- Volume 65, Issue 3, 1984
Volume 65, Issue 3, 1984
- Articles
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- Animal
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The Junctions between the Repetitive and the Short Unique Sequences of the Herpes Simplex Virus Genome Are Determined by the Polypeptide-coding Regions of Two Spliced Immediate-early mRNAs
More LessSUMMARYImmediate-early (IE) mRNAs-4 and -5 of herpes simplex virus type 2 (HSV-2) are transcribed from the IRS/TRS genome regions towards the US region. Each of these spliced mRNAs has an untranslated leader sequence of 249 bases and a single intron of approximately 540 bases which are contained entirely within TRS/IRS sequences. The DNA sequence of the intron largely comprises tandem reiterations of three distinct short sequences. Upstream of the common 5′ mRNA termini the DNA sequence contains regions of homology with the equivalent region of HSV-1. Comparison of the polypeptides encoded by these HSV-2 mRNAs with those of HSV-1 shows blocks of conserved amino acids. The locations of the first initiator ATG triplets of these two HSV-2 mRNAs suggest that the IRS/TRS regions of HSV expand, by gene conversion or by equal though non-homologous crossover, to an extent determined by the functions of the DNA sequences which are duplicated or deleted as a result of the crossover. This mechanism for expansion of repeats may apply to other herpesviruses which have a genome structure similar to that of HSV.
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Differential Substrate Specificity of DNA Polymerase β and of a DNA Polymerase Induced by Herpes Simplex Virus Type 2 towards Thymidine Triphosphate Analogues
More LessSUMMARYSeveral triphosphates of 5-substituted deoxyuridine (dU), such as 5-ethyl-, 5-n-propyl-, 5-n-hexyl- and 5-isopropyldeoxyuridine triphosphates and 5-trifluorothymidine triphosphate are substrates for HeLa cell DNA polymerase β (2′-deoxynucleoside-5′-triphosphate: DNA–deoxynucleotidyltransferase, EC 2.7.7.7) and for a DNA polymerase isolated from HeLa cells infected with herpes simplex virus type 2 (HSV-2) strain 75. At the concentration tested (50 µ m), all these analogues were incorporated more readily into DNA by the virus-coded enzyme than by DNA polymerase β from the host cell. The DNA polymerase coded by HSV-2 showed an affinity for deoxythymidine triphosphate (dTTP) and the analogues studied higher than that of DNA polymerase β. Analogues are preferential substrates for the viral enzyme, since they readily substitute for dTTP during synthesis in vitro. In contrast, arabinosylthymine-5′-triphosphate was readily incorporated into DNA by the host cell DNA polymerase β, but inhibited the DNA polymerase specified by HSV-2.
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Isolation and Characterization of Revertants from Fourteen Herpes Simplex Virus Type 1 (Strain 17) Temperature-sensitive Mutants
More LessSUMMARYThe isolation of spontaneous non-temperature-sensitive revertants of herpes simplex virus type 1 (HSV-1) (strain 17) ts mutants, following a single round of selection at non-permissive temperature, is reported. The distinction between total and incomplete reversion was assessed by the wild-type performance of non-ts revertants in three aspects of HSV infection: (i) virus growth, (ii) induction of HSV-specified enzyme activities (where appropriate) and (iii) the gel profile of infected cell polypeptides. Wild-type behaviour was taken to indicate total reversion, while less than wild-type performance in any test indicated incomplete reversion. Only five of 19 HSV-1 mutants (ts L, U, X, R and G syn) examined for reversion of the ts lesion failed to yield non-ts revertants; these mutants may well contain multiple mutations. Total reversion was obtained with mutants ts K, F, E, S, M, N and I and incomplete reversion with the mutants ts D, T, B, P, A, W, F, H and possibly K. Thus, both total and incomplete revertants of ts F (and possibly ts K) were obtained. Some conclusions concerning the relative importance of individual polypeptide bands have been drawn from these studies. Within our sample in the revertant profiles Vmw 175, 155, 114, 65/64, 43, 40, 39, 30, 28, 21 and 16.5K always returned to wild-type levels; Vmw 273, 100, 67 and 38/37K almost always regained wild-type intensity but polypeptides Vmw 122, 117, 82/81, 57, 51, 27 and 12.5K were frequently not made in wild-type amounts.
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A Study of Interfering Herpes Simplex Virus DNA Preparations Containing Defective Genomes of Either Class I or II and the Identification of Minimal Requirements for Interference
More LessSUMMARYProgeny DNA of herpes simplex virus type 1 (HSV-1) strain ANG from infections involving defective interfering virus particles (DI DNA) has been described to be of low infectivity in transfection assays due to the presence of viral genomes that interfere with plaque formation by infectious standard genomes. In this study it is shown that this observation applies both for DI DNA containing repetitive defective DNA of the class I type and for DI DNA containing class II-type defective DNA. Restriction endonucleases with recognition sites only in one class of repetitive defective DNA could be used to reduce selectively the interfering activity of DI DNA preparations containing the respective defective DNA in abundance. The results obtained directly implicate repetitive defective DNA as an interfering agent. Restriction endonucleases that create monomeric DNA fragments from class II HSV-1 ANG defective DNA did not abolish the interfering activity of DI DNA containing this type of defective DNA in high abundance, indicating that it is not simply the repetitive nature of defective DNA that is required for interference. Certain DNA fragments shorter than the repeat unit of repetitive defective DNA were still capable of causing interference even in the absence of cohesive single-stranded ends. The common location of cis recognition signals responsible for progeny DNA maturation and initiation of DNA replication on one DNA fragment, however, appeared to be a minimal requirement for interference by fragmented defective DNA.
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Epstein—Barr Virus Binding to Virus-carrying Cell Lines is Enhanced in the Presence of C3 and C3d
SUMMARYThe relationship between the receptors for the Epstein—Barr virus (EBV) and the C3d fragment of complement was investigated at the molecular level. In the presence of cell-bound C3, virus binding was enhanced in EBV genome-carrying lines. An identical effect could be elicited by C3d at one-quarter the weight amount; C3b and methylamine-treated C3 had no effect on virus binding. The minimum concentration of C3 which produced significant enhancement was 25 µg/ml. Virus binding increases were observed only after 20 min of complement-cell co-incubation. The response was not noted with EBV-negative lines and was independent of virus strain assayed (B95-8 and P3HR-1). These studies suggest that the binding sites for the two moieties are distinct, although they both involve the same cell surface complex. The two receptors are believed to display cooperativity.
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Comparison of the Pathogenesis of Murine Cytomegalovirus in Lung and Liver Following Intraperitoneal or Intratracheal Infection
More LessSUMMARYThis study compares the pathogenesis of murine cytomegalovirus (MCMV) infections following intraperitoneal (i.p.) and intratracheal (i.t.) inoculation. No deaths were seen in mice given 106 p.f.u. MCMV i.t., whereas 52% mortality occurred among animals given this dose i.p. This difference in mortality was not due to different effects on the lung, since virus titres in this organ on progressive days post-infection were similar for the two routes of inoculation and similar, minor histopathological changes were observed. In contrast, virus titres in the livers of mice inoculated i.p. were 100-fold higher than for those inoculated i.t., and histopathological changes were noticeably greater in the i.p. group. This suggests that the mortality seen following i.p. inoculation may have been due, at least in part, to effects of viral infection on liver function. Parallels between various forms of human cytomegalovirus infections and the types of infections seen following i.t. and i.p. infection with MCMV were observed.
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Molecular Cloning of the Nucleoprotein Gene of Canine Distemper Virus
More LessSUMMARYMessenger RNAs labelled in vivo in Vero cells infected with canine distemper virus were analysed by electrophoresis on 1.5% agarose gels containing 2m-formaldehyde. Seven virus-specific RNA bands could be distinguished which were not sensitive to actinomycin D treatment and were confined to the polyadenylated RNA fraction. The most abundant virus-specific mRNA species had a molecular weight of 0.52 × 106 and its coding capacity was consistent with it being the mRNA for the most abundant virus-specific protein, the nucleoprotein. Polyadenylated RNA of this size class was purified by electrophoresis on a polyacrylamide gel and cloned into the PstI site of plasmid pBR322. A virus-specific clone obtained, clone 224, was then used to select messenger RNA from infected cells. The messenger RNA selected had a molecular weight of 0.52 × 106 and directed the synthesis of only the virus-specific nucleoprotein when used to stimulate a wheat germ cell-free system.
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Studies of Fowl Plague Virus Temperature-sensitive Mutants with Defects in Synthesis of Virion RNA
More LessSUMMARYThree different types of impairment in the synthesis of virion RNA (vRNA) were detected in three groups of temperature-sensitive (ts) mutants of fowl plague virus (FPV) having ts mutations in genes 1, 3 and 5 respectively. Normal synthesis of poly-(A+) cRNA, poly(A−) cRNA and vRNA was observed under non-permissive conditions early in infection in cells infected with the ts 43 mutant having a ts mutation in gene 1 coding for the PB2 protein. However, 4 h after infection synthesis of vRNA ceased, synthesis of poly(A+) cRNA was reduced drastically, but the rate of poly(A−) cRNA synthesis was the same as that in cells infected with wild-type FPV. In cells infected with the ts 166 mutant having a ts mutation in gene 3, coding for the PA polypeptide, a drastic reduction was observed in poly(A+) cRNA synthesis under non-permissive conditions. Synthesis of poly(A−) cRNA was also reduced and synthesis of vRNA was not detected. The ts 60 mutant, having a ts mutation in gene 5 coding for the NP polypeptide, induced synthesis of all types of virus-specific RNA under non-permissive conditions, but the regulation of synthesis of vRNA and poly(A+) cRNA was affected, there being predominant synthesis of RNA segments 5 and 8 late in infection. In cells infected with mutants ts43 and ts 166 synthesis of virus-specific proteins was impaired, which reflected defects in the synthesis of virus-specific RNAs. The data obtained suggest that the PB2 protein may be contained in an enzyme complex responsible for synthesis of vRNA, that different enzyme complexes may be involved in the synthesis of poly(A−) cRNA and vRNA, and that the NP protein plays a significant role in the regulation of vRNA synthesis.
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Biological Activity of a Monoclonal Antibody to a Measles Virus Haemagglutinin Epitope Detected Late in Infection
More LessSUMMARYA hybrid cell line secreting monoclonal antibody 2H9c (mAb 2H9c) with measles virus haemagglutinin (HA) specificity was produced. The mAb 2H9c was non-reactive in haemagglutination inhibition and neutralization assays. A protein of apparent mol. wt. 79000 was immune-precipitated using a Triton X-100/SDS/sodium deoxycholate detergent buffer and two proteins with mol. wt. of 79000 and 69000 were immune-precipitated using 1% Nonidet P40/sodium deoxycholate buffer in SDS-PAGE assays. Similar results were obtained with other anti-HA monoclonal antibodies, supporting the assumption that mAb 2H9c was directed against the HA polypeptide. The indirect immunofluorescence (IFA) staining pattern of acetone-fixed infected cells with mAb 2H9c differed substantially from that with other HA-specific monoclonal antibodies. Kinetic studies revealed that the reactivity of mAb 2H9c lagged behind other HA-specific monoclonal antibodies by 18 to 36 h post-infection in IFA assays. This suggests that mAb 2H9c may be directed against a binding site that arises late in infection, possibly as a result of a conformational alteration of the HA polypeptide.
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The Kinetics of Synthesis of Early Viral Proteins in KB Cells Infected with Wild-type and Transformation-defective Host-range Mutants of Human Adenovirus Type 5
More LessSUMMARYWe have studied the kinetics of early adenovirus type 5 (Ad5) protein synthesis during lytic infection of KB cells by wild-type (wt) and transformation-defective host-range (hr) mutants. Proteins encoded within four early regions were studied: early region 1A (E1A: 1.5 to 4.5 map units, mu), E1B (4.5 to 11.2 mu), E2A (61.6 to 74.9 mu), and E4 (91.4 to 99.1 mu). Synthesis of E1A products, the first to appear during wt lytic infection, was detectable within 2 h after injection, reached a peak within the next hour, then declined to very low levels by 7 h post-infection. Synthesis of E2 and E4 proteins began at about 3 h post-infection, was maximal by 6 h and thereafter declined sharply. The E1B 19K and 58K proteins were first detected around 3 h post-infection and, after reaching maximal levels of expression by 8 h, declined to lower levels by 12 h post-infection. Infections with the E1A mutant hr3 were characterized by greatly depressed levels of early expression of E1B, E2 and E4 polypeptides but protein synthesis from these regions appeared to recover at late times. The pattern of expression exhibited by the E1B mutant hr6 revealed delayed and reduced levels of expression of E1B, E2 and E4 protein synthesis but increased levels of E1A protein synthesis. These results are consistent with the reported role of E1A gene products in the activation of early gene expression and, in addition, suggest that a function encoded in E1B may also influence the expression of Ad5 early genes at early and late times.
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Selective Inhibition of Viral Gene Expression as the Mechanism of the Antiviral Action of PGA1 in Vaccinia Virus-infected Cells
More LessSUMMARYWe have previously shown that prostaglandins of the A series are potent inhibitors of the replication of several animal viruses in cultured cells. In this report we have studied the mechanism of the antiviral action of PGA1 in vaccinia virus-infected mouse L cells, where there is an alteration in both the rate and extent of the synthesis of some virus proteins. When cytoplasmic RNAs from PGA-treated, vaccinia virus-infected cells were translated in cell-free systems, similar selective inhibition of the synthesis of some viral polypeptides was observed. The lack of translation of some viral RNAs was not due to an impairment of the methylation process nor to a difference in ionic requirements. PGA1, even at doses as high as 10 µg/ml, did not exert any direct inhibitory action on transcription in vitro as measured in two cell-free systems, and had no effect on primary transcription-translation of vaccinia virus RNAs when assayed in coupled cell-free systems. Southern blot hybridization analysis of cytoplasmic RNAs to EcoRI restriction fragments of vaccinia DNA showed that PGA1 was able to induce major changes in the pattern of RNA transcripts during the course of viral infection. We propose that changes in the transcription programme of vaccinia virus RNAs could be due either to an alteration of specific viral proteins that regulate transcription by direct binding of PGA1, or to the synthesis and/or activation of a host product that mediates the antiviral action.
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Persistent Infection of Human Fibroblasts by Hepatitis a Virus †
More LessSUMMARYInfection of human embryo fibroblasts with hepatitis A virus (HAV), a picornavirus, leads to an inapparent, persistent infection; cultures can be passed serially with consistent recovery of the virus in the supernatant. All of the cells of a HAV carrier culture are infected and proliferate. Subcultivation under HAV-immune serum cannot achieve a cure or even a reduction in the number of infected cells in HAV carrier cultures. No interferon activity can be detected during HAV infection and persistence. Addition of exogenous interferon eliminates HAV infection in vitro. Persistence of HAV in vitro appears to contradict the clinical course of HAV infection in vivo. The system presented offers the possibility of evaluating the role of immunological injury of HAV-infected cells, an injury which may lead to damage of these cells and to elimination of HAV during an HAV infection in vivo.
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Isolation and Biological Characterization of Mouse LM Fibroblast Variants Resistant to the Cytotoxicity of Polyriboinosinic. Polyribocytidylic Acid after Interferon Treatment
SUMMARYMouse LM fibroblasts growing continuously in the absence of serum have an increased sensitivity to the cytotoxicity of poly(rI).poly(rC) after interferon (IFN) exposure. This has allowed the isolation by an enrichment procedure of several independent and stable variant clones (IFN + I.C)R which are resistant to such a treatment. One of the resistant variants has been more extensively characterized as far as IFN action and IFN production are concerned. It behaves identically to the wild-type parent except for the spontaneous release of low amounts of IFN. The target of the mutation probably resides in a late step in the development of the cytotoxic response as revealed by microinjection techniques. The (IFN + I.C)R variants characterized here thus appear different from mutants in the IFN system isolated so far.
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The Reaction of the Anti-interferon-α Monoclonal Antibody, NK2, with Different Interferons
More LessSUMMARYAnalysis of the neutralization of human interferon-α by the monoclonal antibody NK2 showed, by three different methods, that the avidity constant was about 1010 m −1. A small decrease in the avidity constant (and in the antibody titre) with increasing interferon concentrations was observed, suggesting that the antibody-interferon complex had a little biological activity. The antibody neutralizes the interferon by preventing the binding of interferon to susceptible cells. It does not react with human-γ, mouse-α, monkey, bovine or rabbit interferons.
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Interferon-α Yields from Sendai Virus-induced Human Leukocyte Cultures Are Enhanced by Lowering the Incubation Temperature
More LessSUMMARYIncreased yields of Sendai virus-induced human leukocyte interferon (IFN-α) were obtained by lowering the incubation temperature from the initial 37°C within 1 to 6 h after the addition of virus. Maximal stimulation was found when this was done 2 to 4 h after induction and when the lowered temperature was between 29 and 32°C. Compared to cultures kept at 37°C throughout, the resulting IFN-α yields were enhanced by 2.5- to 3-fold. This was mainly due to prolonged synthesis of IFN-α, which could be observed up to 20 h after induction, whereas in cultures kept at 37°C the production of IFN-α had practically ceased approx. 10 h after the addition of virus.
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Synthesis of M Protein of HVJ (Sendai Virus) in Rat Glial Cells is Selectively Restricted at a Non-permissive Temperature
More LessSUMMARYProduction of HVJ (Sendai virus) wild-type in rat glial cells was characteristically restricted at high temperature. The synthesis of the M protein was selectively decreased in infected cells at 39°C. This temperature-sensitive (ts) character of HVJ was not observed in infection of LLCMK2 cells or chick embryo fibroblast cells. Newcastle disease virus, mumps virus and vesicular stomatitis virus did not show ts growth in glial cells.
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Berne Virus is Not ‘Coronavirus-like’
More LessSUMMARYIn infected embryonic mule skin cells, Berne virus directs the synthesis of two main polypeptides (22K, 20K); in addition, virus-specific proteins with apparent molecular weights of >200K, 80K to 120K, 32K and 17K were detected after radioimmune precipitation. The replication of Berne virus was reduced more than 1000-fold by actinomycin D, when the drug (0.1 to 1.0 µg/ml) was added during the first 8 h after infection; alpha-amanitin (25 µg/ml) produced a similar though less pronounced effect. U.v. preirradiation of the cells for ⩾ 5s led to a dramatic decrease in the production of extracellular virus. The results presented support our suggestion that Berne virus is a representative of a new family of animal viruses.
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