Mar 9, 1974 - W. C. RUSSELL, J. J. SKEHEL AND J. F. WILLIAMS ..... wish to acknowledge the expert technical assistance of Mrs Carol Newman, Mr John.
J. gen. Virol. (I974), 24, 247-259
247
Printed in Great Britain
Characterization of Temperature-sensitive Mutants of Adenovirus Type 5: synthesis of Polypeptides in Infected Cells By W. C. R U S S E L L AND J. J. S K E H E L
Division of Virology, National Institute for Medical Research, Mill Hill, London NW7 IAA AND J. F. W I L L I A M S
Institute of Virology, Church Street, Glasgow, WI (Accepted I9 March 1974) SUMMARY
Cells infected with temperature-sensitive mutants of adenovirus type 5 have been labelled with [asS]-methionine and extracts examined by polyacrylamide gel electrophoresis followed by autoradiography. With the exception of one group of mutants, cells pulse-labelled for short periods (< I h) showed similar patterns of polypeptide synthesis whether they were infected with wild-type virus or with the mutants. However, on longer labelling it was evident that certain polypeptides were relatively less well labelled in the cells infected with the mutants at the restrictive temperature. A pulse-chase experiment suggested that preferential degradation of these polypeptides was occurring presumably by a proteolytic surveillance mechanism which could recognize defective polypeptides. One group of mutants showed no synthesis of capsid polypeptides at the restrictive temperature, in agreement with the results of previous serological investigations.
INTRODUCTION
A series of temperature-sensitive (t~) mutants of adenovirus type 5 has been isolated (Williams et al. 1971) which can be assigned to 17 complementation groups (Williams & Ustacelebi, I97I; Russell, Newman & Williams, i972; J. F. Williams, P. Austin & H. Young, personal communication). Using serological techniques it was found that human cells infected with the various mutants at the restrictive temperature responded in different ways (Russell et al. I972), and four main groups were recognized on the basis of complement fixation tests. Cells infected at the restrictive temperature by mutants of group I showed no major differences in the pattern of production of the capsid antigens; those infe~ted with group II mutants showed no production of capsid antigens; those infected with mutants of group III showed no production of fibre antigen, while cells infected with group IV mutants showed limited production of hexon antigen and variable yields of penton base antigen. This latter group could be further subdivided on the basis of fluorescent antibody studies into mutants which showed no production of hexon antigen and others which showed production of the antigen but with an abnormal distribution in the cell. In cells infected with wild-type virus it has been shown (Russell & Skehel, I972) that the technique of polyacrylamide gel electrophoresis allied to autoradiography is a powerful tool in discerning events at the level of polypeptide synthesis. Thus the synthesis of the virus
248
W. C. R U S S E L L , J. J. S K E H E L A N D J. F. W I L L I A M S
particle polypeptides and at least five other infected cell-specific polypeptides (ICSPs) could be detected. A similar analysis of the polypeptides produced following infection with the ts mutants has been made and the results are reported in this paper. METHODS
Virus. Adenovirus type 5 (Ad 75), both wild type and the temperature-sensitive mutants, were propagated and titrated as described in previous publications (Russell et al. I967; Williams et aL I97t ; Russell et aL I972 ). General plan o f experiments. Cultures of HeLa or HEK cells in monolayers were grown in I oz bottles in Eagle's medium supplemented with antibiotics, Io ~ tryptose phosphate broth and Io ~o calf serum. (All the initial experiments utilized HEK cells, whereas HeLa cells were used in the later experiments. The results from the two cell types were essentially similar although it was noted that the 'switch-over' to later events apparently occurred sooner in the infected HeLa cells.) Cells were then infected by removing medium and adding 0"5 ml of virus to give an added multiplicity of infection of about so to 5o p.f.u./cell. After adsorption for 2 to 3 h, medium (without calf serum) was added to the monolayers to give a final vol. of 4 ml. The infected monolayers and the appropriate controls were incubated either at the permissive temperature of 33 °C or at the restrictive temperature of 38"5 °C. The cells were then 'pulse labelled' at various times after infection with [asS]-methionine as described previously (Russell & Skehel, I972). For the long labelling periods (i.e. > I h) the cells were labelled in Eagle's medium containing 5 ~o of the normal content of methionine (i.e. 4 #g/ml). Electrophoresis was carried out in polyacrylamide gels containing SDS followed by slicing and autoradiography as described elsewhere (Russell & Skehel, I972). Densitometer tracings were obtained with the Joyce-Loebl integrating microdensitometer or with the Unicam gel scanner and areas under peaks were quantitated where the conditions were such that the areas would be expected to be an index of labelling intensity (Fairbanks, Levinthal & Reeder, I965). In some cases gels were stained with Coomassie blue as previously described (Russell, Mclntosh & Skehel, I97i). Nomenclature. The terminology proposed by Ginsberg et al. 0966) for the capsid components (hexon, penton and fibre), by Russell et al. (197I) for the internal components (core-I and core-z proteins) and by Russell & Skehel (I972) for the ICSPs is used in this communication. RESULTS
Polypeptide synthesis in cells infected with wild type virus at 33 °C and 38"5 °C
Previous results have indicated that during the course of infection with wild-type virus there is a clear temporal control of polypeptide synthesis (Russell & Skehel, 1972). Early in infection, even in the presence of uninhibited cellular polypeptide synthesis, the synthesis of ICSP-3 can be detected. Later in infection, after virus DNA synthesis has commenced, the synthesis of the structural components of the virus and also of the other ICSPs can be observed, accompanied by inhibition of cellular polypeptide synthesis. The effect of different temperatures of incubation on these events was examined in the first series of experiments. H E K cells were pulse labelled with [asS]-methionine for 30 min at 8, 12, 16 and 24 h after infection at 33 °C and 38"5 °C. Immediately after labelling the cells were disrupted and the extracts analysed by electrophoresis and autoradiography. Densitometer tracings of the autoradiograms were obtained and four of them are shown in Fig. I. Examination of these indicated that the pattern of labelling of the various polypeptides at both temperatures was
Polypeptides of adenovirus ts mutants
249
16h
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/l/V1
./COre-I/1CSP-4
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A l 6h
33 C
f 24h 33 C
Fig. I. Densitometer tracings of autoradiograms after SDS polyacrylamide gel electrophoresis. Replicate cultures of H E K cells were infected with wild-type virus and incubated at 33 °C and 385 °C. At the post-infection times shown the cells were labelled for 30 rain with [zsS]-methionine, processed and electrophoresed.
similar to that in previous experiments at 37 °C but that the time of 'switch-over' to the later translational pattern was temperature dependent. Thus, the' early' polypeptide ICSP-3 is prominent in the sample of cells pulsed at 26 h after infection at 33 °C whereas at 38"5 °C, the 26 h sample contains relatively less labelled ICSP-3. By 24 h, the synthesis of this polypeptide is less pronounced at both temperatures. A quantitative assessment of these changes was obtained by determining the areas under the peaks corresponding to the various polypeptides and then expressing these as a percentage of the total area attributable to virus polypeptides (Fig. 2). By this method it can be seen that the capsid polypeptides were synthesized earlier at 38"5 °C than at 33 °C and that as the rate of their synthesis increased there was a concomitant decrease in the rate of synthesis of both ICSP-3 and core-I/ICSP-4
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W.C. RUSSELL, J. J. SKEHEL AND J. F. WILLIAMS I
30
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Fig. 2. Temporal appearance and relative stoichiometry of labelled polypeptides in infected HEK cells. The areas under the appropriate peaks in the densitometer tracings of the autoradiograms were computed and expressed as a percentage of the total area attributable to both structural and ICS polypeptides. polypeptide. (Since the electrophoretic mobilities of the latter two polypeptides were similar it was not possible to differentiate them clearly in all cases.) In these experiments it was noted that core-2 polypeptide synthesis increased relative to the others as infection proceeded. In other experiments (e.g. see Fig. 4, Russell & Skehel, I972) it was clear that at later times core-2 polypeptide synthesis decreased relative to capsid polypeptide synthesis. Fig. 2 also shows that once capsid polypeptide synthesis begins then it continues at approximately the same rate for all the polypeptides. On the other hand, there is apparently a more complex temporal relationship between the synthesis of the core polypeptides and the ICSPs and this is masked to some extent by the background of cellular polypeptide synthesis (Bablanian & Russell, I974). The other notable feature of these results is the disparity between the relative amounts of the structural polypeptides which are synthesized compared to the corresponding polypeptides in the virus particle. Table T shows the relative stoichiometry of the structural polypeptides as determined by Coomassie blue staining and from autoradiograms of [asS]-labelled virus after SDS polyacrylamide gel electrophoresis, and although there are some obvious differences in the estimates presumably at least in part due to the differing~methionine contents of the individual proteins (Russell, I97I) it is still apparent that when compared to the structural components synthesized in the infected cell the capsid components are produced in considerable excess to that of the core-2 polypeptide and this component may thus be a limiting factor in virus maturation. In an attempt to provide a useful system to monitor polypeptide synthesis in cells infected with the ts mutants, preliminary experiments were carried out with cells infected with wildtype virus in which labelling with [35S]-methionine was carried out for periods up to 24 h at 33 °C and 38"5 °C. Unexpected differences in the relative labelling of the capsid poly-
Polypeptides o f adenovirus ts mutants
251
Table I. Relative stoichiometry of virus particle polypeptides as seen in purified
virus and in the infected cell r Coomassie staining
Structural polypeptide Hexon Penton base Fibre Core- 1* Core-z
Virus ' ~ Labelled with pS]-methionine
I00 II I2 29 9I
Infected cell polypeptides labelled for 30 min at: ~ 14 h at 38"5 °C
24 h at 33 °C
IO0 43 5o 33 zo
IO0 33 58 74 2I
IO0 lo I9 15 38
* In infected cells it is difficult to separate ICSP-4 and core-I satisfactorily. The areas under the appropriate peaks of the densitometer tracings were computed and expressed relative to the area corresponding to the hexon polypeptide as Ioo. Table 2. Relative stoichiometry of labelled capsid polypeptides in wild-type infected
cells, under different incubation and labelling conditions Relative stoichiometry r
Experiment
Labelling
Temp. (°C)
Hexon (H)
Penton base
Fibre (F)
H/F
I
18-42 h I8-42 h 18-42 h I8-42 h 30 min at 18 h 30 min at 42 h
33 38"5 33 38'5 38'5 38"5
Ioo Ioo loo IOO 1oo IOO
64 74 54 IO8 65 85
8I lO9 71 1I3 79 88
i'2 0'9 I "4 o'9 I"3 i'I
II
The values were derived as described in Table L peptides at the two t e m p e r a t u r e s b e c a m e a p p a r e n t . These are shown in T a b l e 2 where it can be seen t h a t in two separate experiments the labelling of the h e x o n p o l y p e p t i d e s at 38"5 °C is very m u c h r e d u c e d relative to fibre a n d p e n t o n base polypeptides. Shorter 3 o m i n pulses at the beginning a n d end o f the longer labelling p e r i o d s in p a r a l l e l infected cultures nevertheless i n d i c a t e d t h a t the relative s t o i c h i o m e t r y o f the labelling o f the p o l y p e p t i d e s u n d e r these conditions was similar to t h a t observed in the long pulse at 33 °C a n d o f the same o r d e r as the results in Fig. I. These findings suggested t h a t c a t a b o l i s m o f the h e x o n p o l y p e p t i d e was occurring d u r i n g the course o f infection. However, careful e x a m i n a t i o n o f the relevant d e n s i t o m e t e r tracings d i d n o t indicate t h a t cleavage o f the h e x o n p o l y p e p t i d e into any o f the recognized structural a n d I C S p o l y p e p t i d e s was occurring, a n d ' p u l s e c h a s e ' investigations d i d n o t a d d a n y further i n f o r m a t i o n on a possible m e c h a n i s m o f b r e a k d o w n or the p r o d u c t s o f d e g r a d a t i o n . I t was also n o t a b l e t h a t there was considerable v a r i a t i o n in the relative s t o i c h i o m e t r y o f labelling o f the capsid p o l y p e p t i d e s f r o m experiment to e x p e r i m e n t b u t nevertheless the general findings were consistent with the above.
Polypeptide synthesi~ in cells infected with the ts mutants H E K cells were infected with wild-type virus a n d with a n u m b e r o f temperature-sensitive m u t a n t s representing 12 o f the c o m p l e m e n t a t i o n g r o u p s a l r e a d y described (Russell et al. I97z). A s a convenient initial screening p r o c e d u r e the infected cells were labelled f r o m 8 to 36 h post-infection at 33 °C a n d 38"5 °C a n d electrophoresis a n d a u t o r a d i o g r a p h y carried
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W.C. RUSSELL, J. J. SKEHEL AND J. F. WILLIAMS I-5
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Fig. 3 a. For legend see facing page. out in the usual way. Fig. 3 shows the autoradiograms obtained and it can be seen that with the exception of ts 22 infected cells which apparently show less efficient' shut-off' of cellular protein synthesis, there is very little difference in the labelling patterns at 33 °C between those obtained from the mutants and a wild-type virus infection; on the other hand there are a number of differences apparent at 38"5 °C. In a number of cases the total labelling is much less, but more significantly, the relative stoichiometry of the various capsid polypeptides is altered at the higher temperature. For example, in cells infected with ts I7 and t s 13 the hexon and fibre polypeptides respectively appeared to be labelled significantly less than the other capsid polypeptides, when compared to the wild-type infected cells. It is also apparent that cells infected with the ts 36 mutant (complementation group N) do not make the capsid polypeptides at the restrictive temperature but appear to synthesize ICSP-3 polypeptide and possibly some other polypeptides and give a polypeptide pattern very similar to that noted with cells infected with wild-type virus in the presence of cytosine arabinoside (Bablanian & Russell, I974). Cellular polypeptide synthesis is not inhibited]n cells infected with this mutant suggesting that inhibition of cellular polypeptide synthesis is related to a late virus gene function. A more quantitative assessment of these differences
P o l y p e p t i d e s o f adenovirus ts m u t a n t s
253
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