HeLa cell poliovirus infected

Figure 1. Processing of picomavirus. polyprotein by proteolytic enzymes. The numbers in brackets represent molecular weights in thousands. Insert Sodium dodecyl sulfate/ polyacrylamide gel electrophoresis of poliovirus polypeptides, labelled with 5% methionine in infected HeLa cells, a. Infected, labelled at 2.5 hours post infection, b. Labelled in presence of 0.5 niM iodoacetamide. c. Labelled in presence of 0.1 mM tosyl lysine chloromethyl ketone.

Koschei, K., 1971, Release of chromium from labeled HeLa cells after infection by poliovirus, Z. Naturforsch. B26 929. 

HLA virus (5 [xg/ml) and their sensitivity to infection by isolated RNA was analyzed. We found (see Table 5) the cells exposed to HLA virus for one minute at 37° C prior to addition of single- or double-stranded poliovirus RNA yielded 100 times more infective centers after 15 min incubation at 37° C than untreated cells (Breindl and Koch, 1972). This result reveals that the poliovirus coat proteins are able to sensitize HeLa cells to infection by viral RNA. 

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). 

Selected polyamines were tested for their ability to alter the normal replication cycle of two representative RNA viruses Ence-phalomyocardltls (EMC strain MM) and Poliovirus Type I. Mono-layers of L929 and HeLa cells were treated with solutions containing specified polyamines. After Incubation the cultures were washed and then Infected with the RNA virus. After a 24 hours Incubation period, the supernatant fluids were collected and assayed for virus (pfu) content. 

The data given In Tables 5 and 6 are offered to Illustrate the variety of results. Thus for Poliovirus, platinum polyamines can effect an Increase or decrease In the growth of the virus or exhibit no effect at concentrations below that where the HeLa cells are affected. Similar results are found for the polyamines In L929 cells Infected with EMC strain MM. Thus with treatment for 24 hours, polymers 9,11 and 12 suppressed viral replication (Table 6). With the exception of polymer 6, all of the polymers suppressed EMC viral replication when treatment was extended to 48 hours. Again viral replication was affected at polyamlne concentrations well below that necessary to Inhibit the L929 cells. 

The presence of informosomelike RNPs has been demonstrated also in the case of Ehrlich ascites carcinoma cells infected with Sendai virus (Volkova et al., 1969). The cytoplasmic extracts of the cells labeled for 30 minutes with uridine contain virus-specific RNA in the form of particles with a sedimentation coefficient 45S (the sedimentation coefficient of the complete virus equals 57S). The buoyant density of the RNP peak in CsCI equals 1.43 to 1.44 g/cm. Although these properties are compatible with the idea that they are informosome, the particles, and in particular their protein component, should be characterized in more detail before reaching a definite conclusion. Recently SOS virus RNA-containing particles with p = 1.40 g/cm have been found in HeLa cells infected with poliovirus (Huang and Baltimore, 1970), although the authors have some doubts about the reality of these complexes. 

Huang, A. S., and D. Baltimore. 1970. Initiation of polyribosome formation in poliovirus-infected HeLa cells. J. Molec. Biol., 47 275-292. 

The decrease in cellular protein synthesis could result from degradation or inactivation of cellular mRNA. Early studies (19 20), however, showed that cellular mRNA present in polysomes from poliovirus infected HeLa cells had the same sedimentation characteristics as that from uninfected cells. Because no appreciable degradation of cellular mRNA was observed, it was concluded that cellular protein synthesis inhibition does not result from an appreciable increase in endonuclease activity which would degrade cellular mRNA. 

It has been postulated that the 3 -poly(A) sequence on eukaryotic mRNAs mi t be involved in mRNA stability since it has been found that these sequences on mRNAs in HeLa cells (28) are successively degraded with increasing age of mRNA molecules. On this basis Koschei (29) examined whether infection of HeLA cells with poliovirus leads to a more rapid degradation of poly(A) sequences which in turn might explain the shut-off of cellular protein synthesis. No detectable differences were observed in the poly(A) sequences in cellular cytoplasmic RNA from uninfected cells and cells infected with poliovirus for 4 hours. Both amount and size of these sequences were identical. 

The VSY genome codes for only five proteins (33), and soon after infection the five YSY proteins represent the only translation products of the infected cell, since this virus also inhibits cellular protein synthesis (34) The five YSY monocistronic mRNAs serving as templates for these proteins have been relatively well characterized (35) and thus this system lends itself to studies on the mechanism of superinfection. Ehrenfeld and Lund (36) examined the consequences of superinfecting YSY infected HeLA cells at two hours after infection with poliovirus. By I.5 to 2 hours after poliovirus superinfection, virtually all YSY polysomes disaggregated. The pattern of protein synthesis as shown on polyacrylamide gels changed from YSY specific to poliovirus specific within 2.5 hours after infection. All five of the YSY proteins were inhibited to the same extent. 

Another theory to explain the shut-off, proposed by Cooper et al. (4Q) involves the synthesis of viral proteins which have an affinity for the small ribosomal subunit and the 5 end of viral mENA. The viral protein would repress the synthesis of cellular proteins by combining with the 4OS subunit, thereby blocking its link with host mENA. By also binding to the 5 end of viral ENA, the proteins would facilitate the attachment of viral ENA to the 40s subunit and increase the translation of viral ENA. Viral proteins have been found to co-sediment with the 4OS subunits of HeLa cells infected with poliovirus (49)> of Ehrlich ascites tumor cells infected with EMC virus (50) and of L-cells infected with mengovirus (5 ) poliovirus infected cells, the viral proteins co-sedimenting with ribosomes were identified as VPO, VP1 and VP3, all structural proteins (49) Both structural and non-structural proteins were found associated with ribosomes from EMC... 

Most recently Rose et al. (55) have identified an initiation factor that is inactivated after infection of HeLa cells with poliovirus. In their experiments Rose al. (55) took advantage of the finding that translation of YSV mRNA, like host mRNA translation, is inhibited in cells superinfected with poliovirus (52, 56). They prepared extracts from poliovirus-infected and uninfected HeLa cells, and after a preincubation period and RNase treatment to eliminate endogenous mRNA translation, tested the ability of the extracts to translate exogenous poliovirus and 7SV mRNA. Poliovirus mRNA was translated by both extracts, but YSV mRNA was translated only in the extracts from uninfected cells. 

In summaxy, the available evidence suggests that an initiation factor is inactivated after infection of HeLa cells by poliovirus, possibly by a virus-coded factor. The variety of results seen in comparative studies using extracts from infected and uninfected cells in terms of translation of cellular and viral messages is not understood, but may be a property of the virus or cell system under study. 

One way of searching for the presence of inhibitors of polypeptide initiation in infected cells was to add cytoplasmic fractions from virus infected cells to a cell-free system from rabbit reticulocytes. This system initiates the synthesis of new polypeptide chains at a very high rate. Cytoplasm from poliovirus infected HeLa cells, but not from uninfected cells, inhibited protein synthesis in the reticulocyte lysate (59) The inhibitor was isolated and identified as double-stranded (ds) RNA (60). To study the effect of ds RNA on host and viral protein synthesis, a cell-free system from HeLa cells was developed which initiated translation on endogenous cellular or viral mRNA. When added to this system, the ds RNA was found to inhibit the translation of both cellular and viral mRNAs (61). Furthermore, measurement of the amount of ds RNA present in cells early in infection (61, 62) revealed that an insufficient quantity was present to act as a direct agent of protein synthesis inhibition. 

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. 

In the second step, the virus synthesizes an inhibitor that inactivates an initiation factor and by the mid-point of infection, most ribosomes are directed towards translating viral mENA. This two step process could explain the differential effect of such perturbations as exposure to high salt concentrations and histidinol treatment on cellular and viral mENA translation and the inactivation of initiation factor eIF-4B after infection of HeLa cells with poliovirus. 

Polysomes in normal and poliovirus-infected HeLa cells and their relationship to messenger-RNA. Proc. Natl. Acad. Sci. U.S.A. (1963), 4i, 654-661. 

LEIBOWITZ, R. and PENMAN, S. Regulation of protein synthesis in HeLa cells. III. Inhibition during poliovirus infection. 

HUNT, T. and EHRENFELB, E. Gytoplasm from poliovirus-infected HeLa cells inhibits cell-free haemoglobin synthesis. Nature New Biology (1971)> 230. 91-94. 

Under properly defined conditions picomaviruses interrupt host RNA and protein synthesis (1 ) and subvert the cellular machinery to production of viral protein and ENA. By feeding radiolabeled amino acids to virus-infected cells after cessation of host-protein synthesis, viral protein can be selectively labeled. In a pioneering study, which introduced the now widely used SDS-polyacrylamide gel electrophoresis technique. Summers et al. (2) identified some 14 different virus-specified polypeptides in extracts of poliovirus infected HeLa cells. The net mass of these polypeptides exceeded two-fold or more the known coding capacity of the viral genome. 

Approximately 25% of the total polysomes in poliovirus-infected and uninfected HeLa cells are membrane-bound (10). The membrane-bound polysomes are about five times more active in translation per unit mass than free polysomes, as determined by incorporation of amino acids in cell-free extracts (11). The exact role of the membrane in this process has not yet been defined. 

If one examines the pattern of proteins synthesized in cells infected for three to four hours with a picomavirus, the only proteins synthesized are viral, for by this time the synthesis of most host proteins has ceased. In the earliest studies carried out by Summers et al. (I4) in poliovirus-infected HeLa cells, about I4 different virus-specified polypeptides were identified by SDS-polyacrylamide gel electrophoresis. The net mass of these viral polypeptides, however, exceeded the coding capacity of the viral genome by a factor of 2. This finding was incompatible with what was then known about the translation of the small RNA bacteriophages such as Q6 and R17 In these cases, each of the three cistrons was found to have its own initiation and termination signals (15) ... 

Similar results were obtained with mengovirus. In the presence of p-fluorophenylalanine, canavanine and azetidine-2-carboxylic acid, two precursors larger than polypeptide A were detected (22). If these analogues plus ethionine were present, then a small amount of a polypeptide equivalent in size to poliovirus NCVP-00 was observed (23). Figure 4 shows the pattern of labeling obtained from mengovirus-infected L-cells (upper panel) and poliovirus-infected HeLa cells (lower panel) in the presence of the analogues. 

SDS-polyacrylamide gels of lysates of L and HeLa cells pulse labeled with 3H-amino acids in the presence of the amino acid analogues mentioned in the text. The positions of mengovirus capsid proteins are also shown. Upper panel mengovirus infected L-cells pulse labeled for 30 Diin at 6 hr post infection. Lower panel poliovirus infected HeLa cells pulse labeled for 30 min at 3 br post infection. Modified from Paucha ad. (25) ... 

CHATTERJEE, N.K., KOCH, G. and WEISSBACH, H. Initiation of protein synthesis in vivo in poliovirus-infected HeLa cells. Arch. Biochem. Biophys. (l975)> 154 43I-437. 

Comparison of polysomal structures of uninfected and poliovirus infected HeLa cells. Virology (l97l), 44, 259-248. 

Recent evidence points to the presence of protease activity-associated with polysomes and ribosomes when extracts of uninfected cells are assayed (refs. 27 32, Figure j). Characteristic of infection of cells by poliovirus is drastic, rapid inhibition of protein synthesis. Poliovirus infection also depresses the ribosomal protease activity (27, 29, 55) Ribosomes from uninfected cells have been reported to possess an autoproteolytic activity (31, 32), and this has been confiimed by two-dimensional gel analysis (Figure 4) Poliovirus infection of HeLa cells reduces the autoproteolysis of isolated 808 ribosomes markedly (not shown). The inhibition of HeLa cell ribosomal protease activity requires protein synthesis, but proceeds in the presence of guanidine (55) ... 

Figure 6. Left panel Protease activities in poliovirus-infected HeLa cells at various times after infection. Substrate was solid phase-linked poliovirus precursor polypeptides. Right panel Effect of multiplicity of infecting virus on protease activity in poliovirus-infected cells (33) ...

Figure 8. Two-dimensional analysis of proteins in infected HeLa cells. HeLa cells were labelled for 24 hours with (14c) amino acids, washed thoroughly, then infected with poliovirus type 1 Mahoney. Samples were taJcen at sixty min intervals, lysed with 8 M urea, and analyzed by two-dimensional electrophoresis. The first gel was 5% polyacrylamide, 8 M urea, 279 ampholine, pH 5-8, Samples were separated at 500 volts, overnight at 6-7. The gels were sliced, and placed on SHS-polyacrylamide gradient gels, and electrophoresed for 4 5 hours at 20 mA/gel, Gels were stained, dried, and autoradiographed. Gel A uninfected cells Gel B infected with poliovirus for five hours.

Figure 9 Polyacrylajnide SBS gel analyses of poliovirus cytoplasmic polypeptides. Infected HeLa cells were labelled with (55s) methionine, in the presence or absence of carbobenzoxy leucyl chloromethyl ketone. After an appropriate chase period, cells were lysed and proteins resolved. Gel sample identification 1-vii us control 2-addition of carbobenzoxy leucyl chloromethyl ketone (O.l mM) 3-3 emove excess chloromethyl ketone, add 100 jj,g/ml cycloheximide, chase 60 min 4-same as sample 3, but no cycloheximide present. The chloromethyl ketone was supplied by J. Powers, Georgia Institute of Technology, Atlanta, Georgia.

Figure 11. Protease activities detected in extracts of HeLa cells infected with either poliovirus type 1 (Mahoney) or defective-interfering mutants, the latter supplied by A. Nomoto and E. Wimmer, SUNY, Stony Brook, New York.

RNA is the only structure involved in sjnithesis of viral single-stranded progeny RRA (28) ( 52p) j isolated from poliovirus infected HeLa cells (30) and analyzed for the presence of VPg. 

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