Studies on protein and nucleic acid metabolism in virus-infected mammalian cells. 4. The localization of metabolic changes within subcellular fractions of Krebs II mouse-ascites-tumour cells infected with encephalomyocarditis virus

Abstract

Martin, Malec, Sved & Work (1961 b) showed that, during a single growth cycle, infection with encephalomyocarditis virus caused no changes in the total deoxyribonucleic acid, ribonucleic acid or protein of the host ascites-tumour cell. However, by the use of [6-14C]orotic acid and [14C]valine, it was shown that there were substantial changes in the rates of turnover of ribonucleic acid and protein at different times during the cycle of virus growth. In particular, about 5 hr. after infection there was a striking increase in the rate of turnover of ribonucleic acid, and this increase coincided in time with the appearance of new virus. However, this stimulation in turnover appeared to be quantitatively greater than could be accounted for by formation of new virus ribonucleic acid.Methods have now been developed for the welldefined separation of disrupted Krebs II ascitestumour cells into nuclei, mitochondria, microsomes and cell sap (Martin, Malec, Coote & Work, 1961 a). By the use of these methods we have been able to investigate the effect of virus infection on the turnover of the ribonucleic acid and protein of the subcellular components of the host cell, and thus to localize more exactly the sites of metabolic change within the infected cell. This paper describes the result of such a study. METHODSThe origin and propagation of both the Krebs II mouseascites-tumour cells and the encephalomyocarditis virus have been described by Martin et al. (1961 b).Conditions of infection. Sufficient virus was added to suspensions of washed ascites-tumour cells in Earle's medium (2-3 x 107 cells/ml.) to infect all cells (approx. 3 plaque-forming units/cell), and the cells were dispensed in 10 ml. portions into incubation flasks. The virus was allowed to adsorb for 30 min. at room temperature, followed by 15 min. at 360. Suspensions of control (uninfected) cells were treated similarly. The cells were then incubated at 36°as described by Martin et al. (1961 b). * Part 3: Martin, Malec, Coote & Work (1961 a).For the study of protein and nucleic acid turnover, 14C-labelled precursors were added at appropriate intervals during the virus growth cycle to flasks containing infected and control cells; 30 min. later the flasks were removed and diluted with an ice-cold solution of the unlabelled precursor in phosphate-buffered saline b), and the cells sedimented by centrifuging at 120g for 5 min. They were then washed twice with buffered saline and stored in an ice bath until required for disruption.Disruption of Kreb8 11 cells andfractionation of subcellular components. Cells were prepared for disruption by washing with calcium-and magnesium-free buffered saline (Martin et al. 1961 b), then with 0-125M-sucrose-0-075M-KCl solution. The cells were disrupted by a combination of double osmotic shock and Potter homogenizer, as described by . Nuclei, mitochondria, microsomes and cell sap were separated from the tumour-coll homogenates by differential centrifuging in 0-25M-sucrose-0-1M-KCI solution . Preparations of isolated nuclei were us...

Symposium on replication of viral nucleic acids. 3. Replication of mengovirus ribonucleic acid.

“…A report (29) which states that the polymerase from HeLa cells infected with poliovirus is associated with the nuclear fraction is difficult to interpret, since the cells were fractionated after storage at -20 C. Recent data (23, 24) on the distribution of viraldirected synthesis of RNA by Krebs ascites cells inlfected with EMC virus also indicate that it occurs in the cytoplasm. Earlier findings (15,58) which implicated the nucleus as the site of synthesis of viral RNA probably resulted from undetected cytoplasmic contamination of the nuclei (16,23).…”

“…Subsequent studies in which the synthesis of macromolecules was determined by measuring the rate of incorporation of labeled precursors by infected cells confirmed this conclusion. The results of these studies showed that the rate of synthesis of RNA and protein by many strains of mammalian cells is depressed soon after infection with polio- (45,85), mengo- (8,10,31,66), Me (39,75), or EMC virus (58). Inhibition of DNA synthesis, on the other hand, occurs only toward the end of the replicative cycle and, therefore, appears to represent a secondary host response.…”

Accumulation of Poliovirus Proteins in the Host Cell Nucleus

“…It has been well known since the early 1960s that picornaviruses inducean inhibition of host cell transcription [3,21,22] and that virusspecific protein synthesis appears to be respon sible for RNA synthesis shut-off [8]. Since it has been found that cytoplasmic extracts of poliovirus-infected cells can inhibit transcrip tion in uninfected, isolated nuclei [10b], the next step was to see if poliovirus proteins could be demonstrated within the host cell nucleus.…”

Involvement of the Host Cell Nuclear Envelope Membranes in the Replication of Japanese Encephalitis Virus

“…In the case of the small ribonucleic acid RNA viruses, it appears that the cell nucleus plays little or no direct role in viral replication. However, for a wide variety of viral agents such as tobacco mosaic virus (11,19), encephalomyocarditis virus (3,7,16), and influenza virus (13), the possibility that the nucleus has an important function in RNA replication has been suggested.…”