Tamar Ben-Porat

The chemical composition of herpes simplex and pseudorabies viruses.

Abstract Herpes simplex and pseudorabies viruses were purified by differential centrifugation and by equilibrium sedimentation in cesium chloride density gradients. Both viruses occupy approximately the same positions in the gradients and both contain DNA. The DNA of these viruses has a similar base composition and contains a relatively high percentage of guanine and cytosine (74%).

Early functions of the genome of herpesvirus: I. Characterization of the RNA synthesized in cycloheximide-treated, infected cells☆

Abstract The inhibition of cell-specific RNA synthesis induced by infection of rabbit kidney cells with pseudorabies virus does not occur when the infected cells are incubated with cycloheximide. The interference with the processing of ribosomal precursor RNA that normally occurs after infection of cells with the herpesvirus is, however, observed in cycloheximide-treated, infected cells, indicating that in contrast to the inhibition of synthesis of cell-specific mRNA, this interference is not mediated by a viral protein. Virus-specific RNA is transcribed in cycloheximide-treated cells. This RNA is predominantly 26 S and represents an accumulation within the cells of sequences repeatedly transcribed from a relatively small part of the viral genome. Only 25% of the sequences usually transcribed prior to the onset of viral DNA synthesis are transcribed in cycloheximide-treated, infected cells. After the removal of cycloheximide, the cells synthesize virus-specific proteins exhibiting a profile of electrophoretic migration in polyacrylamide gels different from that of the proteins synthesized at any time after infection. These proteins are richer in arginine than the proteins normally synthesized by the infected cells and probably are so-called “early” viral proteins. The results in this paper indicate that the cycloheximide-treated, infected cells synthesize (after the removal of cycloheximide) a larger amount of “early” viral proteins than do the infected cells during a normal infective cycle.

The action of 5-fluorouracil on the nucleic acid metabolism of pseudorabies virus-infected and noninfected rabbit kidney cells☆

Abstract 5-Fluorouracil (FU) inhibits the synthesis of deoxyribonucleic acid (DNA) in rabbit kidney (RK) cells in at least two ways: (1) by preventing the formation of thymidylic acid—this inhibition occurs almost immediately after the addition of FU and is overcome by thymidine; (2) by a mechanism which is not reversible by thymidine and which occurs only after a limited synthesis of DNA has taken place in the FU-treated cells. The experiments in this paper deal mainly with the second type of inhibition (delayed type). The delayed type of inhibition is partially reversed by uridine and cytidine, but only when these nucleosides are added to the cultures simultaneously with FU. It is overcome by infection with pseudorabies (Pr) virus; the incorporation of thymidine- 2-C 14 into the DNA of FU-treated virus-infected cells remains unaffected by a concentration of FU fiftyfold greater than that sufficient to inhibit this incorporation in noninfected cells. The inhibition of the synthesis of thymidylic acid in FU-treated cells is not, however, overcome by virus infection. The synthesis of ribonucleic acid (RNA), as measured by the uptake of uridine- 2-C 14 or adenine- 8-C 14 , is suppressed by FU to a much lesser extent than the synthesis of DNA. This effect is not reversed by virus infection. The implications of these results with respect to the mechanism of inhibition of DNA synthesis by FU are discussed.

The pattern of viral and cellular DNA synthesis in pseudorabies virus-infected cells in the logarithmic phase of growth

Abstract The DNA of pseudorabies (Pr) virus can be separated from the DNA of its host cell by centrifugation in density gradients of cesium chloride. By means of this technique, it was found that in logarithmically growing rabbit kidney (RK) cells infected with Pr virus there is a progressive decrease in the rate of incorporation of thymidine-2-C 14 into cellular DNA, and, concurrently, a progressive increase of its incorporation into viral DNA. The decrease in the rate of incorporation of thymidine-2-C 14 into cellular DNA is due to the inhibition in virus-infected cells of the synthesis of this DNA. The mode of inhibition of cellular DNA synthesis in Pr virus-infected cells was investigated. This inhibition is not due to the failure of the infected cells to enter into the DNA-synthesizing period of their growth cycle, nor is it the result of the degradation of cellular DNA in these cells. There is in the infected cells a shift of the cellular DNA toward the periphery of the nucleus which may be responsible for the inhibition of cellular DNA synthesis.

Synthesis of proteins in cells infected with herpesvirus: V. Viral glycoproteins

Abstract The glycoproteins synthesized by pseudorabies virus-infected rabbit kidney cells have been analyzed. Two types of glycoproteins have been distinguished: (1) glycoproteins that are present primarily in the cytoplasmic fraction of the infected cells and that do not become part of the virions—these glycoproteins are excreted into the culture fluids in relatively large amounts; (2) glycoproteins that are found associated with the nuclei of infected cells—these glycoproteins have an electrophoretic profile similar to that of the glycoproteins associated with viral particles and probably become part of the viral envelope. Nuclear DNA-containing viral particles and mature viral particles possess glyco-proteins with identical electrophoretic profiles; however, the nuclear particles contain fewer of these proteins. These results indicate that the DNA-containing viral particles isolated from the nuclei have the same membrane proteins as mature virus but that these proteins are present in nuclear particles in lesser amounts than in mature particles.

Mechanism of inhibition of cellular DNA synthesis by pseudorabies virus

Abstract The inhibition of cellular DNA synthesis that occurs after infection of rabbit kidney cells with pseudorabies virus is not due to the successful competition of viral DNA with cellular DNA to act as a template for DNA replication. It is also not due to a greater affinity of the DNA polymerase present in virus-infected cells for viral DNA than for cellular DNA. The inhibitory process is arrested by the addition of puromycin to the infected cells. It is concluded that a protein is responsible for the inhibition of the synthesis of cellular DNA in the infected cell.

Phospholipid metabolism of herpesvirus-infected and uninfected rabbit kidney cells

Abstract The incorporation of labeled precursors into the phospholipids of uninfected rabbit kidney cells in stationary phase was studied with the following results: (1) Incorporation of 32 P into cytoplasmic phospholipids is rapid and reaches a plateau by 40 hr of incubation with the isotope; incorporation into the nuclear fraction, however, continues to increase linearly. (2) The different phospholipids become labeled at different rates. (3) The inner nuclear membrane fraction has a different phospholipid composition from the outer nuclear or the cytoplasmic fractions; it contains a significantly larger amount of sphingomyelin. The phospholipid composition of the envelope of purified pseudorabies virions resembles that of the inner nuclear membrane of uninfected cells, offering further evidence that most of the virions acquire their envelope by budding from this membrane. After infection of rabbit kidney cells with pseudorabies virus, there is an increase in the incorporation of 32 P, choline- 3 H, and myo -inositol- 3 H into the phospholipids. An increase in the incorporation of choline- 3 H into the cytoplasmic fractions of the infected cells occurs, but this increase is especially marked in the nuclear fraction. The distribution of the radioactivity incorporated into the different phospholipids is also changed by infection; approximately 2 to 3 times more appears in sphingomyelin in infected than in uninfected cells. Most of the phospholipids which become part of the virions are present in the cell at the time of infection. However, the viral envelope is not derived from unchanged segments of the nuclear membrane preexisting in the cells at the time of infection. The specific activities of the viral envelope and of the inner nuclear membrane of infected cells were compared under various experimental conditions. The results of these experiments indicate that the virions bud from areas on the membrane which have been synthesized after infection or which have experienced an increased rate of turnover of phospholipids.

The synthesis and fate of pseudorabies virus DNA in infected mammalian cells in the stationary phase of growth

Abstract After infection with pseudorabies virus, rabbit kidney cells in the stationary phase of growth do not synthesize cellular DNA; these virus-infected cells synthesize only viral DNA. This DNA is synthesized in excess—at the end of the virus growth cycle, a maximum of approximately 20% of the total viral DNA synthesized by the infected cells is incorporated into mature virus. Viral DNA forms within the infected cells a virus precursor pool from which DNA is withdrawn in a random fashion to be integrated into the virus particles.

Synthesis of proteins in cells infected with herpesvirus: III. Relative amino acid content of various proteins formed after infection☆☆☆

Abstract The proteins synthesized by pseudorabies virus-infected rabbit kidney cells contain more arginine but less methionine, phenylalanine, tyrosine, isoleucine, and lysine relative to leucine than the proteins synthesized by uninfected cells. This change in amino acid composition would be expected on the basis of the difference between the base composition of cellular DNA and of viral DNA. The changes in the patterns of electrophoretic migration of the proteins synthesized at various stages of the infective process were determined. The proteins synthesized by infected and uninfected cells, which migrate to the same positions in polyacrylamide gels, have different average amino acid compositions. Proteins synthesized by infected cells which migrate to different positions in the gels differ from one another in their amino acid composition. Viral proteins synthesized between 4 hours post infection and the end of the infective process which migrate to a given position in the gels have approximately the same amino acid composition. These results indicate that during this period of time, at least, the main viral proteins synthesized by the cells are the same.

MODE OF REPLICATION OF PSEUDORABIES VIRUS DNA.

Pseudorabies (Pr) virus DNA is double-stranded: its buoyant density in cesium chloride is that expected of double-stranded DNA with the same content of guanine and cytosine (73 moles %); furthermore, this DNA does not react with formaldehyde and its buoyant density shows the expected increase after heat denaturation. Pr virus DNA replicates in a semiconservative manner.