains characteristic sequence(s). The L protein of SV5 was detected in purified virions using antiserum directed against an oligopeptide corresponding to the N-terminal region.
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Nucleocapsid gene sequence of a North American isolate of viral haemorrhagic septicaemia virus, a fish rhabdovirus
More LessViral haemorrhagic septicaemia is the most important viral disease of trout in Europe. The causative agent, viral haemorrhagic septicaemia virus (VHSV), a member of the lyssavirus genus of the rhabdoviridae family, was formerly believed to be confined to portions of the European continent; however in 1988, VHSV was isolated from adult chinook (Oncorhynchus tshawytscha) and coho (O. kisutch) salmon returning to two hatcheries in the northwestern part of the State of Washington, U.S.A. Initial fears were that the virus had been imported into North America, perhaps by aquaculture activities. The nucleotide and deduced amino acid sequences of the nucleocapsid (N) gene of one of the North American (Makah) isolates of VHSV were determined and compared with published sequences of a European reference strain of VHSV (07-71) and the Round Butte strain of infectious haematopoietic necrosis virus (IHNV), another salmonid fish rhabdovirus that is enzootic in western North America. The N gene of the Makah isolate of VHSV shared a similarity of 88.433% at the nucleotide level and 94.802% at the amino acid level with the N gene of the European strain of VHSV, and 62.121% amino acid similarity with the N protein of IHNV. Like the European reference isolate, the North American isolate of VHSV showed three domains in the N protein, the central one being the most conserved and the likely site of interaction with genomic RNA. This was also the region of highest similarity with the amino acid sequence of IHNV. The sequence data suggested that the Makah and 07-71 isolates were of independent origin.
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Follow-up of hepatitis C virus infection in chimpanzees: determination of viraemia and specific humoral immune response
More LessChimpanzees were inoculated intravenously with the H strain of hepatitis C virus (HCV), and analysed for viraemia using the polymerase chain reaction and for a humoral immune response using first and second generation anti-HCV ELISAs and an immunoblot assay (4-RIBA). In all seven chimpanzees studied, viraemia occurred several weeks before a significant increase in serum alanine transferase (ALT) activity, whereas the first circulating anti-HCV antibodies became detectable at the time of significant increase in ALT levels, provided the second generation ELISA or 4-RIBA was used. On the basis of the duration of viraemia the chimpanzees studied could be assigned to two different groups: those in which viraemia disappeared in conjunction with or shortly after seroconversion, and those remaining viraemic for many weeks after the appearance of antibodies. The clearance of HCV from the circulation did not correlate with the antibody pattern determined using 4-RIBA, i.e. the HCV-specific assays currently available do not enable us to predict whether an infected chimpanzee will develop persistent viraemia. Only two of the seven chimpanzees analysed developed anti-core protein (c-22) antibodies, which appeared at the same time as the first ALT peak, whereas all animals developed antibodies to the non-structural protein, c-33, and these antibodies persisted.
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Characterization of a new picorna-like virus, himetobi P virus, in planthoppers
More LessPicorna-like virus particles, 29 nm in diameter, were purified from apparently healthy Laodelphax striatellus Fallen. The virus particles had a buoyant density of 1.352 g/ml in CsCl and a sedimentation coefficient of 161 s. The virus capsid proteins consisted of three major polypeptides of M rs 36500, 33000 and 28000, and three minor polypeptides. The virus contained a major ssRNA of M r 2.8 × 106 and was also frequently associated with a minor dsRNA of M r 4×106. The 3′ end of the ssRNA had a poly(A) tract of about 60 adenine residues. The virus has been provisionally named himetobi P virus.
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Further analysis of nucleic acids in purified scrapie prion preparations by improved return refocusing gel electrophoresis
More LessAlthough increasingly unlikely, the possibility of a scrapie-specific nucleic acid carried by infectious prion particles is still unresolved. Return refocusing gel electrophoresis was developed to detect homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. This method was improved with respect to the size range from 13 to 1100 nucleotides (nt) over which analyses could be performed. The yield of nucleic acid, particularly of small DNA oligonucleotides and polyadenylated RNA, was determined after deproteinization and two-phase extraction. Despite extensive nuclease digestions some small polynucleotides remained. Although a scrapie-specific nucleic acid cannot be excluded, the results further define the possible characteristics of a hypothetical molecule. If homogeneous in size, such a molecule would be <80 nt in length at a particle-to-infectivity ratio near unity, if heterogeneous, scrapie-specific nucleic acids would have to include molecules smaller than 240 nt.
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Electrotransfection of protoplasts from tomato, wild tomato, barley and chrysanthemum with tobacco mosaic virus RNA
More LessProtoplasts isolated from tomato, wild tomato, barley and chrysanthemum were electrotransfected with tobacco mosaic virus (TMV) RNA under almost the same optimum electric conditions: five square DC pulses of 50 µs duration at 500 to 800 V/cm, with the protoplasts suspended at 2 × 105/ml in 0.5 m-mannitol containing 100 µm-MgCl2 and 10 to 20 µg/ml TMV RNA. ELISAs of these transfected protoplasts showed that the yields and the growth curves of the virus were quite similar, indicating a lack of host specificity in the initially infected cells of these plants.
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Analysis of the dsRNAs of apple chlorotic leaf spot virus
More LessDouble-stranded RNAs were isolated from plants infected with five different isolates of apple chlorotic leaf spot virus (ACLSV). Analysis by PAGE and by Northern blot hybridization showed that six major species of viral dsRNA of approximately 7.5, 6.4, 5.4, 2.2, 1.1 and 1.0 kbp can be detected in infected plants, irrespective of the ACLSV isolate used. In addition to the dsRNA of 7.5 kbp corresponding to the full-length genome, the size and position on the genome of the 2.2 and 1.1 kbp species indicate that these are very probably double-stranded forms of subgenomic RNAs allowing the expression of the internal open reading frames coding respectively for the ACLSV 50K and coat proteins. The subgenomic messenger for the coat protein was indeed detected in total RNA preparations from infected plants. Surprisingly, the two most abundant dsRNA species, of 6.4 and 5.4 kbp, were found to be 5′-coterminal with the genomic RNA. A model for the expression of the genome of ACLSV and for the production of the molecules 5′-coterminal with the genomic RNA is presented.
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Autocatalytic activity of the tobacco etch virus NIa proteinase in viral and foreign protein sequences
More LessThe small nuclear inclusion (NIa) protein of the tobacco etch virus (TEV) is synthesized initially as part of a genome-derived high M r precursor. The NIa protein releases itself from this genome-derived precursor by self-cleavage, or an autocatalytic processing event. Cleavage between specific glutamine-glycine dipeptides at the N and C termini generates the 430 amino acid or 49000 M r (49K) NIa protein. The requirements of this autocatalytic release, or cis cleavage, were examined by constructing gene cassettes encoding the TEV NIa protein which could be ligated into particular locations in cDNA of the TEV genome and also into foreign gene DNA sequences. Using cell-free transcription and translation systems, polyproteins containing TEV NIa sequences were synthesized and assayed for (i) autocatalysis and (ii) the ability of a functional NIa proteinase, purified from plant tissue, to cleave in bimolecular or trans reactions various artificial polyproteins which contained an inactive form of the NIa proteinase. The NIa self-cleavage events required an active proteinase sequence and a consensus TEV cleavage site sequence at the N and C termini. These results were consistent for NIa protein sequences placed at a foreign TEV cleavage site or in unrelated proteins. Differences were noted in the trans cleavage of these sites.
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Conserved terminal sequences of rice ragged stunt virus genomic RNA
More LessThe 5′- and 3′-terminal nucleotide sequences of the dsRNA genome segments of rice ragged stunt virus (RRSV), a member of the plant Reoviridae, were determined and compared with those published for other viruses in this family. The 5′- and 3′-terminal regions of the RRSV plus strand RNA from all genome segments were found to have the same conserved hexanucleotide (5′ GAUAAA---) and tetranucleotide (---GUGC 3′) sequences, respectively. These conserved terminal sequences were different from those found in viruses in the Phytoreovirus and Fijivirus genera. This result confirms that, as already suggested, RRSV should be placed in a third genus.
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