Alison A. Newton

Changes in nucleic acid content of HeLa cells infected with herpes virus

Abstract The nucleic acid content of HeLa cells was determined at various times after exposure to a high multiplicity of herpes virus. The DNA content rose 6 to 9 hours after infection, before any increase in infective virus, which was just detectable at 12 hours. By 72 hours the cells contained nearly double their normal content of DNA. This increase in DNA was confined to the nucleus. No significant change in RNA content was observed.

THE STRUCTURE OF ORF VIRUS.

Abstract Three methods can be used to show that both upper and lower surfaces of orf virus particles are seen in sodium phosphotungstate-stained electron micrographs. These have demonstrated that the characteristic tubular thread structure of the wall of the particle is in the form of a left-hand spiral coil of a single thread. Within the particle an internal component is demonstrable that is thofght to contain the nucleic acid. This is DNA, and the weight per particle is 2.85 × 10 −16 g, which is equivalent to a molecular weight of 171 × 10 6 per particle. The dry weight of orf is 3.69 × 10 −15 g, and the nitrogen content is 14.77% of the dry weight.

Quantitative cytological studies on HeLa cells infected with herpes virus.

Abstract A cytological study has been made on HeLa cells infected with herpes virus. Two types of specific cellular lesion were recognized: (1) the classic lesions of interphase cells and (2) a lesion of mitosis which rapidly replaces normal mitotic cells and which has a transitory existence only. The results suggest that inhibition of cell division by this virus is characterized either (a) by failure to enter mitosis or (b) by an abnormal mitosis. Microspectrophotometry of Feulgen-stained preparations confirmed that, after a lag period, there is a net increase of the DNA per nucleus in infected cells. At the same time autoradiography has shown that the rate of incorporation of tritiated thymidine is depressed. This paradox may be partly explained if the intracellular deoxyribonucleotide concentration is increased after infection. One factor contributing to such an increase could be breakdown of host nucleic acid as a result of infection. Studies on the loss of label from cells preincubated with tritiated thymidine suggest that such a breakdown may occur.

The effect of some environmental factors on herpes virus grown in HeLa cells.

Abstract The effect of variation of incubation temperature on the various stages in the growth cycle of herpes virus in HeLa cells has been determined. While the adsorption of the virus to the cells was largely temperature independent, the rates of cell penetration by the virus particle and of intracellular viral growth were considerably decreased at temperatures lower than 37°. However, higher yields of infective virus were obtained by incubation at 32° than at 37°. This has been correlated with a rate of viral inactivation which is higher at 37° than at 32°. In addition it has been shown that the pH of the suspending medium is an important factor in maintaining virus infectivity.

Purification and properties of the 'A' antigen associated with Marek's disease virus infections.

Summary The production, purification and properties of the ‘A’ antigen induced by Mareks disease virus in tissue culture were investigated. It was found that although virus multiplied on passage in chick kidney, chick embryo and duck embryo cells the antigen was produced only in chick kidney and duck embryo cultures. The results suggested that the ‘A’ antigen produced by duck embryo cells was a glycoprotein having a mol. wt. in the range 70 000 to 90 000 and was heterogeneous in charge (pI 4.5 to 5.5). A 20-fold purification was achieved by electrophoresis in 5% acrylamide gel with a recovery of 45%. An equally good purification was also possible by chromatography on DEAE-Sephadex A50 and Sephadex G200, although the recovery was only 20% in this case.

Polyamine metabolism in cells infected with herpes simplex virus.

Ifection of LS cells with HSV-1 resulted in an inhibition of spermidine and spermine synthesis from putrescine, possibly through inhibition of host cell protein synthesis. The rate of putrescine uptake increased soon after infection, and later, polyamines were lost from the cells. Inhibition of spermidine and spermine synthesis by methylglyoxal bis (amidinohydrazone) did not affect virus replication.

The Effect of Herpes Virus on HeLa Cells dividing Parasynchronously.

Abstract HeLa cells dividing parasynchronously have been used to study the effect of the HFEM strain of herpes virus upon mitosis. Mitosis of the HeLa cells is inhibited, and this inhibition is prevented by serum containing virus neutralizing antibody. The damage to mitosis occurs rapidly relative to the cycle of virus growth, probably less than 1 hour after virus adsorption. Virus particles which adsorb to, but do not infect, the HeLa cells fail to inhibit mitosis. Similar rates of virus growth were found in both synchronously and asynchronously dividing cells when the former were infected 7 hours before expected division.

The Effects of Herpes Simplex Virus Type 1 on Cellular DNA-dependent RNA Polymerase Activities

An investigation of the activity of nuclear RNA polymerase following infection of LS cells with HSV-1 shows a decline in both major activities. This effect is not entirely due to inhibition of cellular protein synthesis, and the effect of alpha-amanitin-sensitive RNA polymerase is mediated by a protein(s) synthesized in the infected cell. Changes in the properties of this RNA polymerase activity include a reduction in the relative UTP/GTP incorporation ratio and an increased sensitivity to inhibition by actinomycin D, indicating that RNA polymerase II is involved in virus transcription.

Comparison of the activities of HSV-1 and cellular mRNAs as templates for in vitro translation.

Abstract Messenger RNA preparations from HSV-1-infected cells directed the incorporation of [ 35 S]methionine in cell-free translation systems to an extent only 20–25% of that directed by uninfected cell RNA. HSV-1 mRNAs were faithfully translated and the low template activity of the RNA from infected cells could not be explained by the presence of an inhibitor. Measurements of mean ribosome transit times indicated that a reduced rate of polypeptide chain elongation on RNA from infected cells was not responsible for the poor template activity. Initiation of polypeptide chain synthesis on HSV-1 mRNAs on the other hand appeared to be less efficient than initiation on cellular mRNAs, on the basis of in vitro competition experiments. The possibility that HSV-1 mRNAs might be poor templates for protein synthesis is contrary to the situation with a number of other viruses which have been shown to have mRNAs which are particularly efficient relative to cellular mRNAs.

Identification of Polypeptide Precursors to HSV-1 Glycoproteins by Cell-free Translation

Using antisera of limited specificity we have detected among the in vitro translation products of HSV-1 RNA, polypeptides antigenically related to infected cell glycoproteins. The results obtained suggest that non-glycosylated polypeptide precursors of mol. wt. 85,000 and 52,000 correspond to infected cell glycoproteins of mol. wt. 120,000 to 126,000 and 56,000 to 68,000 respectively.