Infected Rabbit Kidney Cells Research Articles

Early functions of the genome of herpesvirus: II. Inhibition of the formation of cell-specific polysomes

During the early stages of infection of rabbit kidney cells with pseudorabies virus, when both cell- and virus-specific proteins are being synthesized, two types of polysomes are present in the infected cells: larger virus-specific polysomes with nascent chains of polypeptides characterized by a low lysine: leucine ratio, and smaller cell-specific polysomes with nascent chains of polypeptides characterized by a high lysine: leucine ratio. This finding demonstrates that virus-specific proteins have a lower content of lysine than cell-specific proteins. The difference between cell- and virus-specific proteins in their relative content of amino acids was used to monitor the types of proteins synthesized by the infected cells in studies on the mechanism controlling the shutoff of host protein synthesis in virus-infected cells. Both RNA and protein synthesis are required in the infected cells to effect the inhibition of cell-specific protein synthesis. It was found that a protein (or proteins) is synthesized in virus-infected cells which is responsible for the disappearance of cell-specific polysomes; the message for this protein is transcribed in cells infected in the presence of cycloheximide and is translated after the removal of cycloheximide.

Phospholipid metabolism of herpesvirus-infected and uninfected rabbit kidney cells

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 32P 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 32P, choline- 3H, and myo-inositol- 3H into the phospholipids. An increase in the incorporation of choline- 3H 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.

Synthesis of proteins in cells infected with herpesvirus: IV. Analysis of the proteins in viral particles isolated from the cytoplasm and the nucleus

Three types of pseudorabies virus particles can be isolated from infected rabbit kidney cells by centrifugation in sucrose density gradients. One type, isolated from the cytoplasm, contains DNA, behaves like extracellular virus, and probably represents a population of mature virus. A second type, isolated from the nucleus, also contains DNA but has a lower sedimentation value and 4-fold lower specific infectivity than cytoplasmic virus. A third type, also isolated from the nucleus, does not contain DNA and is not infectious. Electrophoretic analysis of the proteins present in the infectious nuclear and cytoplasmic viral particles revealed that they contain the same proteins but that the former possess about 30–35% less of at least two proteins normally present in mature virus. These two proteins probably represent proteins of the viral envelope.

Cellular deoxyribonucleic acid synthesis and loss of contact inhibition in irradiated and contact-inhibited cell cultures infected with fibroma virus.

Cultural changes that follow infection of rabbit kidney cells with fibroma virus were studied. Characteristic alterations of cell morphology and development of multilayered piles and cords of cells were found to occur in infected cultures in which cell division was blocked by gamma radiation or by cell crowding and serum deprivation, thus indicating no dependence upon cell division. Fibroma virus infection did not remove blocks to cell division, but it did exert distinct effects upon nuclear deoxyribonucleic acid synthesis in cells blocked by radiation or cell crowding. Use of tritium-labeled thymidine and autoradiography demonstrated that after infection initial inhibition of nuclear incorporation was followed by sharply increased nuclear labeling at a time that coincided with beginning alterations of cell morphology and development of cell piling.

Adenovirus T antigen: production by abortive infection of rabbit kidney cells.

SummaryHigh titered adenovirus type 12 T antigen preparations were produced in LLC-RK-1 cells; this was achieved with little or no virus replication. The T antigens which cross-reacted with B group tumor antibody developed in the same cultures. Infection of the LLC-RK-1 cells with adenovirus type 7 and other representatives of group B failed to yield appreciable amounts of T antigen.

Mechanism of inhibition of cellular DNA synthesis by pseudorabies virus

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.