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J. gen. Virol. (1985), 66, 2161-2170. Printedin Great Britain 2161 Key words: reovirus/translation/double-inJection The Regulation of Translation in Reovirus-infected Cells By A L B E R T O M U N O Z , M I G U E L A N G E L A L O N S O AND LUIS CARRASCO* Departamento de Microbiologia, Centro de Biologia Molecular, Universidad Aut6noma de Madrid and CSIC, Canto Blanco, Madrid-28049, Spain (Accepted 21 June 1985) SUMMARY The regulation of translation in reovirus-infected cells has been investigated by double-infection experiments. Different cell lines were able to translate uncapped encephalomyocarditis (EMC) virus mRNA and capped vesicular stomatitis virus (VSV) mRNAs both early and late during reovirus infection. These results are not fully in agreement with a previously suggested model in which, in the early phase of reovirus infection, only capped mRNAs are translated, whereas in the late phase the cells are modified to translate exclusively uncapped mRNAs. The observations that EMC virus and VSV shut down host protein synthesis more efficiently than reovirus translation in the late phase in double-infections indicate that competition for a messagediscriminatory factor may not be involved in the shut-off of host protein synthesis in these animal virus-infected cells. INTRODUCTION The regulation of translation in virus-infected animal cells has been a matter of intensive research during the last few years. In most animal systems studied, as for example picornavirusinfected cells, there is a potent inhibition of host protein synthesis after viral infection, followed by massive translation of viral mRNAs (Carrasco & Smith, 1976, 1980; Lucas-Lenard, 1979). In other instances, such as in some reovirus-infected cells, host and viral protein synthesis co-exist, with no apparent shut-down of host translation (Walden et al., 1981 ; Detjen et al., 1982). In encephalomyocarditis (EMC) virus-infected cells, inhibition of protein synthesis appears to be related to a modification of membrane permeability which leads to changes in the ionic conditions in the cells, with the result that translation of cellular mRNA is blocked, while viral protein synthesis is still favoured (Carrasco & Smith, 1976; Carrasco, 1977; Lacal & Carrasco, 1982). The suggestion that EMC virus mRNA is preferentially translated in infected cells because it is better able to compete for a limiting factor involved in protein synthesis has also been proposed (Golini et al., 1976). However, the possibility that competition is the cause of the shut-off phenomenon in EMC virus-infected cells has been ruled out for two main reasons: (i) shut-off still occurs even in the absence of viral mRNA production and (ii) if host mRNAs a r e replaced by more efficient viral mRNAs, then no inhibition of total protein synthesis should be observed (Steiner-Pryor & Cooper, 1973; Bablanian, 1972; Holland, 1964; Collins & Roberts, 1972; Carrasco & Smitfa, 1980; Mufioz & Carrasco, 1981). Competition has also been considered to operate in reovirus-infected SC1 cells. Indeed, in a series of publications, Thach and co-workers have developed a kinetic model to explain the behaviour of protein synthesis in reovirus-infected cells (Walden et al., 1981; Brendler et al., 1981 a, b; Godefroy-Colburn & Thach, 1981 ; Detjen et al., 1982). According to this model, viral and cellular mRNAs compete for a message-discriminatory component that participates in translation, before the mRNA binds to the 40S initiation complex. This component has been tentatively identified as initiation factor elF-4A (Ray et al., 1983). Evidence was obtained showing that reovirus mRNAs are poor initiators relative to host mRNAs. Thus, the following order in competition was established: EMC virus > host > reovirus mRNA. Therefore, it was proposed that the translation strategy adopted by reovirus is to overwhelm the cellular synthetic 0000-6557©1985 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 03:14:56 2162 M. MUI~IOZ, M. A. ALONSO AND L. CARRASCO a p p a r a t u s with a large excess of weakly initiating messages ( W a l d e n et al., 1981). In addition, it was o b s e r v e d that the degree of c o m p e t i t i o n b e t w e e n host and viral m R N A largely d e p e n d s on the ionic c o n c e n t r a t i o n in the cell-free system (Brendler et al., 1981a, b). C o n t r a r y to this view, Skup & M i l l w a r d (1980) h a v e suggested that the i n h i b i t i o n o f translation after reovirus infection o f L cells occurs by a m e c h a n i s m similar to t h a t already suggested for poliovirus. The m o d e l proposes t h a t the reovirus m R N A synthesized early in infection is capped, and the infected cells translate this k i n d o f m R N A efficiently, but not u n c a p p e d m R N A s . Late in infection, the viral m R N A p r o d u c e d is u n c a p p e d and it is efficiently translated because the protein-synthesizing m a c h i n e r y has b e e n modified in such a w a y t h a t only u n c a p p e d m R N A s are r e c o g n i z e d (Skup & Millward, 1980; Skup et al., 1981). H o w e v e r , the validity of the reovirus and poliovirus models has b e e n q u e s t i o n e d recently (Detjen et al., 1982; A l o n s o & Carrasco, 1982b). T o investigate these models and also to m e a s u r e to w h a t extent c o m p e t i t i o n plays a part in the regulation o f translation in reovirus-infected cells, we h a v e analysed p r o t e i n synthesis in reovirus-infected cells, s u p e r i n f e c t e d w i t h different a n i m a l viruses w i t h c a p p e d or u n c a p p e d m R N A s . C o n t r a r y to Skup and co-workers (Skup & Millward, 1980; Skup et al., 1981) and in a g r e e m e n t w i t h D e t j e n et al. (1982), the results o b t a i n e d led us to consider t h a t r e o v i r u s - i n f e c t e d H e L a cells are able to translate c a p p e d viral m R N A s late during virus infection. H o w e v e r , in reovirus-infected H e L a cells, no i n h i b i t i o n o f cellular protein synthesis is a p p a r e n t at the t i m e w h e n m o s t viral proteins are synthesized, w h i c h suggests that there is an excess o f translation c o m p o n e n t s and that no c o m p e t i t i o n b e t w e e n cellular and viral m R N A s is established in intact infected cells. M o r e o v e r , superinfection o f these cells w i t h E M C virus shuts d o w n cellular protein synthesis m o r e efficiently t h a n reovirus translation. T h i s is a n o t h e r a r g u m e n t against c o m p e t i t i o n playing a part in the shut-off p h e n o m e n o n , since, as suggested by W a l d e n et al. (1981), host m R N A s are m o r e efficient c o m p e t i t o r s t h a n reovirus m R N A s . METHODS Cells and viruses. HeLa cells and BHK-21 cells were grown in Dulbecco's modified Eagle's medium (E4D) supplemented with l0 % calf serum (E4D10 medium) and incubated at 37 °C in a 5 ~ CO2 atmosphere. Poliovirus type 1 was grown on HeLa cells in E4D medium supplemented with 2 ~ newborn calf serum (E4D2). Vesicular stomatitis virus (VSV) was grown in BHK-21 cells in the same medium. EMC virus was grown on L929 cells in a mixture of Eagle's medium, phosphate-buffered saline (PBS) and E4D medium (80 : 15 : 5) supplemented with 1 newborn calf serum. In all cases, the fraction obtained after removal of cell debris by low-speed centrifugation was used as the source of virus. Reovirus type 3 Dearing strain was purchased from the American Type Culture Collection. Virus infection, measurement and analysis of protein synthesis. Cells grown on 24-well plates (Falcon Plastics) containing 1 ml E4D10 were infected with the indicated virus at the m.o.i, described in each experiment. After 1 h incubation at 37 °C, the medium was removed and 1 ml E4D2 was added. Time of virus addition was considered as 1 h, and 0 time was taken as the time when the virus was removed. Incubation at 37 °C was continued until the labelling period. For this purpose, 0.5 ml methionine-free E4Dl medium and 5.4 gCi [35S]methionine (1100 Ci/mmol, 5.4 mCi/ml; Amersham) were added to the cells for a 1 h pulse. At the end of the pulse period, the ceils were washed with 1 ml PBS and dissolved in 200 gl 0.02 M-NaOH plus 1~ SDS and 200 gl sample buffer (62-5 mMTris-HC1 pH 6.8, 2 ~ SDS, 0.1 M-dithiothreitol, 17~ glycerol and 0.024~ bromophenol blue as indicator). Each sample was sonicated to reduce viscosity and heated to 90°C for 5 min. Ten ktl was applied to a 15~ polyacrylamide gel and run overnight at 30 V. Fluorography of the gel was carried out with 2,5-diphenyloxazole at 20~o (w/w) in DMSO. The dried gels were exposed using XS-5 X-ray films (Kodak). Ten gl more of each sample was precipitated with 1 ml 10~ TCA and filtered through GF/C glass fibre filters in a Millipore apparatus. Toluene POPOP PPO (2-5 ml) was added to the dried filters and the radioactivity retained was estimated in an Intertechnique scintillation spectrometer. Measurement of S6Rb÷ content. HeLa cells grown in E4D10 medium were placed in 280 gl of a mixture of methionine-free E4D1 medium :E4D 10 medium (3 : 1), 0.2 gCi 86Rb+ (1 mCi/ml; Amersham) was added, and the cells were incubated for 17 h at 37 °C. Virus infection was then carried out while the 86Rb+ concentration was maintained constant. At the times indicated, the cells were pulse-labelled with 0.14 gCi [35S]methionine. After 1 h incubation, the medium was removed and the cells were washed three times with 1 ml PBS, and 0.5 ml 5 ~ TCA was added to extract the 86Rb+ from the cells. The radioactivity of 0.4 ml of the TCA extract was determined by estimating the Cerenkov radiation in a liquid scintillation counter. The level of protein synthesis was estimated in parallel cultures as described above. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 03:14:56 - 2163 Translation regulation in virus-infected cells HeLa cells Time I 3 5 7 9 1| 1 3 1 5 1 7 1 9 2 1 2 3 I (h p.i.) 2 // |(a)' ' ' ~0~- ' ' o/~..,~,, o / . /, .... , \ °%, / ", ~,, × ",.__] ",{ -o,-- 4 L cells I Time x I 3 5 7 911131517192123 ~0 (h p.i.) I I t I I o I 04 tJ .E -12 3~ ~ ez :~ ,, ..6 30 :'" ~ ~o~O \ /o \ ,, o 'e 4 "--o._ ~ ~'. °~ 2 0 0 I 4 I 8 I I ° - - o - -o I 12 16 20 24 Time (h) Fig. 1. Kinetics of protein synthesis ( O ) and 86Rb+ content ( 0 ) in different reovirus-infected cells. Cells were grown on 24-well Linbro dishes. Time of reovirus infection (20 p.f.u./cell) was considered as - 1 h. Proteins were labelled by 1 h pulses with 5 IXCi [35S]methionine and processed as indicated in Methods. The S6Rb+ content was estimated in parallel cultures also as described in Methods. Bands corresponding to reovirus proteins (2, Ix, a) are indicated. (a) HeLa cells; (b) L cells. RESULTS Protein synthesis and 86Rb+ content in reovirus-infected cells The reovirus genome comprises ten different pieces of dsRNA, each one coding for a single protein (Joklik, 1981). These different proteins have been classified into groups according to size. Infection of HeLa cells with reovirus type 3 led to massive synthesis of reovirus proteins starting from about 6 h post-infection and continuing at very high levels for several hours. It is striking that infection did not lead to inhibition of total protein synthesis as occurs with most animal viruses, but instead total protein synthesis was stimulated almost 100~o compared to uninfected cells. Also, cellular protein synthesis continued unabated for long periods of time, indicating that cellular and viral protein synthesis co-exist (Fig. 1). However, infection of L cells by reovirus interfered with cellular protein synthesis from about 10 h post-infection, and from 15 h onwards the cell translated viral m R N A s exclusively (Fig. 1). Analysis of membrane permeability to monovalent cations in these cells indicated that a close parallel exists between variations in protein synthesis and modifications in S6Rb+ content. For HeLa cells, there was an increase in S6Rb+ until 11 h post-infection, followed by a gradual decline, but even at 20 h postinfection the S6Rb+ content was similar to that in uninfected cells (Fig. 1). Analysis of 86Rb+ uptake by very short pulses (15, 30 and 60 s) indicates a rapid increase in the uptake of this cation, which was maximal (250~ of control) at 11 h post-infection (results not shown). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 03:14:56 2164 M. MUI~IOZ, M. A. ALONSO AND L. CARRASCO (a) f Timep.i. reovirus (h) (b) I 16 17 18 19 20 21 23 24 I I 17 18 19 20 21 23 24 Fig. 2. Effect of VSV superinfection on protein synthesis in reovirus-infected HeLa cells. Cells grown on 24-well Linbro dishes were infected with reovirus (20 p.f.u./cell) at - 1 h. Superinfection with VSV (100 p.f.u./cell) was at + 15 h. Bands corresponding to VSV (L, G, N, NS, M) and reovirus (2, ~t, a) proteins are indicated. (a) Reovirus-infected cells; (b) reovirus-infected cells superinfected with VSV at +15h. Theoretically, the higher uptake of this cation should be compensated by a higher release of sodium ions, since at that time there is no loss of other cellular metabolites. The behaviour of reovirus-infected L cells was similar to that of HeLa cells during the first 13 h of infection. After that time, a drastic release of 86Rb+ occurred, accompanied by a cessation of synthesis of several reovirus proteins. However, the synthesis of other reovirus proteins was particularly resistant to these ionic modifications (Fig. 1). Protein synthesis in HeLa cells infected with reovirus and superinfected with VSV, poliovirus or E M C virus According to the model proposed by Skup and co-workers (Skup & Millward, 1980; Skup et al., 1981) for regulation of translation in reovirus-infected cells, a modification in the proteinsynthesizing apparatus late in reovirus infection takes place, thus preventing the cell from translating capped m R N A s . To test this, reovirus-infected HeLa cells were superinfected late in infection with VSV, a virus that possesses typical capped m R N A , after which the synthesis of VSV proteins was analysed. Fig. 2 shows that VSV m R N A s were translated in these doubleinfected cells, suggesting that the protein-synthesizing machinery is in fact able to translate capped m R N A s . As regards shut-offof protein synthesis by VSV in this system, it is worth noting that cellular m R N A translation was shut down before reovirus protein synthesis. Twenty h after reovirus infection, which corresponds to 5 h after VSV superinfection, no cellular proteins were made, and there was then a significant decrease in reovirus protein synthesis (Fig. 2). Since reovirus m R N A is a weaker competitor than cellular m R N A (Walden et al., 1981), this result does not support the idea that the interference of VSV with translation is carried out by competition with preexisting m R N A s . Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 03:14:56 Translation regulation in virus-infected cells 2165 Poliovirus added at + 1 h I Time p.i. reovirus (h) I Guanidine i + Guanidine I 34 f 1 567810 34567810 41! o I I I .~~...~ (a)_ ×2 o-- 6.1 I Poliovirus added at + 15 h ! 1 - Time p.i. I reovirus (h) 17 Guanidine 18 19 20212224 I J + Guanidine 17181920212224 I ~0 i 5 o\o,x,k 8 "11 k._._ ._= "~ 6 I • I °-"-r~o 7 9 [ I I 11 (b) I - 54 2 411 I I I 17 19 21 23 25 Time after reovirus infection (h) Fig. 3. Effect of guanidine on protein synthesis in reovirus-infected HeLa cells superinfected with poliovirus. Cells grown on 24-wellLinbro dishes were infected with reovirus (20 p.f.u./cell) at - 1 h, and superinfected with poliovirus (75 p.f.u./cell) at either (a) + 1 or (b) + 15 h. Cells were treated with guanidine (3 m~,l) where indicated from the beginning of poliovirus infection until the end of the labelling period. Bands corresponding to some poliovirus (NCPVla, NCPVlb, NCPV2, VPO, VP3) and reovirus (2, p., a) proteins are indicated. O, Untreated cells; Q, guanidine-treated cells. The translation of uncapped m R N A s in reovirus-infected cells was analysed by superinfection with two different animal viruses, poliovirus and E M C viruses. Both are thought to block protein synthesis by different mechanisms (Jen et al., 1980; Detjen et al., 1981). According to the model suggested for reovirus-infected L cells, the translation of reovirus uncapped m R N A s is blocked early during infection, and thus is only possible in the late phase (Skup & Millward, 1980; Skup et al., 1981). Fig. 3 and 4 indicate that this does not hold for picornavirus m R N A translation in reovirus-infected HeLa cells, because poliovirus and E M C virus m R N A s were translated both early and late during infection. Poliovirus protein synthesis is blocked in interferon-treated cells (Mufioz & Carrasco, 1983, 1984) and also by the presence of guanidine (Fig. 3) (Lacal & Carrasco, 1983). However, even though viral protein and R N A synthesis is profoundly inhibited under those conditions, the shut-off of protein synthesis still occurs (Mufioz & Carrasco, 1983; Mufioz et al., 1983). Analysis of the blockade by poliovirus of host and reovirus protein synthesis late in reovirus infection of HeLa cells and in cells treated with guanidine leads to a number of conclusions. First, both host and reovirus translation are blocked by poliovirus infection in control cells, but host protein synthesis is more sensitive. This raises the question that if reovirus and poliovirus block protein synthesis by a mechanism similar to that already suggested (Skup & Millward, 1980; Skup et al., 1981) why is reovirus protein Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 03:14:56 2166 M. MUI~OZ, M. A. ALONSO AND L. CARRASCO EMC virus superinfection h after reovirus infection Timep.i. EMC virus (h) 1 3 4 5 6 +llh +lh Control I I 13456 I I I 1 3456 A D E e, F Y I I I I I I I 2 I 3 I 4 5 I ~3 x ¢-~ o ¢x .=. 2-0 1 I ~ . 6 Time after EMC virus infection (h) Fig. 4. Protein synthesis in reovirus-infected HeLa cells superinfected with EMC virus. Cells grown on 24-well Linbro dishes were infected with reovirus (10 p.f.u./cell) at - 1 h, and superinfected with EMC virus (100 p.f.u./cell) at either + 1 (O) or + 11 (Ak)h. Cells infected with EMC virus only were taken as control (0). Bands corresponding to some EMC virus (A, D, E, e, ct, ),, H, I) and reovirus (2, ~t, a) proteins are indicated. synthesis inhibited? In addition, we know that E M C virus and poliovirus translation are compatible in double-infected cells (Alonso & Carrasco, 1982a). On the other hand, inhibition of poliovirus replication by means of guanidine still allows the shut-off of protein synthesis to take place. However, it is particularly clear in guanidine-treated cells that the inhibition of host protein synthesis occurs before the inhibition of reovirus protein synthesis. Moreover, not all reovirus proteins behave in the same way, because some of them are more resistant to inhibition than others. This result is difficult to explain if we assume that poliovirus destroys a factor involved in cap recognition, necessary for the translation of capped reovirus m R N A s (Rose et al., 1978). The pattern of translation after E M C virus superinfection was similar, i.e. E M C virus protein synthesis occurred early and late during reovirus infection of H e L a cells, and shut-offof host protein synthesis occurred before that of reovirus protein synthesis (Fig. 4). Three h after E M C virus superinfection of reovirus-infected H e L a cells (14 h post-infection) no host Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 03:14:56 Translation regulation in virus-infected cells 2167 EMC virus superinfection at + 1 h 1 HEp-2 I 3T6 I I Time p.i. 4689 reovirus (h) Vero I 4689 I BHK-21 I 4689 I L I 4689 I l 46810 : .... , . ~ - - y 7: - I EMC virus superinfection at + 11 h Time p.i. reovirus (h) HEp-2 r - - I 14161819 3# 3T6 [ - - ] 14161819 :-:~ Vero I I 14161819 BHK-21 i I 14161819 le~;' 'Z ..... ' L I I 14161819 IilU~ z # "I Fig. 5. Effect of EMC virus superinfection on protein synthesis in different reovirus-infected mammalian cells. Cells grown on 24-wellLinbro dishes were infected with reovirus (20 p.f.u./cell) at - 1 h, and superinfected with EMC virus (100 p.f.u./cell) at either + 1 or + 11 h. Bands corresponding to EMC virus (A, D, E, e, F, a, y, H, I) and reovirus (2, ~t, a) proteins are indicated. translation was evident, whereas reovirus protein synthesis continued unabated. This indicates once again that E M C virus does not block protein synthesis by competition for a messagediscriminatory factor. Comparison of different cell lines infected with reovirus and superinfected with E M C virus or V S V The inhibition of host translation after reovirus infection differed according to the cell type analysed. For some cells like HeLa (Fig. 1) or SC1 (Walden et al., 1981) there was hardly any inhibition of cellular protein synthesis, whereas for others like L cells or B H K cells (Fig. 1) the blockade of host translation was noticeable. The model suggested by Skup and Millward was based on experiments in L cells (Skup & Millward, 1980; Skup et al., 1981). It was therefore of interest to compare the behaviour of different cell lines infected with reovirus, as regards the translation of capped or uncapped m R N A s both early and late during infection. Since poliovirus only infects cells of simian origin, we chose E M C virus as representative of a virus with an uncapped m R N A and VSV to analyse capped m R N A translation in cells superinfected with these viruses. Fig. 5 shows that, on superinfection with E M C virus of a variety of cell lines Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 03:14:56 2168 M. MUI~IOZ, M. A. ALONSO A N D L. C A R R A S C O VSV superinfection at + 1 h HEp-2 I Time p.i. 4689 reovirus (h) 3T6 1 I Vero I I 4689 BHK-21 I L I - - ] 4689 4689 46810 --L --G --N --NS --M VSV superinfection at + 11 h I I HEp-2 I Time p.i. 14161819 reovirus (h) 3T6 I I 14161819 Vero I I 14161819 BHK-21 I I 14161819 L I I 14161819 I L N G~--- NS M Fig. 6. Effect of VSV superinfection on protein synthesis in different reovirus-infected mammalian cells. Cells grown on 24-wellLinbro dishes were infected with reovirus (20 p.f,u./cell) and superinfected with VSV (100 p.f.u./cell) at either + 1 or + 11 h. Bands corresponding to VSV (L, G, N, NS, M) and reovirus (,~, ~t, ~) proteins are indicated. previously infected with reovirus, expression of E M C virus proteins occurred early during infection. This leads to the conclusion that in none of the cell lines tested, including L cells, was there any restriction of translation of uncapped E M C virus m R N A . On the other hand, in some of the cell lines tested, such as L, HEp-2 and BHK-21 cells, no E M C virus proteins were detected late in infection. This might be either because this uncapped m R N A is not translated in these cells or, more likely, because viral replication cannot occur in these cells in the late phase of reovirus infection. Similarly, VSV protein synthesis was observed in all cell lines analysed early in infection at levels comparabLe to control uninfected ceils, but once again no VSV proteins were detected late during reovirus infection of L cells, whereas some inhibition was observed in HEp-2 and BHK21 cells (Fig. 6). These findings that capped m R N A s are translated late in infection in some cell lines and that uncapped EMC virus m R N A is translated early in reovirus infection in all cell lines tested do not support the idea of changes in the protein-synthesizing apparatus in every type of reovirus-infected cell, although it might operate in L cells if it is proven that the restriction of VSV replication is at the translational level. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 03:14:56 Translation regulation in virus-infected cells 2169 DISCUSSION Two different models have been put forward to account for the regulation of translation in reovirus-infected cells. One suggests that early during reovirus infection the infected cells preferentially translate capped mRNAs, whereas in the late phase only uncapped m R N A s are translated. The reason for this specificity could be that the protein-synthesizing machinery is modified perhaps in a way similar to that suggested for poliovirus-infected cells (Skup et al., 1981). Indeed, cell-free systems obtained late in infection from reovirus-infected cells translate uncapped m R N A s exclusively, a result very similar to that obtained for poliovirus-infected cells (Skup et al., 1981). However, the translation of capped viral m R N A s in intact poliovirus or reovirus-infected cells occurs during the course of superinfection (Alonso & Carrasco, 1982b). This suggests that the results in vitro cannot be directly equated with the situation in vivo. The other model suggested for the regulation of translation after reovirus infection indicates that viral and cellular m R N A s compete for a message-discriminatory factor which is present in the cell in limiting amounts (Walden et al., 1981; Brendler et al., 1981a, b; Ray et al., 1983). Although host m R N A s are better competitors than reovirus mRNAs, viral protein synthesis occurs because the viral m R N A s are made in larger quantities (Walden et al., 1981). If this were so, then superinfection with EMC virus or VSV should shut down reovirus protein synthesis more effectively than host translation. Clearly, the results obtained in doubly infected cells show that the contrary is true, i.e. host translation is more sensitive to inhibition than reovirus protein synthesis. In fact, after EMC virus or VSV superinfection of reovirus-infected cells, there was a drastic decline of total protein synthesis which is hardly explicable by weak m R N A s replacing stronger mRNAs, as the competition model suggests. As regards the possibility that reovirus-infected cells exclusively translate capped m R N A s early during infection and switch to a cap-independent mechanism at late times, the experiments reported in the present work suggest that this is not so for all cell lines analysed. Thus, mouse cells such as L and 3T6 cells efficiently translate uncapped EMC virus RNA early during reovirus infection. It might still be argued that the lack of ability of L cell extracts to translate uncapped m R N A only holds for reovirus m R N A s and not for picornavirus RNA. Besides, HEp-2, BHK-21 and L cells did not efficiently support the replication of EMC virus or VSV when they were superinfected late in the reovirus cycle. The argument that in these cell lines there is less VSV protein synthesis because these cells only translate uncapped reovirus m R N A s needs to be substantiated by experiments designed to measure the step of VSV replication inhibited late during reovirus infection of those cells. An interesting finding was that the 86Rb+ content paralleled the changes observed in protein synthesis both in HeLa and L cells infected with reoviruses. For L cells there was a marked decrease in total protein synthesis from 13 h post-infection, at the same time as a decrease in 86Rb+ ions occurred. This indicates a loss of potassium ions from the cellular cytoplasm and most probably entry of sodium ions. Curiously enough, the synthesis of some reovirus proteins was more resistant than others to these ionic modifications. The excellent technical assistance of Ms M. A. R a m o s is acknowledged. A.M. was the holder of a FIS fellowship. This work was supported by grants from C A I C Y T and Fundaci6n Cientifica de la Asociaci6n Espafiola contra el Cancer. REFERENCES ALONSO, M. A. & eARRASeO, L. (1982a). Protein synthesis in HeLa cells double-infected with encaphalomyocarditis virus and poliovirus. Journal of General Virology 61, 15-24. ALONSO, M. A. & eARRASeO, L. (1982b). 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