When NIH/3T3 mouse fibroblasts were infected with the Moloney strain of murine leukemia virus, part of the viral genome RNA molecules were detected in polyribosomes of the infected cells early in the infectious cycle. The binding appears to be specific, since we could demonstrate the release of viral RNA from polyribosomes with EDTA. Moreover, when infection occurred in the presence of cycloheximide, most viral RNA molecules were detected in the free cytoplasm. Size analysis on polyribosomal viral RNA molecules indicated that two size class molecules, 38S and 23S, are present in polyribosomes at 3 h after infection. Analysis of the polyriboadenylate [poly(rA)] content of viral RNA extracted from infected polyribosomes demonstrated that such molecules bind with greatest abundance at 3 h after infection, as has been detected with total viral RNA. No molecules lacking poly(rA) stretches could be detected in polyribosomes. Furthermore, when a similar analysis was performed on unbound molecules present in the free cytoplasm, identical results were obtained. We conclude that no selection towards poly(rA)-containing viral molecules is evident on binding to polyribosomes. These findings suggest that the incoming viral genome of the Moloney strain of murine leukemia virus may serve as a messenger for the synthesis of one or more virus-specific proteins early after infection of mouse fibroblasts. MATERIALS AND METHODS Cells and viruses. NIH/3T3 mouse fibroblasts were used as control cells throughout this study. NIH/ 3T3 cells chronically infected with MLV [NIH/ 3T3(MLV)] were used as a source for infecting virus particles. Unless otherwise indicated, the cells were grown in 100-mm dishes (Nunc, Roskilde, Denmark)

SUMMARYRadioactively labelled virus particles of intracellular origin were isolated from the cytoplasmic fraction of disrupted NIH/3T3 cells chronically infected with Moloney murine leukaemia virus [NIH/3T3 (MLV)]. Interferon (IFN) treatment for 48 h, which arrested more than 90 % of virus release, resulted in a remarkable accumulation of these intracellular virions. However, no major effect of such treatment was apparent on their structural properties. Transmission electron microscopic examination revealed that these intracellular virions were located within cytoplasmic vacuoles. IFN treatment resulted in a considerable increase in the number of viruscontaining vacuoles, as well as the total number of vacuolar virions. It seems that IFN inhibits the final release of vacuolar virions from the cells, thus leading to their intracellular accumulation.

The effect of interferon (IFN) on the adsorption, penetration and subsequent binding of the incoming genome RNA of Moloney murine leukemia virus (MLV) to polyribosomes, was studied in NIH/3T3 mouse fibroblasts. Virus adsorption was assayed by determining reverse transcriptase activity in the inoculating virus stock and in the cell membrane fraction before and after 45 minutes of infection. Both measurements suggested that IFN had no effect on virus adsorption. Virus penetration was determined by measuring the amount of viral RNA in the cell cytoplasm at 45 minutes after infection. This amount was remarkably lower in IFN-treated than in untreated cells. This reduction was not due to inhibition of a possible induction of endogenous viral genetic information by the penetrating virions, but was proved to be a direct effect of IFN on virus penetration, which was related to the IFN-induced antiviral state. The effect of IFN on binding of parental genome RNA to polyribosomes was then investigated by analysing Crt hybridization kinetics of polyribosomal viral RNA at different time intervals after infection. While in untreated control cells maximal binding occurred at 3 hours postinfection, this maximal binding was observed in IFN-treated cells at 5 hours postinfection. The distribution of viral RNA molecules between sub-cytoplasmic fractions at 3 hours after infection was, in IFN-treated cells, significantly different from that observed in the untreated cells.

Postmitochondrial cytoplasmic extracts, prepared from uninfected NIH/3T3 cells as well as from chronically or exogenously infected with murine leukemia virus (MLV), were found to stimulate the endogenous reaction of purified MLV reverse transcriptase. No such stimulation was observed with the exogenous reaction of this enzyme, using poly (rA) oligo (dT) as an exogenous template-primer. While the stimulatory capacity of extracts from uninfected and chronically infected cells was comparable, that of the exogenously infected cells was much more powerful in this respect. The stimulatory activity could be destroyed by trypsin, indicating that it was excerted by a protein. In uninfected and chronically infected cells this protein was found to be of a short functional life time under conditions blocking continuous protein synthesis. However the mRNA coding for this factor was found in these cells to be stable. On the other hand, the increased stimulatory activity, observed in extract of exogenously infected cells, was independent on protein synthesis and therefore was attributed to a protein apparently introduced into the cells by the penetrating virions. Experiments with monospecific antibodies against MLV proteins suggested that p30 is an important accessory for reverse transcriptase activity and that the cytoplasmic stimulatory factor might be also related to p 30.