HeLa cells, competent

In this review I have outlined several theories that have been proposed to explain the mechanism by which picornaviruses inhibit cellular protein synthesis. Some theories seem less likely than others. Inhibition by ds ENA, for example, is no longer thought to be a likely possibility. In cell-free extracts ds ENA inhibits both cellular and viral mRNA translation (61). The inhibitor of cellular protein synthesis would be expected to be selective in its inhibitory activity. It is also apparent that picornavirus infection does not result in the degradation or alteration of cellular mENA (9> 27, 29 51). So,.too, experiments demonstrating that protein synthesis inhibition takes place in the absence of significant viral ENA synthesis (I4) tend to weaken the argument that protein synthesis inhibition results from direct competition of viral mENA with cellular ENA for initiation factor eIE-4D (47) As mentioned earlier, superinfection with poliovirus of cells infected with VSV prevents VSY mENA translation (J2, 56). In lysates from uninfected HeLa cells, however, 7SY mENA translation is favored over poliovirus mENA translation when both mENA species are present in equimolar saturating concentrations (55) If competition were a major cause of cellular protein synthesis inhibition, one would have expected poliovirus mENA to out-compete VSV mENA in cell-free translation, not the contrary. 

The virus-specific RNA had all the characteristics of messenger RNA it formed polysomes and competed actively in this respect with messenger RNA of the HeLa cells and completely expelled it from the sites of protein synthesis approximately 3 h after infection. 

Finally, SECM studies on single HeLa cells have also focused on their interaction with silver nanoparticles. The detection scheme relied on the interaction of an iridium hexachloride couple with silver nanoparticles. When the microelectrode approached the HeLa cells, two mechanisms competed with IrCV regeneration from IrClg by the silver nanoparticles adsorbed on the ceU surface IrCl " diffusing across the intracellular space and IrCI transported in the intracellular lumen where it could subsequently be reduced by an intracellular redox species or silver nanoparticles segregated in the nucleus (Eigure 12.23). 

G. Competence of Hela Cells for Infection by Viral RNA at Different Stages... 

Fig. 8. Kinetics of adsorption and penetration of single- and double-stranded RNA. Aliquots of viral RNA or RF-RNA were added to 0.2 ml samples of competent HeLa cells (pre-exposed to 10% DMSO and 160 xg/ml DEAE-dextran) and incubated at 37° C. At the times indicated, the interaction of RNA and cells was stopped by adding 1.5 ml of indicator cells or 1.5 ml of indicator cells containing 20 (xg/ml ribonuclease. — RF-RNA RF-RNA plus RNase o---o viral RNA viral...

The viral RNA infectivity is increased when poly-L-ornithine and methylated albumine are added to HeLa cells either 30-5 minutes before or 5 30 minutes after the addition of RNA. In contrast, DEAE-dextran only stimulates cellular competence if it is added shortly before the viral RNA. If the cells are kept at 37° C, they lose the competence for infection induced by DEAE-dextran and DMSO rapidly and this cannot be restored completely by adding DEAE-dextran once more together with the RNA (Tabled). However, when the cells are cooled to 0° C after exposure to DEAE-dextran and DMSO for one min at 37° C, they remain competent for infection by viral RNA for up to 48 hours (Koch, 1971b). 

The loss of cell competence for infection by viral RNA and RF-RNA at 3 7° C is more pronounced when the cells are incubated in the absence of DMSO. Polycations are toxic for cells, therefore it was of interest to study the effect of DEAE-dextran in our system. The viability of the cells after different periods of incubation with DEAE-dextran was determined by counting the number of cells which were stained by trypan blue. HeLa cells (2.6 X 10 /ml) were incubated with I60 [xg DEAE-dextran for 30 minutes at 37° C in Eagle s medium without and with 10% DMSO. In the absence of DMSO, 38% of the cells lost their viability, but in the presence of 10% DMSO only 12% became trypan blue-positive. Thus, DMSO enables the cells to tolerate higher concentrations of DEAE-dextran. 

These results show that the competence of HeLa cells for infection by viral RNA does not change as dramatically during growth as the competence of bacteria for transformation. Only the two initial stages in the interaction of nucleic acids with host cells are equal in both systems, i.e. adsorption and penetration. Thereafter, quite different nucleic acid-dependent events occur, i.e. transforming DNA is incorporated in the host cell genome, viral RNA has to act as mRNA to induce a cycle of virus replication with concomitant interference with the metabolism of the host cell. 

Table 8. Competence of HeLa cells pre-exposed to VP4 and/or DEAE-dextran for infection by viral RNA and RF-RNA...

Breindl and Koch, 1972). When these two viral peptide fractions were analyzed for their capability to increase the cellular competence for RNA infection, it was observed that VP4 was the most active coat peptide in this respect (Tables). It stimulated the competence of HeLa cells for infection by viral RNA and RF-RNA more than 1000-fold compared to untreated cells in concentrations as low as 10 [J g/nil. 

In further experiments (Koch, unpubhshed), the effect of DEAE-dextran and VP4 on the competence of HeLa cells for infection was compared. It was found that the VP4 induced competence was stable at 37° C for 20 min, whereas 90% of the competence induced by DEAE-dextran was lost within 10 min. As shown in Table 8 there is no synergistic effect between VP4 and DEAE-dextran, indicating that both act in a similar way. Preincubation of viral RNA with VP4 at room temperature or at 37° C neither significantly increases nor decreases the biological activity of either component. 

Preincubation of HeLa cells with actinomycin (0.4 to 2.0 (xg/ml) for 150 min at 37° C before exposure to polycations increases the infectivity of viral RNA up to 3-fold (Koch et al., 1967) (Table2). Inhibition of host cell RNA synthesis results in an accumulation of free ribosomes inside the cells which provide the invading viral RNA with a greater chance of finding free ribosomes and thus of acting as mRNA. Incubation of cells with actinomycin has the opposite effect on the infectivity of RF-RNA. The polycation-induced competence of actinomycin-treated cells for RF-RNA is only 10% that of normal cells. This agrees with the h q)othesis that host cell functions are required to melt double-stranded RNA or to use it as a template for the synthesis of new viral RNA. 

Evidence that there are differences between HeLa cells infected by viral RNA and those infected by RF-RNA in initial stages was also obtained in studies with phleomycin. Although the infectivity of viral RNA is ten times more sensitive to exposure to phleomycin than the infectivity of RF-RNA, preincubation of cells with phleomycin has the reverse effect. A short incubation (1 to 30 min) of HeLa cells with phleomycin (10-20 irg/ml) reduces their competence for infection by both viral RNA and RF-RNA to 5%. But subsequent incubation of these cells in the absence of phleomycin for 30 to 60 min before the addition of RNA restores the competence for viral RNA but does not alter the low competence for RF-RNA (Koch, 197I a). 

Comparable results were obtained when macromolecular synthesis in HeLa cells was inhibited by infection with the DNA-containing frog virus 3 (Koch, unpublished) the competence of cells for viral RNA was reversibly affected by infection with frog virus 3 whereas the competence for RF-RNA was not restored within 120 minutes of incubation at 37° C. Cellular competence for infection by viral RNA and RF-RNA is enhanced by various polycations, by DMSO and by VP4 in differing degrees. Thus, cellular competence for RF-RNA is maximal when the cells are sensitized by DEAE-dextran alone (Koch and Bishop, 1968), and cannot be further stimulated by poly-L-omithine or DMSO (Koch, unpublished). However, cellular competence for infection by viral RNA is 5 to 10 times higher when cells are exposed to DEAE-dextran and DMSO or DEAE-dextran and poly-L-ornithine than to DEAE-dextran alone (Koch, 1971b, and unpublished). 

Breindl, M., Koch, G. Competence of suspended HeLa Cells for infection by inactivated poliovirus particles and by isolated viral RNA. Virology 48, 136-144 (1972). 

Superinfection with poliovirus has a striking effect on VSV-in-fected HeLa-Ss cells the evidence in these experiments was quite convincing that preformed VSV mRNA was not translated following infection with poliovirus (Ehrenfeld and Lund, 1977 Trachsel et al., 1980). Of course, the effect of VSV on cellular protein synthesis could not be determined in this experiment because poliovirus itself drastically inhibits cellular protein synthesis. As mentioned above, the ascendency of the mRNAs of two viruses may also depend on the type of cell which is doubly infected with the two competing viruses (Otto and Lucas-Lenard, 1980). 

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