The replication of mengovirus was studied in two strains of Novikoff (rat) hepatoma cells propagated in vitro. The replicative cycle in both strains required 6.5 to 7 hr. Infection resulted in a marked depression of ribonucleic acid (RNA) and protein synthesis by strain NISI-63. Inhibition of RNA synthesis was reflected by a decrease in the deoxyribonucleic acid (DNA)-dependent RNA polymerase activity of isolated nuclei. Mengovirus had no effect on either protein or RNA synthesis or on the DNAdependent RNA polymerase activity of a second strain, NlSl-67. The time course of viral-induced synthesis of RNA by cells was studied in cells treated with actinomycin D. It was first detectable between 2.5 and 3 hr after infection and continued until 6.5 to 7 hr. The formation of mature virus was estimated biochemically by measuring the amount of RNA synthesized as a result of viral infection which was resistant to degradation by ribonuclease in the presence of deoxycholate. Approximately 70% of the deoxycholate-ribonuclease-resistant RNA was located in mature virus, and the remainder was double-stranded. The formation of mature virus began about 45 min after viral-directed (actinomycin-resistant) synthesis of RNA was detectable in the cell, and only about 18 to 20% of the total RNA synthesized was incorporated into virus. Release of virus from cells began about 1 hr after maturation was first detectable. Release of virus from cells was accompanied by a loss of a large proportion of their cytoplasmic RNA and protein.

It was confirmed that mice bearing many transplantable tumors are infected with a virus-like agent which causes a 5- to 10-fold elevation in the plasma lactic dehydrogenase activity of infected mice without tumors. The agent is non-identical with the polyoma virus and without effect on adult rats and hamsters. Maximum titers of 109 to 1010 ID50 per ml of plasma were observed within 36 hours after infection. Subsequently the titer decreased to 105 to 107 ID50 per ml and remained constant thereafter. The plasma lactic dehydrogenase reached maximum activity about 96 hours after infection and remained elevated indefinitely. The virus was present in feces and in a variety of tissues and organs in relatively high concentrations. Liver and spleen yielded the highest titers.Five electrophoretically distinct forms (isozymes) of lactic dehydrogenase were separated from mouse tissues. Infection of mice resulted in an increase of the slowest migrating isozyme in the plasma. Liver, spleen, and erythrocytes were each found to contain only this isozyme while other tissues and organs contained mixtures of lactic dehydrogenase isozymes. The plasma of tumor-bearing mice contained more of the slowest migrating isozyme than infected mice without tumors.

The tricarboxylic acid cycle has received general acceptance as a mechanism to explain the oxidation of acetic acid by animal tissues. The data obtained in studies with bacteria do not satisfy many of the criteria on which the cycle was based in animal tissues (Krebs, 1948-1949). In the preceding report, Saz and Krampitz (1954) critically evaluated this problem as applied to bacteria. Techniques were described which permit the isolatioga of intermediates of acetate oxidation in the absence of added carriers. These intermediates were in approximately complete isotopic equilibrium with each other, with the recovered acetate, and with the respiratory carbon dioxide. These data were in contrast with the results obtained with carrier experiments employing Micrococcus lydeikticu (Saz and Krampitz, 1950; Ajl and Kamen, 1950) and Escherichia coli (Swim and Krampitz, 1950; Ajl and Kamen, 1950). In the latter case, isotope from acetate-2-C4 was not incorporated into a-ketoglutarate to a significant extent. The carrier succinate, on the other hand, was highly radioactive, and the carboxyl carbons were in almost complete isotopic equilibrium with the respiratory carbon dioxide. These data could be interpreted as evidence that the tricarboxylic acid cycle is of no quantitative importance as a mechani for the oxidation of acetate by E. coli and that the major mechanism involves acetate condensation to succinate (Thunberg condensation) and the other reactions of the dicarboxylic acid cycle. It has been shown by Saz and Krampitz (1954) that nonequilibration between metabolic a-ketoglutarate and that added as a carrier can account for the observed results, and, therefore, the evidence for the dicarboxylic acid cycle was unsatisfactory. The results of experiments I This work was supported by a grant (Contract no. AT(30-1)-1050) from the Atomic Energy Commission. The radioactive isotope used in these studies was obtained on allocation from the Atomic Energy Commission.

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