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Volume 66,
Issue 10,
1985
Volume 66, Issue 10, 1985
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Paramyxovirus Antigens in Osteoclasts from Paget’s Bone Tissue Detected by Monoclonal Antibodies
More LessSUMMARYThe fluorescent antibody technique using both monoclonal and specific polyclonal virus antibodies was applied to investigate the nature of the inclusions seen in the abnormal osteoclasts associated with Paget’s bone disease. The results show that antigens of measles virus, simian virus 5 (SV5) and human parainfluenza virus type 3 (PF3) could be detected in the osteoclasts but not in control bone cells. Measles and SV5 nucleoprotein (NP) and haemagglutinin-neuraminidase (HN) antigens were apparently present in all the cases of Paget’s disease examined, whereas PF3 NP and HN antigens were present only in some of the cases. These investigations suggest that paramyxoviruses may play a role in the aetiology of the bone disease.
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Two Distinct Subtypes of Human Respiratory Syncytial Virus
More LessSUMMARYAntigenic variation of human respiratory syncytial (RS) virus strains was analysed using a collection of nine, six, six, nine and one monoclonal antibodies respectively directed against the large glycoprotein (G), fusion protein (F), matrix protein (M), nucleoprotein (NP) and phosphoprotein (P) components of the Long strain of RS virus. A comparison was made with seven other strains isolated during different years in radioimmune precipitation analyses and immune fluorescence tests. Two different subtypes of the virus were demonstrable. Subtype A included the prototype strains Long and A2 and virus isolates from 1973, 1983 and 1984; subtype B included four virus strains isolated in successive years from 1979 to 1982. Subtype A viruses reacted with all the antibodies, whereas subtype B viruses showed different epitope characteristics in four structural components. The number of altered epitopes were 5/6, 1/2, 2/6 and 1/6 in the G, F, M and NP components, respectively. It is concluded that the two subtypes have evolved separately. The finding of two subtypes may explain previously observed strain variations in neutralization tests, and gives a new perspective on the immunobiology of RS virus.
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Antigenic Sites on the ERA Rabies Virus Nucleoprotein and Non-structural Protein
More LessSUMMARYThirty-one monoclonal antibodies, specific for either the nucleoprotein (N) or the non-structural protein (NS; nucleocapsid-associated protein) of the nucleocapsid of the ERA strain of rabies virus, were used to investigate the topography of antigenic sites on the nucleocapsid complex. Based on the results of a competitive enzyme immunoabsorbent assay using these antibodies, five spatially distinct antigenic sites were defined: three on the N protein (groups N I, N II and N III) and two on the NS protein (groups NS I and NS II). Antigenic variations among various street and laboratory strains of rabies virus were analysed by indirect immunofluorescence assay with the monoclonal antibodies specific for the nucleocapsid. Some correlation between the natural nucleocapsid variation and the antigenic topographical map was observed.
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A cAMP-independent Serine/Threonine Kinase Activity Is Associated with the mos Sequences of ts110 Moloney Murine Sarcoma Virus-encoded P85gag-mos
More LessSUMMARYTwo proteins, termed P85gag-mos and P58gag, are encoded by the temperature-sensitive transformation mutant, ts110 Moloney murine sarcoma virus (MuSV). Based on temperature-shift studies, P85gag-mos is believed to be important for the transforming potential of ts110 MuSV and has been found to be associated with a thermolabile kinase activity that phosphorylates both P85gag-mos and P58gag in immune complexes. Modifications of the original kinase assay conditions are reported here that have allowed a 30-fold increase in the specific activity of P85gag-mos phosphorylated in vitro. The in vitro P85gag-mos-phosphorylating activity was found to be unresponsive to 10 µm-cAMP or 10 µm-cGMP. Addition of 1 mm-pyrophosphate, a known phosphatase inhibitor, to the reaction mixture resulted in an increased yield of phosphorylated P85gag-mos and P58gag; the molar phosphate incorporation per mole of P85gag-mos increased from 0.032 to 0.9, whereas the specific activity of in vitro-phosphorylated P58gag increased 18-fold, from 0.013 to 0.234. pH curves of the in vitro kinase reaction further confirmed the presence of phosphatase activity; in the absence of pyrophosphate, a sharp optimum at pH 4 to 5 was observed, whereas it shifted broadly to pH 7.0 in the presence of pyrophosphate. Under the latter conditions, several experiments were performed in order to determine if the kinase was associated with either gag or mos sequences of P85gag-mos. Antisera directed against p15, p12 and p30 sequences of the gag protein region of P85gag-mos yielded immune complexes that allowed phosphorylation in vitro of P85gag-mos. No phosphorylating activity was detected in immune complexes containing MuSV-124-encoded P62gag. An anti-mos serum generated against a synthetic peptide representing the predicted v-mos amino acid residues 37 to 55 recognizes P85gag-mos and allowed phosphorylation of P85gag-mos in vitro in the absence of P58gag. Peptide mapping of both phosphorylated P85gag-mos and P58gag, by using a combination of Cleveland and Western/immunoperoxidase techniques, demonstrated that P85gag-mos became phosphorylated not only on gag sequences, but also at the N-terminal portion of v-mos. Phosphoamino acid analyses of P85gag-mos and P58gag phosphorylated in vitro under these modified conditions yielded predominantly phosphoserine and lesser amounts of phosphothreonine. Metabolically 32P-labelled P85gag-mos and P58gag were also found to contain phosphoserine and phosphothreonine. Based on these results, we conclude that a cAMP-independent, serine/threonine protein kinase activity is associated with the mos sequences of P85gag-mos.
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Hormonal Regulation of a Polyoma Virus Middle-size T-Antigen Gene Linked to Growth Hormone Control Sequences
More LessSUMMARYWe constructed a recombinant DNA gene containing sequences from the 5′ flanking region of a dexamethasone-inducible rat growth hormone gene linked to the coding region of the polyoma virus transforming protein, the middle-size T (MT) antigen. We used this gene to derive cell lines in which the expression of the MT antigen could be regulated by dexamethasone. We transfected mouse NIH 3T3 cells and isolated transformed foci from cultures grown in the presence or absence of dexamethasone. The frequency of focus formation and the size of the transformed foci were increased in the presence of dexamethasone. Several transformants showed regulated expression of the MT antigen: the levels of polyoma-specific RNA, MT protein and MT-associated kinase activity were increased two- to fivefold in cells grown in the presence of dexamethasone. These results show that 248 base pairs of rat growth hormone DNA, including the first 237 base pairs upstream from the major transcription initiation site, contain promoter activity and a regulatory element required for glucocorticoid induction. This region of the rat growth hormone can be used to regulate expression of the polyoma MT antigen gene. In some cell lines regulated expression of the MT antigen was accompanied by regulated expression of transformed cell growth properties. The minimum level of the MT antigen required for expression of transformation was considerably less than the level found in a polyoma MT-transformed cell line. Increasing the level of the MT antigen led to increased expression of transformation, assayed by morphology, focus formation and growth in agar.
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The Regulation of Translation in Reovirus-infected Cells
More LessSUMMARYThe regulation of translation in reovirus-infected cells has been investigated by double-infection experiments. Different cell lines were able to translate uncapped encephalomyocarditis (EMC) virus mRNA and capped vesicular stomatitis virus (VSV) mRNAs both early and late during reovirus infection. These results are not fully in agreement with a previously suggested model in which, in the early phase of reovirus infection, only capped mRNAs are translated, whereas in the late phase the cells are modified to translate exclusively uncapped mRNAs. The observations that EMC virus and VSV shut down host protein synthesis more efficiently than reovirus translation in the late phase in double-infections indicate that competition for a message-discriminatory factor may not be involved in the shut-off of host protein synthesis in these animal virus-infected cells.
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Ectromelia Virus-induced Changes in Primary Cultures of Mouse Hepatocytes
D. N. Lees and J. StephenSUMMARYMouse hepatocytes were isolated by collagenase perfusion, maintained in non-proliferating monolayer culture and shown to retain liver cell function as judged by gluconeogenesis for 15 to 18 h. Such cells could be infected with and support the replication of a virulent strain of ectromelia virus. Virus antigen and characteristic cytoplasmic ‘B’-type poxvirus inclusion bodies were demonstrated by immunofluorescence in virtually all cells. By electron microscopy it was shown that ‘B’-type inclusions were the site of virus replication, and that the biogenesis of ectromelia virus and ultrastructural changes in hepatocytes were similar to those observed in infected mouse livers. Early cell rounding effects, a normal characteristic of poxvirus infections in tissue culture cells, were not seen in ectromelia-infected hepatocytes, although late degenerative changes did occur. Pulse-labelling of hepatocyte cultures with [35S]methionine showed that ectromelia virus inhibited the rise in protein synthesis seen in controls and imposed a gradual decline in host protein synthesis to an extent and at a rate significantly different from that in mouse L929 cells. Gluconeogenesis was inhibited by ectromelia virus infection of hepatocytes.
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Demonstration of the Colinearity of Human Cytomegalovirus Genomes and Construction of Restriction Maps of Unknown Isolates Using Cloned Subgenomic Fragments
More LessSUMMARYIn this study, we have established the colinearity of human cytomegalovirus (HCMV) genomes using stringent conditions of DNA-DNA filter hybridization of HCMV HindIII fragments and cosmid-cloned AD169 strain HCMV DNA fragments. Large cosmid-cloned fragments of AD169 DNA were used for the preparation of radioactive probes by nick translation. These probes were hybridized to HindIII digests of DNA from three fresh isolates of HCMV and to that of the Davis strain. Using published HindIII restriction maps for the AD169 strain as a reference, the results obtained by hybridization allowed us to construct HindIII restriction maps for the genomes of the three fresh isolates. Confirmation of our methodology was found in the correspondence between the HindIII map we constructed for the Davis strain and that published previously. Furthermore, it is shown that variation in the restriction profiles of the unique regions of the genome are due to the absence or gain of restriction sites, and not to a rearrangement of fragments. This technique allows rapid construction of physical maps of the DNA of any fresh isolate for a given restriction enzyme provided the corresponding restriction map of a strain to be used as reference is available.
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Asynchronous Expression of the Immediate-Early Protein of Herpesvirus Saimiri in Populations of Productively Infected Cells
More LessSUMMARYThe time course of virus replication in cultures of permissive cells infected with high multiplicities of herpesvirus saimiri (HVS), a gammaherpesvirus, is protracted relative to the replication of herpes simplex virus (HSV), an alphaherpesvirus, under similar conditions. The basis for this difference was investigated by quantitative immunofluorescence microscopy exploiting monoclonal antibodies specific to the HVS 52 000 mol. wt. immediate-early polypeptide (IE 52K) and to delayed-early (DE 51K, DE 110K) and late (130K and capsid proteins) gene products to measure the timing of gene expression in individual cells of infected cultures. The timing of the transitions from IE to DE and from DE to late protein synthesis occurred at proportionately different intervals in the growth cycle of HVS, relative to that of HSV. In particular, the DE to late transition occurred relatively later in HVS infections. However, asynchrony in the events leading to the expression of the first class of virus proteins (IE 52K) was the main source of the extended course of HVS replication in populations of infected cells. This asynchrony was not modified significantly by infection at different stages of the host cell-cycle and was reduced, but not overcome, by very high applied multiplicities of infection. Double-antibody staining revealed a positive correlation between the accumulation of high concentrations of parental virus particles at perinuclear sites and early detection of HVS IE 52K gene expression. Both of these events remained sensitive to a microtubule poison (colcemid) for many hours after infection with HVS, whereas the rapid and synchronous expression of the IE 175K protein (ICP4) in HSV-1-infected cells was insensitive to post-infection exposures to this drug. We conclude that significant differences in early stages of virus entry and intracellular processing which precede immediate-early gene expression are largely responsible for differences between the replicative cycles of these representatives of gamma- and alphaherpesviruses in cultures of permissive cells.
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Differences in Humoral Immunogenicity between Herpes Simplex Virus Types 1 and 2
More LessSUMMARYInfection of NMRI mice with increasing doses of six different strains of herpes simplex virus type 1 (HSV-1) induced increasing levels of neutralizing antibodies. In contrast, three strains of HSV-2, irrespective of the dose, induced only marginal antibody responses. Only strain HG 52 (HSV-2) at high doses of infection stimulated antibody formation. The virus content of some organs in 6- to 8-week-old mice appeared to be lower after HSV-2 than after HSV-1 infection. Application of immune-modulating drugs [silica or poly(I).poly(C) coupled via l-lysine to CM-cellulose] resulted in little augmentation of antibody formation if compared to HSV-1 infection. Secondary infections with HSV-1 or HSV-2 after a primary dose of HSV-1 were followed by a marked booster response. In contrast, a primary infection with HSV-2 suppressed secondary responses of HSV-1 and HSV-2, thus indicating fundamental differences between the antibody-stimulating potency of HSV-1 and HSV-2 strains.
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Inhibition of Herpes Simplex Virus Replication in vitro by Human Cytotoxic T Cell Clones and Natural Killer Cell Clones
More LessSUMMARYThe abilities of human cytotoxic T cell (CTL) clones and natural killer (NK) cell clones to inhibit the replication of herpes simplex virus (HSV) in vitro were shown. The specificities of clones inhibiting HSV replication were the same as those of cytotoxicity in HSV type specificity and HLA restriction, i.e. HSV type 1 (HSV-1) and HSV type 2 (HSV-2) common CTL clones inhibited the replication of both HSV-1 and HSV-2 in autologous cells, but not in allogeneic cells. HSV-1-specific CTL clones inhibited the replication of HSV-1 in autologous cells; however, the replication of HSV-2 was not inhibited. NK cell clones inhibited the replication of HSV-1 and HSV-2 in autologous and allogeneic cells. This is the first demonstration that human virus-specific CTL clones and NK cell clones directly limit virus replication in vitro.
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Inhibition of Replication of Herpes Simplex Virus in Mouse Macrophages by Interferons
More LessSUMMARYThe replication of herpes simplex virus (HSV) type 1 in macrophages grown from spleen cells of mouse strains susceptible to HSV infection in vivo was very sensitive to interferon (IFN). Different types of mouse IFN (α, β, γ) exhibited similar antiviral activities. However, treatment of cells with IFN-γ in combination with IFN-α or IFN-β resulted in a synergistic inhibition of virus growth. As shown by assaying HSV DNA polymerase, IFN inhibited expression of the β-genes. Inhibition of enzyme induction correlated well with the reduction of viral yield. Induction of HSV DNA polymerase was delayed by IFN in a dose-dependent manner. These results show that IFN inhibits HSV replication at an early step prior to or during the synthesis of β-proteins.
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Identification of the Products of a Varicella-Zoster Virus Glycoprotein Gene
More LessSUMMARYTwo of the genes identified from the previously published DNA sequence of the US component of the varicella-zoster virus (VZV) genome were predicted to encode membrane proteins with polypeptide molecular weights of 39 000 (39K) and 70K. A rabbit antiserum directed against a unique peptide containing the seven amino acid residues at the carboxy terminus of the 39K gene product specifically precipitated glycoproteins with apparent molecular weights of 55K and 45K from VZV-infected cells labelled with [3H]mannose. The complete inhibition of precipitation of gp55 by free peptide and the partial inhibition of precipitation of gp45 support the conclusion that the 39K gene encodes gp55 and perhaps gp45. The number of VZV genes currently thought to encode glycoproteins is discussed in view of this finding.
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A Comparison of the Acid-soluble Polypeptides of Five Herpesviruses
More LessSUMMARYThe polypeptides soluble in 0.25 m-HCl were extracted from the nuclei of BHK cells infected with herpes simplex virus type 1 or type 2 and separated by SDS-PAGE. Seventeen polypeptides were detectable in each extract of which 10 type 1 and nine type 2 polypeptides were reproducibly effectively extracted. In cells infected with bovine mammillitis virus, pseudorabies virus or equine herpesvirus type 1, at least 12, 13 and eight polypeptides respectively were acid-soluble. In addition to histones, three other cellular polypeptides were present in sizeable quantities in the acid extracts and could obscure other acid-soluble viral polypeptides. Possible relationships between some polypeptides of the five herpesviruses are discussed.
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Glucocorticoid-mediated Establishment of an Antiviral State Coincident with Other Glucocorticoid-induced Biochemical Activities in L929 Cells
More LessSUMMARYIncubation of L929 cells with three different glucocorticoids, hydrocortisone, dexamethasone and triamcinolone acetonide, rendered the cells unable to support plaque formation by several unrelated DNA and RNA viruses. The establishment of this antiviral state by dexamethasone coincided with an inhibition of cell growth and induction of glutamine synthetase activity. These steroid-mediated activities occurred only in cultures of L929 cells and not in cultures of rabbit skin or rat glioma cells.
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Sequencing of Coronavirus IBV Genomic RNA: Three Open Reading Frames in the 5′ ‘Unique’ Region of mRNA D
More LessSUMMARYThe nucleotide sequence of a genomic cDNA clone corresponding to the 5′ terminal domain of mRNA D of the Beaudette strain of infectious bronchitis virus (IBV) has been determined. This region contains three open reading frames which predict polypeptides of molecular weights 6700 s(6.7K), 7.4K and 12.4K. The predicted 12.4K polypeptide has a codon usage very similar to that predicted for the products of the IBV nucleocapsid, membrane and spike genes. The sequence also predicts a hydrophobic, potentially membrane-anchoring, region in the N terminal half of the 12.4K polypeptide, and a hydrophilic C terminus.
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The Genome Structure of a New Chicken Virus Identifies It as a Parvovirus
More LessSUMMARYThe nucleic acid of chicken parvovirus-like particles showed sensitivity to DNase and S1 nuclease treatment and resistance to digestion with RNase. Viral DNA readily served as a template for self-primed conversion in vitro into a double-stranded form of about 5200 base pairs. There was no evidence for encapsidation of strands of opposite polarities. These findings confirm the taxonomic classification of chicken parvovirus-like particles as fowl parvovirus type 1 within the Parvovirus genus of the Parvoviridae.
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Synthesis of Proviral DNA in Inbred Mouse-derived Clones of Cells Expressing Different Fv-1 Phenotypes
More LessSUMMARYFormation of proviral DNAs by B-tropic murine leukaemia viruses (MLVs) was examined in N-type and dually permissive mutant cells derived from two inbred mouse strains, DDD and G, both of which are N-type. In the N-type cells, formation of circular proviral DNA was strongly suppressed relative to that of linear DNA. Mutation resulting in loss of the N-type Fv-1 restriction resulted in efficient formation of circular DNA by the previously restricted B-tropic MLV. This showed that Fv-1 restriction and inhibition of closed circular DNA formation were controlled by the same gene. The efficiency of formation of circular proviral DNA by the defective Kirsten murine sarcoma virus was determined by the tropism of the helper virus.
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Mutagen-directed Attenuation of Rift Valley Fever Virus as a Method for Vaccine Development
More LessSUMMARYSerial mutagenesis with 5-fluorouracil has been employed to derive an attenuated strain of Rift Valley fever virus for use as a live virus vaccine.
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A Hypothesis Accounting for the Effect of the Host Cell on Neutralization-resistant Virus
More LessSUMMARYEvidence is presented showing that a monkey anti-enterovirus 71 immune serum contains several antibody populations which differ in their mode of function. One population reduces infectivity, although inefficiently, by interactions at exposed antigenic sites and can be detected by measuring residual virus infectivity after mixtures of virus and antibody have been allowed to interact. Another antibody population, which is unaffected by the immunosorbent Staphylococcus aureus (Cowan I strain), appears to attach to its antigenic site(s) only after interactions between enterovirus 71 and host cells have already begun. In view of the transience of (presumed) conformational changes in the invading viruses, demonstration of this type of antibody activity requires a particular host cell system. This second type of antibody neutralization could be detected on RD cells but not on green monkey kidney cells.
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