Genetic analysis of adenovirus type 2 IV. Coordinate regulation of polypeptides 80K, Illa, and V

76, 709-724 (1977)

VIROLOGY

Genetic IV. Coordinate

JOSEPH Dhpartement

WEBER,’

de Microbiologic,

Analysis

of Adenovirus

Regulation of Polypeptides

MICHEL UniversitP

BEGIN,2

de Sherbrooke, Que’bec, Canada

Accepted

JlH

September

Type 2 80K, Illa, and V

AND ERIC Centre Hospitalier 5N4

B. CARSTENS Universitaire,

Sherbrooke.

15,1976

An adenovirus type 2 temperature-sensitive mutant, ts3, deficient in virion assembly was studied by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electron microscopy. A total of 28 virus-induced polypeptides could be detected, four of which were precursor proteins. At the nonpermissive temperature, ts3 failed to synthesize a newly identified nonstructural polypeptide 80K. Polypeptide V was absent, but it may have been replaced by an arginine-rich polypeptide which was oversynthesized and migrated as a 50K band regardless of temperature. Similarly, polypeptide 55K (= IVa,) was also oversynthesized and migrated slower than WT-55K (= 1Va.J. In addition, several polypeptides were synthesized at increased or decreased levels, compared with WT. Polypeptide IIIa was sometimes resolved into three bands (66K, 67K, 68K) in WT, while ts3-IIIa lacked the 66K and 68K components. Polypeptide 36K was found to be rich in arginine. Consistent with the notion that the processing of PVII into VII requires virion assembly, no processing of ts3-PVII could be observed. By contrast, the cleavage products, VI and VIII, appeared normally, thus disputing the virion assembly requirement previously postulated for these polypeptides. Electron microscopy of ts3-infected cells revealed two hitherto undescribed intranuclear structures, possible tubular forms in addition to normal virions at the permissive temperature, and roughly spherical, core-like structures of 80-100 nm at the restrictive temperature. These results suggest that the pleiotropic effects of the ts3 mutation arrest virus assembly at a corelike structure. INTRODUCTION

Adenovirus type 2 (Ad2) has a doublestranded DNA genome of 23 x 106 daltons theoretically capable of coding for 1.2 x lo6 daltons of protein or for approximately 3050 polypeptides. The virion contains at least 14 polypeptides (Ishibashi and Maizel, 1974; Everitt et al., 1975; Weber, 1976); 6 of these (II, III, IIIa, IV, V, IX) appear to be primary gene products (Anderson et al., 1974; Lewis et al., 1975; Oberg et al., 19751, while 7 (IVa, or 55K, VI, VII, VIII, X, XI, XII) seem to be cleavage products from I Author to whom reprint requests should be addressed. 2 Present address: Departement de Microbiologic, Faculte de Medecine Veterinaire, Universite de Montreal, C.P. 5000, St. Hyacinthe, Quebec, Canada.

higher molecular weight precursor proteins (Weber, 1976). Virion polypeptide IVa, cannot as yet be classified in this way. Of the remaining 18 polypeptides reported in the literature, 6 (44K, 15K, 72K, 15.5K, 19K, 11K; Lewis et al., 1976) to 13 (72K, 67K, 60K, 42-50K, 35K, 26.5K, 19K, 18.5K, 17.5K, 14.5-16K, 14.5K, 12.5K, 10.5K; Saborio and Oberg, 1976) are detected early in infection by selected early mRNA programmed cell-free protein synthesis or in uiuo synthesis, while 5 (lOOK, 95K, 50K, 14K, 11K) are observed. late in lytic infection (Lewis et al., 1975; Oberg et al., 1975; Anderson et al., 1974; Weber et al., 1976). Although the functions of some of these virion and virus-induced proteins are beginning to be elucidated (Levine et aZ., 1974; Everitt et al., 1975; Marusyk et al., 1975; Robinson and Bel709

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN

0042-6822

710

WEBER,

BEGIN

lett, 1974), which remains to be learned. This paper is part of a series which attempts to study the Ad2 polypeptides in terms of their function through the use of temperature-sensitive mutants. We have previously reported that the ts3 mutant of Ad2 was temperature sensitive for the formation of virion particles (Weber et al., 1975). In this communication we examine the phenotype of ts3 in detail. We find that the virion polypeptides IIIa and V are either absent or modified. A new virusinduced nonstructural polypeptide 80K, is shown to be completely missing in ts3infected cells. Further quantitative perturbations of virus-induced polypeptides will also be documented. (These results already form part of a report presented at the Cologne workshop on the Molecular Biology of Adenoviruses, February 1976). MATERIALS

AND

METHODS

Cells and virus. KB, CV1, or HTC (SV40-induced hamster tumor cell line) cells were grown in monolayer culture in Dulbecco modified minimal essential medium (DMEM). Wild-type (WT) adenovirus type 2 and ts3, referred to as ts235 in paper I of this series (Begin and Weber, 1975), were propagated and purified as described previously (Weber, 1976). SV40 was the generous gift of Dr. Christopher Lomax of this department. Marker virus for the electrophoresis experiments was prepared by labeling tsl grown at 33” in the presence of 25 &!i/ml of 13Slmethionine. The polypeptide composition of this virus consists of the precursor proteins, Va, Vb, and PVII, in addition to the normal virion polypeptides (Weber, 1976). Labeling of infected cells. Confluent monolayers of cells in petri dishes or Linbro plates were infected with virus in 50 or 25 ~1 of Tris-buffered saline (TBS), respectively. Mock infections consisted of TBS only. The cultures were gently agitated at intervals for 1 hr at 33”, then DMEM containing 2.5% calf serum and 0.4 mM arginine was added and incubated at the Labeling with stated temperatures. [35S]methionine (25 PCilml) (about 250 Ci/ mmol, Amersham-Searle), was performed

AND

CARSTENS

in DMEM, containing l/20 the normal concentration of methionine. Labeling with ]3Hlglucosamine consisted in simply adding 10 #Nrnl (2.4 Ci/mmol) to complete DMEM, while in the case of [14C]arginine, 25 $Zi/ml (277 Ci/mmol) was added to DMEM which did not contain the extra 0.4 mM arginine. Unless otherwise stated, the cells were labeled for 2 hr at 33” and 1 hr at 39”. At the end of the pulse, the cells were rinsed once with TBS and the culture was either lysed immediately or chased by the addition of complete medium containing a loo-fold excess of cold methionine (3 mg/ ml). At the end of the pulse or chase, either the cells were collected into 0.1 ml of sample solution (0.05 M Tris, pH 6.8; 1% SDS (Matheson-Coleman); 10% glycerol; 0.1% mercaptoethanol; 0.001% phenol red), or an equal volume of 2x sample solution was added to the entire culture (cells and medium), then the samples were boiled for l-2 min and stored at -20“. Prior to electrophoresis, the samples were thawed and briefly dipped into boiling water. Cell fractionation. The cells were washed twice with TBS and once with distilled water. One-tenth milliliter of 0.1% Tween-80 in Tris-EDTA buffer 0.01 M (pH 7.6) was added to the cell pellet and vigorously agitated on a Vortex. The nuclei and cytoplasm were separated by centrifugation for 30 set in a Beckman B microfuge. The cytoplasmic fraction in the supernatant was lyophilized and dissolved in SDS sample solution. The nuclear pellet was resuspended in Tris-EDTA buffer, 0.5 n-&f (pH 7.5), in a final concentration of 0.1% Triton X-100 and 0.3 mg/ml of phenyland incubated methylsulfonylfluoride, overnight at 4”. These Triton-washed nuclei were centrifuged as above, and dissolved in SDS sample solution. SDS-Polyacrylamide

gel electrophore-

sis. The gel system described by Maize1 (1969) was used. The gels (0.75 mm thick, 10.5 cm high) were formed in the Hoefer apparatus (Hoefer Scientific, San Francisco), consisting of a 12.5% resolving gel and a 5% stacking gel with an acrylamidebisacrylamide ratio of 30:0.24 and 30:0.8, respectively. The buffer system consisted of Tris-HCl (pH 8.9). Some gels were also

REGULATION

OF ADENOVIRUS

run without the stacking gel with apparently equally good resolution, The slabs were generally run at 30 mA/slab, stained with Coomassie brilliant blue, destained, dried in UUCUO, and autoradiographed with Kodak RP-oxomat X-ray film. Some of the gels containing low 3sS counts per minute or tritium were treated with Z,&diphenyloxazol (PPO) as described by Bonner and Laskey t 1974) and exposed at - 85”. Electron microscopy. Infected cells were incubated at 33 or 39”, washed, and fixed using a modification of a previously described method (Carstens and Marusyk, 1975). Fixation was carried out in situ at 4” with 3.5% g~utaraldehyde in phosphate buffer. The cells were then scraped off the petri, pelleted, and postfixed in 1% os-

2 PROTEINS

711

mium tetroxide for 1 hr. Samples were dehydrated in acetone, embedded in Epon 812, and polymerized at 80”. Thin sections were stained with uranyl acetate followed by lead citrate. RESULTS

Time Cottrse of ~s~-~~du~~d Protein thesis

Syn-

Preliminary experiments have indicated that the course of infection progresses differently in ts3-infected cells at the nonpermissive temperature. To examine this observation in terms of viral ~l~ptide synthesis, KB cells were infected with WT or ts3 at 39” and pulse-labeled with [““Slmethionine for 1 hr at various times

20 ts

WT

ts

WT

ts

WT

M

I”

WT

ts

I

FIG. 1. Comparison of the time course of WT and ts3 polypeptide synthesis. Infected cells at 39” were pulse labeled for I hr with 13”S]methionine at 9, 11, 13, 20, and 30 hr postinfection. The cultures were solubilized in SDS sample solution and analyzed by SDS-polyaerylamide gel electrophoresis on 12.5% slabs as described in Materials and Methods. M, mock-infected cells; V, consists of purified tsl virions as marker. The points to the right of bands indicate polypeptides of interest.

712

WEBER,

BEGIN

AND CARSTENS

after infection. Following the labeling, the cells were immediately lysed in SDS sample solution and later analyzed on SDSpolyacrylamide slab gels (Fig. 1). The results show a number of differences between ts3 and WT: (a) polypeptides PVII and IX are consistently present in greater amounts in ts3 than WT; (b) a new polypeptide band, 22K, is present in ts3 during the middle of the infection cycle (at 20 hr);

(c) polypeptide V is greatly reduced at 20 and 30 hr, while 50K is greatly increased very early (11 hr); (d) polypeptide IIIa is reduced early and in the middle phase; (e) polypeptides 95K and 11K are greatly increased at 20 hr, and 8 polypeptide 80K, a newly observed, infected cell-specific component, is absent throughout the infection cycle (see Table 1 for an inventory of Ad2 polypeptides). The ts3 mutation thus re-

TABLE Ad%INDUCED

Band designation”

Molecular weightb

PROTEINS

DETECTED

36K

120,000 100,000 95,000 85,000 80,000 72,000 66,000 62,000 60,000 55,000 50,000 48,500 46,000 36,000 32,000 26,000 24,000 23,000 20,000 19,000 18,500 14,000 13,000 12,000

LATE

Fatet;ieT:ypPulse

II 100K (IIal 95K III 80K 72K IIIa IV IVal (IVal IVa2 (55K IVb) 50K V 46K

1

+ + + + + + + + + + + + + + + + +

Chase’ I U D U U U U U U I U U I U U D D I D I I U I U D I I I

IN LYTIC

INFECTION

Remarks

Hexon Nonstructural Precursor protein” Penton base Nonstructural” DNA-binding protein’ PeripentonaP Fiber, glycosylated’ Virion component Virion component Empty shell virion component” Minor core protein DNA-binding protein’ Moves slower in chase”, ’ Minor protein” Presumed precursor to VI” Precursor protein” Ninemer Precursor to VII Cleavage intermediate of PVII Major core protein Nonstructural’s’ Ninemel” Nineme?’ Nonstructural”’ i Internal virion component”, * Internal virion component+ ” Internal virion componenCv* ”

32K Va (27K) Vb (26K) VI PVII (Via) (VIb) VII 14K VIII IX 11K 11,000 X 6,500 XI 6,000 5,000 XII u The polypeptides are designated as in Weber (1976); designations in brackets are synonymous terms used by other authors. 6 As determined previously (Weber, 19761. c The letters indicate the relative quantity of protein judged from autoradiograms as follows: I, increases; D, decreases; U, unchanged. d Weber (1976). c This paper. ‘Levine et al. (1974). u Everitt et al. (1975). h Ishibashi and Maize1 (1974). i Lewis et al. (1975). j Anderson et al. (1974).

REGULATION

OF ADENOVIRUS

veals a complex pattern of changes not entirely surprising in view of its inability to produce virion particles (Weber et al., 1975). Stability

of ts34nduced Polypeptides

The stability and processing of ts&induced polypeptides were measured by a typical pulse-chase experiment. WT, ts3, and mock-infected KB cells at 39” were 2hr 1 WT

PULSE TS

2hr MilWT

2 PROTEINS

713

pulse labeled for 2 hr with [35Slmethionine at 20 hr postinfection and either lysed in SDS sample solution immediately or after a 2- or 24-hr chase in excess cold methionine. The pattern of polypeptide bands obtained by electrophoresis is seen in Fig. 2. In addition to the changes already enumerated above for the pulse-labeled polypeptides, the superior resolution of this gel reveals that whereas WT-IIIa can be reCHASE TS

24hr MiIWT

CHASE TS

Ml

FIG. 2. Stability of ts3-induced polypeptides. Infected cells at 39” were labeled from 20 to 22 hr postinfection with [YSlmethionine and either solubilized with SDS or chased with 100x methionine for 2 or 24 hr. Approximately 3500 cpm of each sample was analyzed by SDS-gel electrophoresis. The dried gel was exposed for 54 days. M, mock-infected cells.

714

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BEGIN AND CARSTENS

solved into three bands (66K, 67K, 68K; also, see Fig. ll), the same region in ts3 only exhibits a single band (67K). Differences in this region have been repeatedly observed, yet due to the ambiguity of IIIa in the WT, interpretation of the mutant dictates caution. 80K remains consistently absent in the chase. 55K appears during the chase in greater quantity and moves slower than its WT counterpart. 50K is stable, while the small amount of V present in the pulse slowly disappears during the chase. Va (27K) appears to be processed normally into VI, while Vb (26K) diminishes in the long chase and VIII appears concomitantly. The cleavage of PVII into VII is almost completely inhibited, although the new 22K band is unstable, vanishing completely in the long chase. The nonstructural 11K polypeptide fails to disappear in the chase as it normally does in WT. In the region between V and Va, there are minor polypeptide bands which are reduced in ts3 during the pulse (in particular 36K and 32K), although one of them, 36K, reappears after the 24-hr chase. The significance of these bands, however, is at present difficult to assess. The principal differences distinguishing ts3 from WT appeared thus to reside in polypeptides 80K, IIIa, and V. To examine the possibility of rapid postsynthesis degradation of these polypeptides, the experiment was repeated using short labeling periods of 5, 10, and 20 min, followed by immediate lysis of the labeled cells. The gel patterns (Fig. 3) show clearly that 80K is absent and IIIa is reduced, even in the 5min pulse. This suggests that these polypeptides are either not synthesized at the normal rate or their rate of degradation exceeds the rate of synthesis. Both polypeptides 50K and V are labeled in these short pulses in ts3, while in the WT only V is labeled. These results are in agreement with those seen in the 2-hr pulse in Fig. 2, suggesting that polypeptide V is unstable in ts3. The effect of multiplicity on these proteins was also examined (results not shown) and we find the differences between WT and ts3 during pulse labeling gradually diminishing with increasing

MINUTES 5 ‘WT

ts

PULSED 10 fv’d-‘WT

20 ts fd

FIG. 3. Infected cell polypeptide synthesis during short-pulse labeling with [3”S1met. WT, ts3, and control cells (Ml at 39” were labeled for 5, 10, and 20 min at 20 hr postinfection and immediately lysed in SDS sample solution and prepared for electrophoresis. Each well was loaded with 1000 cpm and the gel was exposed for 42 days.

multiplicity of ts3 from 5 to 50 PFU/cell. During the chase, however, the mutant profile of ts3 is reestablished. Polypeptides 50K and 36K Are ArginineRich

As the above results show a pleiotropic pattern of temperature-sensitive effects, and in particular, the reduced synthesis and instability of basic protein V, we reexamined the polypeptide labeling pattern with [‘4Clarginine. WT and ts3-infected cells were labeled at 20-21 hr postinfection with either 25 &X/ml of [*4Clarginine or 25 &i/ml of [YS]methionine and prepared for SDS-polyacrylamide gel electrophoresis as usual. Following electrophoresis, the gel was impregnated with PPO, dried, and exposed at -85” for 7 and 20 days, as described in Materials and Methods. Figure 4 confirms the reduced synthesis of polypeptide V and the greatly increased synthesis of 50K, which is apparently also rich in arginine. The 36K and 22K polypep-

REGULATION

OF

ADENOVIRUS

715

2 PROTEINS

glycosylated. Since this polypeptide may be absent or modified in ts3-infected cells, we checked if it could be labeled with [3H]glucosamine. Figure 5 shows the labeling pattern obtained at 20-22 hr postinfection with WI, ts3, and mock-infected cells. It would appear that two bands are labeled in the WT IIIa-IV region, while only the faster moving band is clearly labeled in the ts3. Although this would confirm the reduction in the synthesis of ts3IIIa that we observe with the [Wlmethionine label, the poor resolution between IIIa and IV dictates caution in this interpretation. The increased radioactivity after the 24-hr chase indicates that glycosylation is not very rapid, the greater part of it taking place after the 2-hr pulse.

nm

c1

50-a 36-

-PVII 2hr Pulse

24hr Chase

UU-II

TS

WT

TS

WT

FIG. 4. Comparison of polypeptide labeling with [“Clarginine and [““Slmethionine of ts3 and WTinfected KB cells at 39”. The cells were labeled at 2021 hr postinfection and prepared for electrophoresis. The gel was impregnated with PPO and exposed at -85” for 7 days (left three columns) or 20 days (right three columns) as described in Materials and Methods.

tides, which have been noted above, are evidently also rich in arginine. Since 95K is not visibly labeled with arginine, we can exclude a precursor-product relationship between 95K and 50K, 36K. Glycosylation

of Adenovirus

Polypeptides

Ishibashi and Maize1 (1974) have shown that the fiber (IV) of adenovirus is glycosylated. Their separation of virus polypeptides on cylindrical gels could not have been expected to resolve the closely migrating polypeptides IIIa and IV. It has been suggested that IIIa may be important in neutralization and uncoating of adenovirus (Everitt et aZ., 1975). It would not be surprising if IIIa, like the fiber, were also

FIG. 5. Glycosylation of adenovirus polypeptides. WT, ts3, and mock-infected (M) cells at 39 were labeled with [“Hlglucosamine at 20-22 hr postinfection, and either solubilized in SDS sample solution, or washed and incubated for 24 hr in the absence of label. Approximately 2000 cpm were loaded in each well for electrophoresis. The gel was impregnated with PPO and exposed for 50 days. V consists of [z’;‘Slmet-labeled, purified tsl virions as marker.

716

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BEGIN

The glycosylation of polypeptide IV confirms Ishibashi and Maizel’s original observation (1974). In addition to polypeptide IV, and possibly polypeptide IIIa, we also saw labeling of IX, 22K, and a band which comigrates with IVa,. IVa, is a minor structural polypeptide which we have identified previously to appear after a chase in infected cells as 56K (Weber, 1976). The comparative oversynthesis of IV and IX of ts3 already seen with [35Slmethionine (Figs. 2, 3, 61, is also clearly visible here with [3Hlglucosamine. Although not easily discerned, we have repeatedly seen a [3Hlglucosamine-labeled band comigrating with the X-XII complex. Since this latter band appears only during a long chase, it could be identical with one or more of the X-XII complex.

3 C Pulse ? 33C Chase ,- 4hr II 46hr wTts

FIG. 6. Stability with WT and ts3 at (left panel), or first and chased for 0, 4,

MWTts

I ; M

AND

CARSTENS

Temperature-Shift

Experiments

In order to further explore the nature of the ts3 defect, a series of temperature shift-up and shit%-down experiments was conducted. Cultures infected at 39” were pulse labeled with [%]methionine at 2021 hr postinfection, then shifted to 33” and chased for 4 or 46 hr (Fig. 6, left panel). Alternatively, cultures infected at 39” were shified to 33” at 21 hr postinfection, then after 1 h pulse labeled from 22-24 hr postinfection and chased for 0, 4, and 43 hr (Fig. 6, right panel). In general, the polypeptides shown to be affected by ts3 remain affected whether synthesized at 39” and shifted down, or whether synthesized at 33” shortly after shifting the culture down from 39”. Under these conditions, a

39c 4 33c ,m, WTts

33C 4hr MWTts

Chase ,, 43hr MWTts

1 , M

and synthesis of polypeptides after temperature shift-down. KB cell cultures infected 39” were either pulse labeled at 20-21 hr, shifted-down to 33”, and chased for 4 or 46 hr shifted down to 33” at 21 hr and then labeled with [35S1met, from 22-24 hr postinfection and 43 hr (right panel).

REGULATION

OF

ADENOVIRUS

polypeptide in the region of 36K is very prominent in ts3 labeled at 33”, but appears to be unstable during the chase. Polypeptides 50K and IX return to a quantitatively normal rate of synthesis at 33”. Polypeptide V presents a special problem. It appears that in ts3, polypeptide V might be present and stable at 33” whether synthesized before or after the shift-down, if we assume this identity for the band migrating slightly slower than WT-V. That this may in fact represent polypeptide V is supported by the observation that during a 33” infection, we also see this slower migrating band (results not shown). It should be noted that in both temperature-shift experiments, the processing of Va and PVII is clearly visible. As we have shown before, the processing of P-VII into VII takes place during the maturation of “young virions” into infectious “old virions” (Weber, 1976). The synthesis of mature virions after the shift-down in ts3 thus may proceed normally. The present data are insufficient by themselves to warrant speculation as to the meaning of the observation that some ts3 polypeptides are affected even if synthesized after a shift to 33”. The Virion

Polypeptides

717

2 PROTEINS

WT ts -

-II IOOK

IVa

VIII -IX

of ts3

WT and ts3 virus labeled with [YSlmethionine at 33” was purified by CsCl centrifugation and analyzed by SDSgel electrophoresis (Fig. 7). Two important differences are visible between WT and ts3. First, as already suggested above, the polypeptide migrating slightly slower than WT-V is probably related to V. Whether this represents a somewhat longer precursor form of V or an otherwise altered V remains to be determined. Evidently this change in V does not alter the infectivity and may not even be related to the temperature-sensitive mutation causing the other observed changes. The second stable change is the altered migration of polypeptide IVa,. We have already shown above that 55K is oversynthesized by ts3 at 39 (see Figs. 2, 6). The following circumstantial evidence suggests that the cleavage product 55K and the structural polypeptide IVa, are identical: (a) the ts3 altera-

-XXI XII FIG. 7. The virion polypeptides of ts3. WT and ts3 grown in the presence of [““SIrnet at 33” was purified by one cycle of CsCl gradient centrifugation and analyzed by SDS-gel electrophoresis. Then 22,000 cpm were applied to each well and the gel was exposed for 5 days.

tion in mobility is observed in both the in uiuo 55K and the viral IVa,; (b) the molecular weight (Weber, 1976) and the stoichiometry of 55K and IVa, agree; (c) 55K is only observed in infected cells when virion assembly begins (unpublished results). Apparently the oversynthesis (Fig. 6) and the altered migration are changes which are present even at 33”, as in the case of polypeptide V. Distribution of Polypeptides lar Fractions

Since

particles

and

in Subcellu-

several

virus-in-

718

WEBER,

BEGIN

duced polypeptides are not produced in ts3infected cells, we examined the integrity of protein transport and compartmentalization. WT, ts3, and mock-infected cells at 39 were labeled at 20-21 hr postinfection with [35Slmethionine and processed or chased for 24 hr. The nucleus and cytoplasm were separated with Tween-80. The nuclear fraction was treated with 0.1% Triton X100 in the presence of 300 pug/ml of phenylmethylsulfonylfluoride (PMFS), an inhibitor of proteolytic enzymes, for 18 hr at 4”. The incubation mixture was clarified by centrifugation in the Beckman microfuge for 20 set, and the Triton-wash supernatant and nuclear pellet were subjected to SDS-gel electrophoresis. The cytoplasmic Cytoplasm Ihr Pulse 24hrChase ‘WT ts M”WT ts M’

AND

CARSTENS

fraction in the 1-hr pulse contains mostly cellular proteins, while the 24-hr chase contains principally polypeptides II, lOOK, III, IV, and IX (Fig. 8, left panel). The Triton-washed nuclei show the differences already enumerated between WT and ts3. It is evident that lOOK, 95K, 72K, 36K, Vb, P-VII, and 11K are present specifically in the nucleus. 80K is thus localized in the nucleus and, as usual, is absent in ts3. Similarly V is also absent in ts3, being replaced by the slower migrating 50K. Under these conditions, IIIa could not be resolved from IV. Interestingly, the normal processing of 95K is efficiently inhibited by PMFS in the WT, while in ts3, 95K is not recovered in either the nuclear or cytoplas-

Nuclei Pulse “mts

Triton-wash Chase

Pulse Chase --WT ts M WT ts

M

FIG. 8. Subcellular distribution of virus polypeptides. Infected and control cells were pulse labeled with [Yllmethionine at 20 hr postinfection and some cultures were chased for 24 hr subsequently. The nucleus and cytoplasm were separated by treatment with 0.1% Tween-80 for 30 min at room temperature and centrifuged, and the supernatant, containing the cytoplasmic fraction, was applied on a gel, while the pellet was extracted with 0.1% Triton X-100 containing phenylmethylsulfonylfluoride (300 pg/ml) for 18 hr at 4”. The mixture was centrifuged and the supernatant and nuclei-containing pellet were analyzed on SDS gel by applying about 3000 cpm per well. The gel was impregnated with PPO and exposed for 30 days. V consists of [35Slmethionine-labeled, purified tsl virions as marker.

REGULATION

OF

ADENOVIRUS

mic fractions. The absence of 95K in ts3 is all the more remarkable since the analysis of total cell proteins has shown it to be over-synthesized in ts3. The inhibitor, PMFS, also efficiently prevents the appearance of cleavage products VI and VII. The Triton washing of the nuclei apparently solubilizes very little radioactivity. Since the same polypeptides are present as in the cytoplasmic fraction, it is possible that these represent contaminants on the external surface of the nuclei, rather than solubilization from the interior of the nucleus itself. Complementation

of ts3 by SV40

The restricted growth of adenoviruses in monkey cells can be facilitated by coinfection with SV40. Although the defect has been identified to be at the level of translation, the exact mechanism remains unknown (Eron et al., 1975; Fox and Baum, 1974; Hashimoto et al., 1973; Klessig and Anderson, 1975). Some ts mutants of Ad5 have been shown to be complemented by SV40 in monkey cells at the nonpermissive temperature (Williams et al., 1974; Levine et al., 1974). To throw additional light on the nature of the ts3 defect, appropriate complementation experiments were performed in CV, cells. Table 2 shows that SV40 complements the ts3 defect very efficiently at the nonpermissive temperature: This implies that the function defective in ts3 is the same one which SV40 normally supplies, thereby allowing the synthesis of the late adenovirus proteins. Complementation by integrated information in T-antigen-positive SV40 tumor

cells (HTC) was also tested. No complementation was observable (Table 2) with either WT or ts3. Coinfection of HTC cells with SV40 and WT or ts3 was also negative. This is not surprising as SV40 fails to grow in HTC cells. These results indicate that the expression of late SV40 functions are required for the complement&ion of adenovirus. Levine et al. (1974) have also found that SV40-transformed hamster cells failed to complement the early mutant, 5ts125. Electron

Virus

ts3 ts + sv40 WT WT + SV40

OF ts3

KB

Microscopy

Figures 9 and 10 show the morphology of ts&infected cells at 33 and 39”, respectively. Several differences are apparent. The nuclei infected at 33” (Fig. 9a) exhibit a virus-induced tubular structure. They consist of two parallel filaments crossed railroad-track fashion by bars at regular intervals (Fig. 9b). The diameter of the tubes appears to be somewhat smaller than that of vu-ions. Similar structures have also been observed by Dr. Dennis Brown, Institute of Genetics, Cologne, in very late stages of infection with WT Ad2 (personal communication). Virus particles are not assembled at 39”, but an electron-dense, core-like structure of approximately 80-100 nm fills the nucleus of infected cells (Fig. lOa). Figure lob shows a higher magnification of these structures. In comparison with that at 33”, the cytoplasm at 39” appears to be filled with amorphous electron-dense material, which may represent untransported viral proteins. The material is always found massed in the vicinity of the nuclear mem-

TABLE COMPLEMENTATIONO

719

2 PROTEINS

2 IN KB,

BY SV40

33”

39”

-------------33”

2.4 x 10R 3.6 x 10’ -

9.6 x lo” 3.2 x 10” -

1.1 3.2 1.0 4.8

x x x x

CV-1,

OR HTCb

cv-1

5.7 2.8 4.1 4.1

x x x x

__..-HTC

------------_-33”

39 104 10’ 10” 10”

CELLS

10” 10’ 104 108

2.5 6.0 2.5 1.1

x x x x

39” lo4 lo4 10“ 1w

2.0 6.0 5.6 2.2

x x x x

lo” lo4 lo” 165

a Cells were singly infected with 2-5 PFU/cell or doubly infected with 2-5 PFUicell each of Ad2 or SV40 viruses, and the yields were titrated by plaque assay on KB cells and expressed as plaque-forming units per milliliter (PFU/ml). b HTC is an established line of cells derived from an SVQO-induced Syrian hamster tumor.

720

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AND

FIGURE

CARSTENS

9

REGULATION

OF

ADENOVIRUS

brane. Extensive vacuolation of the cytoplasm and a contracted appearance of the nucleus are typical features of ts3 infection at 39”. If the electron-dense material in the cytoplasm is indeed composed of viral proteins, this would appear to be in conflict with our finding (Fig. 8) that Tween-80prepared cytoplasmic supernatant contains little of viral proteins. We feel, however, that nucleus-cytoplasm separations should be viewed with caution in the face of these electron microscopy results. It may be concluded, however, that the electron-dense core-like structures, which, incidentally, are present in numbers expected for viral particles, represent the cores of the virus and the stage at which virus maturation is arrested in ts3. DISCUSSION

Figure 11 is a composite diagrammatic summary of the polypeptide pattern observed in ts3-infected cells at the nonpermissive temperature. Ts3 suffers from two major perturbations: (1) 80K, IIIa, and V are absent or altered; (2) 95K, IV, 55K (= IVa,), 50K, P-VII, and IX are synthesized in increased quantities (see Table 1 for an inventory of Ad2 polypeptides). The present results strengthen previous evidence which suggested that 95K is a nonstructural precursor protein which is cleaved into as yet unidentified products (Weber, 1976). 80K has been identified as a new, virus-induced nonstructural polypeptide which may not be essential for normal infectivity. Although not discussed by the authors, both 95K (Atkins et al., 1975) and 80K (Lewis et al., 1975) are visible minor components of in vitro-translated, preselected adenovirus mRNA. 80K appears to map onto EcoRI A fragment (Lewis et al., 1975). We have also identified infected cellspecific polypeptides 36K and 22K, both arginine-rich, and polypeptide 32K. Polypeptides 36K and 32K may be identical to two similar-size polypeptides frequently seen in overloaded polyacrylamide gels of top component particles (Weber, 1976; Ish-

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ibashi and Maize& 1974). Furthermore, 36K which we have shown to be a basic protein is probably identical to the 38K obtained by in vitro translation of selected mRNA and mapped on the adjacent EcoRI fragments F and D (Lewis et al., 1975). It may, in fact, also be identical to the 35K early polypeptide, recently identified by Saborio and Oberg (1976). Basic polypeptide 22K, present in ts3 in variable quantity, has occasionally been observed in WT-infected cells after a short pulse-labeling period; during a chase in WT it is quantitatively processed into VII. Based on this observation, we suggest that 22K is in fact a species of P-VII, the presence of which in ts3 is for some reason especially favored. Evidently, the failure of processing of P-VII, as shown by results obtained with tsl (Weber, 19761, is not sufficient for the accumulation of 22K. As noted previously (Weber, 19761, polypeptide 55K appears during the chase as a cleavage product of an unknown precursor protein. It corn&rates with virion polypeptide IVa,, suggesting identity. Surprisingly, IVa, appears in the in vitro translation system as a primary gene product (Lewis et al., 1975), implying a virus-coded protease distinct, at least in activity, from the protease acting on Va, Vb, and P-VII. In a previous communication (Weber, 1976), we reported the concomitant inhibition by tsl of the processing of Va, Vb, and P-VII into VI, VIII, and VII. The processing of these polypeptides, as well as the appearance of cleavage products X, XI, and XII, was shown to take place during the maturation of “young” virions into “old” virions. Ts3 has confirmed our hypothesis that the processing of P-VII into VII is dependent on particle assembly and thus represents true “maturation” cleavage. By contrast, the processing of Va and Vb and the appearance of VI and VIII is not dependent on particle assembly, as it appears to take place normally in ts3-infected cells. In this connection, it should be noted

FIG. 9. Electron micrograph of HEp2 cells infected for 72 hr with ts3 at 33”. (a) Note the tubular structures in the nucleus. (b) Higher magnification of the intranuclear tubular structures shown above.

722

WEBER,

BEGIN

AND

FIGURE

CARSTENS

10

REGULATION

OF

ADENOVIRUS

36 32,

VlbVII-

22 PVII

llX7=

FIG. 11. Composite idealized artist’s drawing of polyacrylamide gel pattern of WT and ts3-induced polypeptides in cells infected at the nonpermissive temperature. All the differences, which cannot be demonstrated in a single gel, are represented: polypeptides 80K, 68K, and 66K (components of IIIal, V, VIb, VII, X, XI, XII are absent; polypeptides 95K, IV, 55K, 50K, 22K, IX are present in amounts greater than in WT; polypeptide 36K is reduced. Although polypeptides X, XI, and XII were sometimes resolved in the WT (gels not shown), they were never seen in ts3.

that while ts3 produces a normal amount of hexon polypeptides, the amount of immunologically reactive hexon antigen is somewhat reduced (Weber et al., 1975). This is also true for the fiber. Since hexon and fiber are produced in great excess during adenovirus infection, while the nine-

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mer polypeptide VI is synthesized in limiting quantity, evidently sufficient hexon antigen is assembled to consume all available VI, thereby stabilizing it. Evidence, which will be published elsewhere, suggests that the stability of polypeptide VI is dependent on the assembly of hexon antigens. The absence of polypeptide V and presence of an equal amount of arginine-rich polypeptide migrating slightly slower than V, i.e., 50K, strongly suggest the possibility that we are in fact looking at a modified or larger “V” in ts3. That this might be the case is supported by the observation that this altered “V” is also observable at the permissive temperature as well as in ts3 virions synthesized at the permissive temperature. Alternative explanations are, however, also possible. The possibility that this might be the precursor to V is unlikely since V is obtained as a primary product in the in vitro protein-synthesizing system (Anderson et al., 1974; Lewis et al., 1975; Eron et al., 1975; Oberg et al., 1975). Shiroki et al. (1972) isolated Ad12 ts mutants, several of which failed to synthesize groups of two antigens. Thus, it was shown by serology and SDS-gel electrophoresis that pairs of capsid proteins, like fiber-penton-base, hexon-penton-base, or hexon-fiber, were not synthesized in some ts mutants. Similar results were reported for the fiber-hexon pair of antigens with Ad31 ts mutants (Suzuki et al., 1972). Assuming single mutations, this would lead us to conclude that regulatory mutations are involved. Although multiple mutations cannot as yet be excluded in ts3, the several perturbations observed also argue for the role of some sort of regulatory event which links these perturbations and, particularly, polypeptides IIIa, 80K, and V. The physical mapping of ts3 is in progress, and we hope that it will help to resolve the nature of the ts3 mutation.

FIG. 10. Electron micrograph of HEp2 cells infected for 55 hr with ts3 at 39”. (a) Note absence of typical virus particles and presence of virus-induced core-like particles. The cytoplasm contains electron-dense material not present at 33” or in WT-infected cells. (b) Higher magnification of the core-like structures.

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ACKNOWLEDGMENTS The technical assistance of Mr. Ken Harrison, Ms. Lise Imbeault, Ms. Halyna Marusyk, and Mr. Pierre Magny is gratefully acknowledged. M. B. was the recipient of a studentship from the Conseil de la Recherche Medicale du Quebec. A part of this work has been taken from a thesis submitted by M. B. for the partial fulfillment of the requirements for the Ph.D. degree. J. W. is Research Scholar of the Medical Research Council of Canada and recipient of a grant from the National Cancer Institute of Canada and Grant No. MT4164 from the Medical Research Council of Canada. REFERENCES ANDERSON, C. W., LEWIS, J. B., ATKINS, J. F., and GESTELAND, R. F. (19’74). Cell-free synthesis of adenovirus 2 proteins programmed by fractionated messenger RNA: A comparison of polypeptide products and messenger RNA lengths. Proc. Nat. Acad. Sci. USA 71, 2756-2760. ATKINS, J. F., LEWIS, J. B., ANDERSON, C. W., and GESTELAND, R. F. (1975). Enhanced differential synthesis of proteins in a mammalian cell-free system by addition of polyamines. J. Biol. Chem. 249, 6331-6338. BEGIN, M., and WEBER, J. (1975). Genetic analysis of adenovirus type 2. I. Isolation and genetic characterization of temperature-sensitive mutants. J. Virol. 15, 1-7. BONNER, W. M., and LASKEY, A. (1974). A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biothem. 46, 83-88. CARSTENS, E. B., and MARUSYK, R. G. (1975). An image analysis study of adenovirus type 5-induced crystalline inclusions. J. Gen. Virol. 29, 249-256. ERON, L., WESTPHAL, H., and KHOURY, G. (1975). Post-transcriptional restriction of human adenovirus expression in monkey cells. J. Viral. 15, 1256-1261. EVERITT, E., LUTTER, L., and PHILIP~ON, L. (1975). Structural proteins of adenoviruses. XII. Location and neighbor relationship among proteins of adenovirion type 2 as revealed by enzymatic iodination, immunoprecipitation and chemical crosslinking. Virology 67, 197-208. Fox, R. I., and BAUM, S. G. (1974). Post-transcriptional block to adenovirus replication in nonpermissive monkey cells. Virology 60, 45-53. HASHIMOTO, K., NAKAJIMA, K., ODA, K., and SHI~0~0, H. (1973). Complementation of translational defect for growth of human adenovirus type 2 in simian cells by a simian virus 40-induced factor. J. Mol. Biol. 81, 207-223. ISHIBASHI, M., and MAIZEL, J. V., JR. (1974). The polypeptides of adenovirus. VI. Early and late glycopolypeptides. Virology 58, 345-361. ISHIBASHI, M., and MAIZEL, J. V. (1974). The poly-

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