Expression of Vaccinia Virus Early mRNA in Ehrlich Ascites Tumor Cells

Abstract

A method for preparing detergent cytoskeletons from uninfected or vaccinia-virus-infected cells is described. This method resulted in the fractionation of the cytoplasmic compartment into soluble and cytoskeletal fractions. More than 85% of small molecules and tRNA were released from the cytoskeleton and recovered into the soluble fraction. The cytoskeletal fraction contained about 40% of the cytoplasmic proteins and 6?':,, of total cytoplasmic RNA. Similar values were obtained for vaccinia-virus-infected cells. In contrast, whereas 85 ?,, of the cellular polysomes and poly(A)-rich RNA remained associated with the cytoskeleton in uninfected cells, more than 40% of vaccinia early mRNA engaged into polysomes was found to be not associated with the cytoskeleton. A similar partition was found when the viral R N A was labeled for 15 min at 5 min and 20 min after the infection or when varying the duration of the pulse. Soluble and cytoskeletal viral polysomes were found to synthesize a similar set of proteins after translation in vitro of the corresponding mRNA. The fate of rapidly labeled cellular poly(A)-rich RNA upon vaccinia virus infection was followed by a glucosaminei uridine chase procedure, and also that of relatively stable poly(A)-rich RNA after long-term labeling and chase. In both cases no release of poly(A)-rich RNA from the cytoskeleton occurred after vaccinia virus infection. These experiments reveal that cellular mRNA remains associated to the cytoskeleton in EAT cells infected with vaccinia virus (early period) whereas at least 40% of the vaccinia early polysomes are not associated with the cytoskeleton. A model for vaccinia early mRNA metabolism is presented, which may account for the rapid shut-off of host protein synthesis.The cytoplasm of animal cells contains several types of protein filaments constituting the cytoskeleton. These include the microtubules, intermediate and microfilaments and also a microtrabecular lattice [I] although the existence of the latter is questioned [2]. One important observation of these electron microscopy studies was that the so-called free polysomes (i.e. not membrane-bound) were indeed seen as being bound to the microtrabecular lattice [3]. This observation was supported by a biochemical analysis of the detergent cytoskeletons prepared from HeLa cells: it was concluded that messenger is translated only when bound to the cytoskeletal framework [4,5]. Although the detailed mechanism by which cellular polysomes are bound to this structure is still obscure, Penman and co-workers extended their conclusion to the case of poliovirus [6] and cells infected with vesicular stomatitis virus [5]: the viral inRNA involved in translation was found associated with the cytoskeleton. This conclusion was recently confirmed for the case of KB cells infected with adenovirus [ 7 ] and for the BSC-1 cells infected with SV40 [8]. The general conclusion of these studies is that no unique class of cellular or viral mRNA is translated while bound to the cytoskeleton, with one exception [9]...

Interaction between mRNA, ribosomes and the cytoskeleton

“…Furthermore, association of mRNAs with the cytoskeleton and their subsequent translation are not tightly coupled in that attachment to the cytoskeleton alone is not sufficient for translation. Thus in influenza-and vaccinia-virus-infected cells host mRNAs are associated with the cytoskeleton although they are not translated (Lemieux & Beaud, 1982;Katze et al, 1989). This demonstrates that association of mRNA with the cytoskeleton and then its translation occur as independent events.…”

“…shown that up to 40% of viral mRNAs are associated with polysomes in the free fraction released by gentle treatment with non-ionic detergent (Lemieux & Beaud, 1982). The consensus of most results, therefore, is that free, cytosolic polysomes do exist.…”

“…277 where disruption of the cytoskeleton does not occur, the host mRNAs are retained on the cytoskeleton (Katze et al, 1989). There is also evidence to suggest that during viral infection there is retention of some host mRNAs, such as those for actin and tubulin, in the cell matrix fraction even though these are not translated (Lemieux & Beaud, 1982;Bonneau et al, 1985;Katze et al, 1989). It is likely that these two mRNAs are cytoskeletalbound even during viral infection without being actively translated.…”

“…70 % of cellular mRNA has been recovered in a socalled 'cytoskeletal-bound' form either retained in the cell matrix or released from such structures by Triton X-1 00/deoxycholate treatment. This has been observed in a variety of cells such as HeLa cells (Lenk & Penman, 1979;van Venrooij et al, 1981), myoblasts (Bag & Praminik, 1987;Meadus et al, 1990), virusinfected cells (Lenk & Penman, 1979;Cervera et al, 1981;van Venrooij et al, 1981;Lemieux & Beaud, 1982;Bonneau et al, 1985;Katze et al, 1989) and oocytes (Moon et al, 1983;Jeffery, 1984;Yisraeli & Melton, 1988). However, as with the analysis of polysome distribution, the main limitation of these experiments has been the definition of the insoluble cell matrix as a cytoskeletal fraction and any mRNAs associated with it or with material released by Triton X-100/deoxycholate treatment as cytoskeletalbound.…”

Noncoding RNA of U87 host gene is associated with ribosomes and is relatively resistant to nonsense-mediated decay

“…Drugs treatment, cell fractionation, and RNA isolation Cells were incubated with cycloheximide (20 μg/ml), puromycin (200 μg/ml) or actinomycin D (5 μg/ml) for time indicated. HeLa cells were fractionated into nuclear and cytoplasmic fractions as described (Lemieux and Beaud, 1982). Nuclear RNA was isolated by extraction with hot (65°C) phenol saturated with sodium acetate, pH 5.0.…”