Broadly active inhibitor of viruses spontaneously produced by many cell types in culture.

A broadly active inhibitor of viruses (vaccinia, polio 1, and vesicular stomatitis) was found in the culture fluid from many types of normal human and mouse cells in culture. Virus plaque-inhibiting activity appeared in culture fluids within a few hour after incubation of cultures with fresh medium. Peak inhibitory activity occurred within 24 h. Blockade of cellular ribonucleic acid or protein synthesis decreased appearance of the inhibitor, thereby substantiating that it is a cell-produced viral inhibitor. Inhibition of virus required the simultaneous presence of inhibitor, virus, and cells (due to the reversible nature of the inhibition of virus attachment and penetration, as shown in the accompanying paper [T. K. Hughes et al., Infect Immun. 32:454-457, 1981]). The degree of inhibitory activity depended on the animal species of origin of the inhibitor, the cell type used for assay, and the virus type used for challenge. No cell species barrier against inhibitor action was found. Strong inhibition of multicycle yields of vesicular stomatitis virus and Sindbis virus was caused by low doses of inhibitor. These specific characteristics of the present inhibitor separate it from commonly recognized inhibitors. Possible biological significance of the inhibitor is discussed.

Nuclear and cytoplasmic deoxyribonucleic acid polymerases from rat nephroma cells.

We have examined the DNA polymerases found in a rat nephroma cell line. Using DEAE- and DNA-cellulose chromatography, we have found two major cytoplasmic DNA polymerases and one major and three minor DNA polymerases from the nucleus. The enzymes were all purified, characterized, and distinguished from each other by several criteria. The enzyme require, for maximal activity, a natural or synthetic double-stranded DNA, four deoxynucleoside, triphosphates, and magnesium. They are inhibited to varying degrees by sodium pyrophosphate, ethidium bromide, and rho-chloromercuribenzoate.

Growth characteristics of Kilham rat virus and its effect on cellular cellular macromolecular synthesis.

Kilham rat virus (KRV) is adsorbed into the rat nephroma cell within 1 hr after infection. There follows a latent period of about 12 hr during which less than 1% of the input infectious virus can be accounted for. New infectious virions can be detected at about 12 hr and the maximal yield of virus is attained by 23 hr after infection. The increase in final virus yield is about 200-fold over that found in the latent period. During this 23-hr period of virus growth, the rate of protein synthesis remains 75 to 100% of that in the uninfected cell. Ribonucleic acid (RNA) synthesis during this period is maintained at 100 to 150% of that found in the control cells. The addition of the inhibitor of deoxyribonucleic acid (DNA) synthesis, 5-fluoro-deoxyuridine (FUDR), up to 8 hr after infection completely suppresses virus production. After 8 hr, viral DNA production has started and FUDR inhibition progressively decreases until by 23 hr the addition of the inhibitor no longer causes a reduced virus yield. Viral DNA synthesis once initiated is required for the remainder of the 23-hr virus cycle. Viral DNA synthesis probably begins about 4 hr before the production of infectious virions. In the KRV-infected cells, DNA synthesis decreased sharply for 6 to 7 hr after infection in comparison to the uninfected cell. At 7 to 8 hr after infection, DNA synthesis in the infected cell increased and was maintained at a higher level than in the control cells for the rest of the virus growth period.