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Catalogs, Library↗
Biomedical subjects
M C Timbury
Publications and source records attributed to M C Timbury.
Complementation with ts mutants of herpes simplex virus.
Tests to detect complementation between ts mutants of herpes simplex virus types 1 and 2 infectious centre and yield of virus assay were investigated. Progeny analysis of both intratypic and intertypic complementation showed a considerable proportion of recombinant or ts+ virus in the progeny; this was more marked in the infectious centre tests. Virus of intermediate temperature-sensitivity was produced in intratypic as well as intertypic complementation. Reduction in the input multiplicity of one of the two mutants in the test to extremely low levels did not prevent complementation, suggesting that non-infectious particles probably contribute to complementation. Demonstration of virus DNA synthesis in mixedly-infected cells at the non-permissive temperature was used to detect complementation between DNA-negative mutants.
Animals↗
Recombination in herpes simplex virus: mapping of mutations and analysis of intertypic recombinants.
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Base Sequence↗
Physical mapping of paar mutations of herpes simplex virus type 1 and type 2 by intertypic marker rescue.
Mutations (paar) in herpes simplex virus (HSV) which confer resistance to phosphonoacetic acid involve genes associated with virus-induced DNA polymerase activity. Two mutants of HSV (HSV-1 tsH and HSV-2 ts6) produce a thermolabile DNA polymerase activity. In this study, the ts lesions present in these mutants and those present in two independent phosphonoacetic acid-resistant mutants of HSV-1 and HSV-2 (paar-1 and paar-2) have been physically mapped by restriction endonuclease analysis of recombinants produced between HSV-1 and HSV-2 by intertypic marker rescue. All four mutations mapped within a 3.3-kilobase pair region around map unit 40. The accuracy of the method is reflected by the mapping results for tsH and paar-2, which were found to lie in the same 1.3-kilobase pair region. paar-1 was found to lie to the right of ts6. Virus-induced DNA polymerase is thought to have a molecular weight of 150,000, necessitating a gene with a coding capacity of 4.6 kilobase pairs. The four mutations mapped in this study all lie within a region smaller than this, but the results do not yet prove that all four lesions reside in this or any single gene.
Chromosome Mapping↗
Mutant of herpes simplex virus type 2 with temperature-sensitive lesions affecting virion thermostability and DNase activity: identification of the lethal mutation and physical mapping of the nuc-lesion.
We had previously shown that a temperature-sensitive (ts) mutant of herpes simplex virus type 2 strain HG52, ts13, induced a heat-labile DNase activity in infected cells (B. Francke, H. Moss, M. C. Timbury, and J. Hay, J. Virol. 26:209-213, 1978). Earlier work indicated that the mutant also possessed temperature-sensitive infectivity (I. W. Halliburton and M. C. Timbury, J. Gen. Virol. 30:207-221, 1976). In this study temperature-stable revertants of ts13 have been isolated; examination of them revealed that ts13 is a double mutant, with genetically distinct temperature-sensitive lesions affecting nuclease activity and particle stability. The lethal mutation, in the cell system studied, is the latter. Revertants, which all maintain the nuclease lesion, grew well at a high temperature. Physical mapping of the nuclease lesion placed it between 0.12 and 0.21 (fractional length) on the virus genome, quite distant from the lethal mutation at 0.64 to 0.70.
Animals↗
The fine structure of cells infected with temperature-sensitive mutants of herpes simplex virus type 2.
The fine structure of cells infected with the HG 52 strain of herpes simplex virus type 2 and 13 temperature-sensitive mutants derived from it was investigated. In cells infected with the wild-type virus, development of virions appeared to be similar to that described in previous reports. However there were two exceptions to this: (1) capsid envelopment apparently occurred de novo in the nucleus; (2) densely staining vacuolar accumulations were seen, frequently surrounding virus capsids. The 13 temperature-sensitive mutants of the virus were divided into three classes according to the type of capsid, if any, produced in cells infected and maintained at the non-permissive temperature. Class I mutants produced no capsids, Class II mutants produced empty and partial-cored capsids and Class III mutants produced empty, partial- and dense-cored capsids. Cellular alterations were also determined. Membranous tubular structures, previously unreported for herpes simplex virus, were observed in cells infected with Class III mutants and very occasionally with wild-type virus at the non-permissive temperature. Cytoplasmic particles were also found, but could not be correlated with any particular class of mutant.
Capsid↗
Alkaline DNase activity in cells infected with a temperature-sensitive mutant of herpes simplex virus type 2.
BHK cells infected with the temperature-sensitive mutant ts13 of herpes simplex virus type 2 at a nonpermissive temperature lack the alkaline nuclease activity, which is induced by the mutant at a permissive temperature and by wild-type virus at either temperature. For ts13, enzyme activity could be induced by a temperature shift to permissive conditions, but not in the presence of cycloheximide. After a shift from permissive to nonpermissive conditions in the presence of cycloheximide, the activity was stable in wild-type, but not in mutant-infected, cells. After extensive purification, the wild-type nuclease was fourfold more heat stable in the presence of substrate than was the mutant enzyme. Mixtures of both purified enzymes showed the predicted intermediate stabilities. The results strongly suggest that the enzyme is virus coded and that the mutant possesses a lesion in the structural gene of the enzyme.
Cell Line↗
Collaborative complementation study of temperature-sensitive mutants of herpes simplex virus types 1 and 2.
Twenty-three complementation groups of herpes simplex virus type 1 (HSV-1) and 20 of HSV-2 were identified by qualitative and quantitative complementation analysis from among 43 temperature-sensitive (ts) mutants of HSV-1 and 29 ts mutants of HSV-2 which had been isolated independently in 10 laboratories.
Genes, Viral↗
Recombinants between herpes simplex virus types 1 and 2: analyses of genome structures and expression of immediate early polypeptides.
Recombinants between temperature-sensitive mutants of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) were constructed. Using restriction endonucleases, we analyzed the genome composition of 17 intertypic recombinants and detected crossovers in every region of the genome. The virion DNA of one recombinant appeared to be largely "frozen" in two of the four possible genome arrangements of HSV. Knowledge of the genome structures of recombinants enabled us to physically map immediate early polypeptides. We present evidence that the immediate early polypeptide Vmw IE 110 of HSV-1 and its functionally equivalent polypeptide, Vmw IE 118, of HSV-2 may map in the repetitive sequences bounding the long unique region of HSV.
DNA Restriction Enzymes↗
Physical mapping of herpes simplex virus-induced polypeptides.
Analysis of the polypeptides induced by 29 herpes simplex virus type 1/type 2 intertypic recombinants and correlation of the data with the crossover points in the recombinant DNAs have enabled the map positions of many polypeptides to be deduced. These include 25 polypeptides which label with [35S]methionine, 11 which label with [32P]orthophosphate, and 4 which label with [14C]glucosamine. Together with the data of Preston et al. (J. Virol., in press) on the mapping of five immediate-early polypeptides, the results show that representatives of four groups of proteins--immediate-early, late, phosphorylated, and glycosylated--map in both long and short regions. The functional organization of the herpes simplex virus genome does not therefore restrict any of these four groups to either the long or the short region.
Cell Line↗
Physical mapping of herpes simplex virus-coded functions and polypeptides by marker rescue and analysis of HSV-1/HSV-2 intertypic recombinants.
A number of temperature-sensitive (ts) mutants and one pyrimidine deoxyribonucleoside kinase-deficient mutant of herpes simplex virus (HSV), have been located on the physical map of the genome by means of marker rescue experiments and by the analysis of the crossover points in intertypic recombinants between HSV types 1 and 2. The physical map is compared to the genetic map and certain anomalies identified. Analysis of infected-cell polypeptides specified by intertypic recombinants has allowed tentative map co-ordinates to be assigned to the structural genes (or genes which cause post-translational modification) for many of the polypeptides. Immediate-early, phosphorylated, glycosylated and structural as well as non-structural polypeptides have been analysed in this way and it can be concluded that there is no restriction of any of these groups of polypeptides to either the long or the short regions of the genome. One of the recombinants, 2853, is at least partially "frozen" in one orientation of the long region. This orientation is also the one which exhibits a minimum number of crossovers in three other recombinants.
Chromosome Mapping↗
Complementation of ts mutants by a herpes simplex virus ts-transformed cell line.
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Cell Line↗
Temperature-sensitive mutants of herpes simplex virus type 2: a provisional linkage map based on recombination analysis.
Thirteen ts mutants of type 2 herpes simplex virus were backcrossed to a syncytial but not temperature-sensitive mutant of wild-type virus. This was an attempt to introduce a third marker, syncytial plaque morphology or syn, into at least some of the ts mutants. Three ts mutants carrying the syn marker were obtained but only one, ts 9, was satisfactory for genetic experiments. Three-factor crosses were carried out between ts 9 syn and the mutants which determined the order of eleven ts mutations relative to both the ts 9 mutation and the syn mutation. A provisional linkage map based both on the order derived from the three-factor crosses and on map distances from recombination frequencies has been prepared: it contains nine ts mutations and the syn mutation.
Cell Line↗
Temperature-sensitive mutants of herpes simplex virus type 2: description of three new complementation groups and studies on the inhibition of host cell DNA synthesis.
Three new complementation groups of type 2 herpes simplex virus are described bringing the total number of complementation groups characterized to 13. Of the three new groups, ts 11 fails to make virus DNA at non-permissive temperature (38 degrees C) whereas ts 12 and ts 13 synthesize only very small amounts of virus or cellular DNA at 38 degrees C. ts 11, like ts 9 (Halliburton & Timbury, 1973) fails to switch off host cell DNA synthesis at 38 degrees C. That this is a failure to switch off cell DNA rather than a stimulation of cell DNA synthesis was confirmed in experiments using resting cells. Both the inability to make virus DNA and the inability to switch off cell DNA are reversed in temperature shift-down experiments with cells infected with ts 9 or ts 11. In temperature shift-up experiments, cellular DNA synthesis is inhibited after the shift but virus DNA is only made in very small amounts, probably due to the continuing functioning of a protein made at permissive temperature (31 degrees C) before the shift but which cannot be made at 38 degrees C. The shift-down experiments and the fact that ts 9 and ts 11 complement one another, suggest that the switch-off of host cell DNA synthesis may involve more than one virus specified function. U.v. irradiated virus fails to switch off host cell DNA synthesis.
Cell Line↗
Herpesvirus proteins: DNA polymerase and pyrimidine deoxynucleoside kinase activities in temperature-sensitive mutants of herpes simplex virus type 2.
Eleven temperature-sensitive mutants of herpes simplex virus type 2 strain HG52 were examined for ability to induce DNA polymerase activity in BHK 21/C13 cells. All mutants induced DNA polymerase at a permissive temperature, (31 degrees C) and all DNA-positive mutants at a non-permissive temperature (38 degrees C). Three DNA-negative mutants induced no DNA polymerase (ts 6, ts 9) or very little DNA polymerase (ts 11), at a non-permissive temperature, while ts 1, also DNA negative, induced a little more DNA polymerase than wild-type, often at both temperatures. The DNA polymerase induced by ts 6 at 31 degrees C was temperature-sensitive in vivo, but only slightly so in vitro. These results were confirmed immunologically and suggest that HSV-2 codes for at least part of a DNA polymerase activity, necessary for infection, and that full expression of this enzyme involves at least three viral genes.
Antigens, Viral↗
Collaborative study of temperature-sensitive mutants of herpes simplex virus type 2.
Eighteen complementation groups were identified by complementation tests and by phenotype from twenty temperature-sensitive mutants isolated independently in Glasgow, Scotland, and Houston, Tex.
Genetic Complementation Test↗