Genetic analysis of adenovirus type 2 VI. A temperature-sensitive mutant defective for DNA encapsidation

VIROLOGY

81, 126-137 (1977)

Genetic

Analysis

VI. A Temperature-Sensitive

of Adenovirus

Mutant

G. KHITTOO Dt!partement

de Microbiologic,

Defective

AND

Type 2 for DNA Encapsidation

J. WEBER’

lJniuersit6 de Sherbrooke, Centre Hospitalier Quhbec, JlH 5N4, Canada

Uniuersitaire,

Sherbrooke,

Accepted March 28,1977 H2ts4 is a mutant defective in a late function. Centrifugation experiments in C&l showed that at the nonpermissive temperature (39”) the mutant could produce only top components (TC). When examined in an electron microscope these TC appeared to be morphologically normal. Pulse-chase experiments revealed that proteins synthesized at 39” failed to assemble effkiently into virions when a shift-down to 33” was done immediately after the pulse. DNA encapsidation was completely inhibited when a chase was done at 39” prior to the shift-down to 33”. When [35Slmethionine-labeled polypeptides synthesized by infected cells were analyzed on sodium dodecyl sulfate-polyacrylamide gels it was found that at 39” ts4 was defective in the processing of PVII. The postcleavage polypeptides VII, VIII, X, XI, and XII were virtually absent. Temperature-shift experiments showed that processing was temperature sensitive in the shift-up experiments only. Electrophoresis of purified TC grown at 39” showed that the TC of ts4 differ from those of the wild type (wt) only in the slightly higher levels of polypeptides VIII, XI, and XII in the tsl-TC. Polypeptide X was absent from both wt and ts4-TC. INTRODUCTION

to be heterogeneous in length, varying from 15% of to full viral genome length. The incomplete particles do not arise from the degradation of complete virions (Rosenwirth et al., 1974). Rather, several authors have proposed on the basis of labeling kinetics, that the incomplete particles are intermediates in virus maturation (Sundquist et al., 1973; Ishibashi and Maizel, 1974; Weber, 1976). Unfortunately, the definite establishment of the role of incomplete particles is made difficult by the large excess of capsid proteins which are never assembled into virions (Green, 1962). Added to this difficulty, recent findings show’that some of the incomplete particles may be generated in vitro by the CsCl centrifugation and only partially resemble the putative in uioo particles as demonstrated by Ficoll gradient centrifugation (Edvardson et al., 1976). In this communication we examine the properties of ts4, a mutant which accumulates incomplete particles (referred to as top components) at the nonpermissive temperature. We demonstrate that the ts4

Incomplete particles of adenoviruses which band at lighter densities in CsCl than mature infectious particles have been described and partially characterized for several adenovirus serotypes (Smith, 1965; Maize1 et al., 1968; Mak, 1971; Sundquist et al., 1973; Wade11 et al., 1973; Schaller and Yohn, 1974; Burlingham et c-d., 1974; Rosenwirth et al., 1974). These particles band at lower densities than complete adenovirions when centrifuged in cesium chloride. Although the proportion of incomplete particles varies with the type of adenovirus, it has been estimated that these particles constitute more than 30% of all viral particles synthesized by Ad3 (Prage et al., 1972). It has been shown that the incomplete particles may contain viral DNA fragments (Wade11 et al., 1973; Burlingham et al., 1974). More recently (Daniell, 1976) the DNA contained in particles of low density was characterized and found ’ Author dressed.

to whom reprint

requests

should be ad126

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

ISSN 0042-6822

ADENOVIRUS

MUTANT

FAILS

block is effected in a late stage of maturation thereby preventing normal packaging of viral DNA. MATERIALS

AND

METHODS

Cells. HEp-2 or KB cells obtained from Flow Laboratories (Rockville, Md.) were grown in Dulbecco’s modification of minimal essential medium (DMEM) as described by Weber (1976). Experiments were carried out in confluent monolayers of cells grown in 5-cm Falcon plastic petri dishes or 24-well Linbro plates (Bellco Glass Incorporated, Vineland, N.J.). Virus. The wild-type (wt) Ad2 used in all experiments was the parental strain from which ts4 was isolated. Begin and Weber (1975) described the isolation and preliminary genetic characterization of the ts mutants of Ad2. The new nomenclature of these mutants was also described (Weber et al,, 1975); accordingly ts4 is the same as ts500. The stock of ts4 used in all experiments was obtained from a plaque isolate after two passages in KB cells. Virus infection. Infection was done on confluent monolayers of cells by adding 0.2 ml of virus suspension (in DMEM) per petri dish, while in Linbro plates the volume was 0.05 ml per well. The multiplicity of infection was maintained between 5 and 10 plaque forming units/cell. The inoculum was absorbed for about 1 hr at both 33 and 39” with frequent tilting of the dishes to enhance uniform distribution of virus over the cells. Mock infections consisted of DMEM only. Radioactive labeling. Labeling of the viral DNA with [3H]thymidine was done as described by Weber et al. (1975). [35SlMethionine was added to the virus- or mock-infected cells 20 hr postinfection (p.i.1 at 39” and 40 hr p.i. at 33”. Prior to labeling the cells were incubated in DMEM without methionine (0.5 hr at 39 and 1 hr at 33”). After that period the medium was replaced with fresh medium containing 20 &i/ml of ]35S]methionine (about 250 Ci/mmol, Amersham/Searle). The volumes of radioactive medium used were 0.05 ml for Linbro plates and 0.2 ml for petri dishes. The labeling period varied from 1 to 3 hr at 39” and 1.5 to 5 hr at 33”.

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At the end of the pulse the cells were rinsed with Tris-buffered saline and the cultures either lysed immediately or chased in medium containing a loo-fold excess of cold methionine (3 mg/ml). Virus and top components. Virus was purified from infected cells by freezethawing three times and sonication for 20 set, followed by low speed centrifugation (1500 rpm for 5 min) to remove the cell debris. The supernatant was extracted with trichlorofluoroethane (Freon 113, DuPont of Canada, Ltd.) as described by Green et al. (1967). The clear supernatant or part of it could then be layered on a preformed CsCl gradient (1.2 to 1.5 g/ml). Centrifugation was done in 12-ml polyallomer tubes using the B60 International ultracentrifuge and an SB283 rotor, for 1.5 hr at 35,000 rpm and 4”. After centrifugation, fractions were collected from the bottom of the tubes. The density of some fractions was determined through refractive index measurements. This allowed the separation of top components (1.30 g/cm31 from virions (1.34 g/cm3). For further purification the top component and virion bands were separately pipetted out, diluted to 4 ml in a buffer (0.05 M Tris-HCl, pH 8.11, layered over a CsCl linear gradient, and centrifuged for 1.5 hr as described above. Radioactivity of the fractions obtained was measured by transferring aliquots on fiberglass filter disks (Whatman GF-81) and washing with icecold 5% trichloroacetic acid followed by ethanol. The disks were dried and counted in a Beckman liquid scintillation spectrometer. Dialysis of the various bands in CsCl was done against a cocktail (10% glycerol; 0.01 M Tris-HCl, pH 7.4; 0.01 M MgCl*, and 0.5% of n-butanol) as described by Sundquist et al. (1973). After dialysis, samples required for electrophoresis were lyophilized. Electron microscopy. Dialyzed virions and top components were negatively stained on Formvar grids (stabilized with carbon) using 2% phosphotungstic acid, pH 7.0 to 7.2, and examined in a Philips 300 electron microscope. SDS-polyacrylamide-gel electrophoresis. Virus-infected or mock-infected cells

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growing in Linbro plates were lysed in the following way: The medium was removed at the end of the pulse or chase and 0.1 ml of SDS-sample solution was added (0.05 M Tris, pH 6.8; 1% SDS, Matheson-Coleman; 10% glycerol; 0.1% mercaptoethanol; 0.001% phenol red). About 3 min later the cells were scraped with the heat-blunted end of a Pasteur pipet, transferred to a small glass vial, and boiled for 2 min. When cooled, the samples were ready to be loaded onto the gel for electrophoresis. Lyophilized samples were also dissolved in the SDS-sample solution by boiling for 2 min. Both acrylamide and bisacrylamide were purchased from Eastman Kodak. The polyacrylamide-gel system used was essentially that described by Maize1 (1971). RESULTS

Production of Virions nents (TC)

and Top Compo-

When Ads-infected human cells are lysed late in the lytic cycle and the cell extract is layered over a CsCl solution and centrifuged, several light-scattering bands are observed (Ishibashi and Maize& 1974; Burlingham et al., 1974). The ability of ts4 to form such bands at the permissive and nonpermissive temperatures was measured as follows. Confluent monolayers of HEp-2 cells were infected with wt and ts4

AND

WEBER

in the presence of [35Slmethionine and cultured for 36 hr at 39” and 70 hr at 33”. Then the cells were lysed and the cell lysate was layered over a linear CsCl gradient (1.2 to 1.5 g/cm31 and centrifuged for 1.5 hr. The centrifuge tubes were photographed and fractionated and the cold TCA-precipitable radioactivity in each fraction was measured. Figure 1 displays the band patterns observed in the centrifuge tubes. It is clear that ts4 is unable to produce virions banding at 1.34 g/cm3 at 39”. Instead, there is an accumulation of TC (at density 1.30 g/ cm3). The wt virus gives rise to the same band patterns at both temperatures. It is to be noted that ts4 accumulates more TC than wt even at 33”. Figure 2 shows the radioactive profiles of wt and ts4 grown at 39” in the presence of ts4. The superimposed patterns confirm the inability of ts4 to produce virions at 39”. The DNA content of incomplete particles of Ad2 and Ad3 has been analyzed in detail (Burlingham et al., 1974; Daniell, 1976). To compare the DNA associated with TC of ts4 and wt viral bands produced at 39”, the infected cells were labeled with 13H]thymidine and then prepared for centrifugation in a CsCl gradient as usual. Figure 3 shows the distribution of 13Hlthymidine in both wt and ts4 viral bands. These results show that very little DNA is associated with TC of ts4. At 33 however, the same patterns of radioactive

FIG. 1. CsCl band patterns of wt and ts4 grown at 33 and 39”. HEp-2 cells infection of 5 to 10 PFUkell) at both temperatures. At 39” the cells were labeled (p.i.1 with [%Slmethionine (20 &i/ml) followed by a 13-hr chase. The cells at hr p.i., followed by a 25-hr chase. The virus was then harvested, layered over ml), and centrifuged for 1.5 hr (at 35,000 rpm and 4”, using a B60 International rotor).

were infected (multiplicity of from 20 to 23 hr postinfection 33” were labeled from 40 to 45 a CsCl gradient (1.2 to 1.5 gl ultracentrifuge and an SB283

ADENOVIRUS

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TO ENCAPSIDATE

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DNA

-

1.5

-

1.4 5

0

-13

En P. = YI

-12: 0

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LO

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FIG. 2. Radioactive profiles of wt and ts4 grown at 39” and centrifuged in CsCl. The labeling and centrifugation were done as described in Fig. 1. The centrifuge tubes were fractionated from the bottom and the TCA-precipitable radioactivity in each fraction was measured. Through refractive index measurements the density of every fifth fraction was determined. The arrows indicate the density regions where the virions (1.34 g/cm31 and the TC (1.30 g/ cm? banded. (A-A) Wild type; (A-A), ts4.

thymidine were found for both wt and ts4, with most of the radioactivity in the region of density 1.34 g/cm3 (results not shown). Electron

Microscopy

In an attempt to discern any morphological abnormalities in the TCs of ts4, samples from the CsCl gradients were examined under the electron microscope. Figure 4 shows the electron micrographs of the various particles examined. There do not seem to be any obvious morphological differences between the TC produced by wt and ts4 at 39”. Attempts

at “Curing”

ts4 TCs

In order to answer the question of whether the TC produced by ts4 at 39” were “dead ends” or could be chased into mature virus, a series of temperature-shift experiments was conducted. HEp-2 cells

FIG. 3. DNA associated with the viral bands in CsCl. HEp-2 cells, infected with wt and ts4 were labeled with 13H]thymidine (5 &i/ml at 15. hr postinfection) and grown at 39” for a total of 37 hr. The cell lysates were extracted with fluorocarbon and centrifuged in a CsCl gradient as usual. The tubes were fractionated and the TCA-precipitable radioactivity was measured.

were infected with wt and ts4 and incubated at 33 and 39“. At 20 hr postinfection, the cells at 39” were labeled for 3 hr with ts4 while those at 33” were labeled for 5 hr at 40 hr postinfection. After labeling, the radioactive medium was removed and some cells from both temperatures were frozen in Tris buffer. Fresh medium containing a loo-fold excess of methionine was added to the rest of the cells. The petri dishes incubated at 33” were transferred to 39” while the petri dishes incubated at 39 were transferred to 33”. At different times after the temperature-shifts, petri dishes were removed and the cells were frozen in Tris buffer. The cells were then lysed and centrifuged in a CsCl density gradient. The tubes were fractionated and the radioactivity in each fraction measured. The results of these experiments are shown in Figs. 5 and 6. In the shift-down experiments (Fig. 5) we note that, during the pulse, there is not much radioactive material at the viral or TC region in both wt and ts4. However, during the chase at 33” there is a large accumulation of virions in the wt, whereas only a very small amount of radioactive material sediments to the viral region of density 1.34 g/cm3. At all

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the cultures were chased for 13 hr. At the end of the chase at 39”, the shift-down experiments were carried out as described above, except that the centrifuge tubes were photographed before being fractionated. The pictures of the centrifuge tubes (Fig. 7) show that in ts4, as the length of the chase at 33” increases, the viral band at 1.34 g/cm3 intensifies although there is still a greater accumulation of the TC. In the case of the wt, the TC band remains stable while the virions accumulate in the proportion to the length of the chase. Figure 8 shows the radioactive profiles obtained by fractionating the centrifuge tubes. The striking feature is that the radioactive profiles of ts4 and wt are mirror images of each other. This indicates that only a negligible amount of virions formed during the shift-down of ts4 is radioactive. This implies that the viral bands observed during the chases at 33” are mostly “cold” virus synthesized de novo and that they did not arise from radioactive top components synthesized at 39”. Therefore it seems that once ts4 assembles TC at 39” they fail to encapsidate DNA even after a shift to the permissive temperature. SDS-Polyacrylamide-Gel FIG. 4. Electron micrographs of wt virus, wt-TC and tsCTC. Wild type and ts4 were grown at 39”. The virus and TC bands obtained after CsCl centrifugation were dialyzed and negatively stained with 2% phosphotungstic acid (pH 7.0 to 7.2). (A) Wild-type virus; (B) wt-TC; (C) tsb-TC. x 103,200.

times when ts4 was grown there was more material in the TC region than in the viral region. This indicates that ts4 is very inefficient in the production of mature virions once the TC are assembled at 39”. Figure 6 shows the radioactive profiles of the shiftup experiments. Although in ts4 there are some virions formed during the chase at 39”, there is a much larger accumulation of TC. This shows that the ts4 defect interferes with the assembly of mature viral structures even if synthesized at 33”. A modification of the temperature-shiftdown experiments was introduced to enable most of the labeled proteins to assemble into TC or virions at 39”: After labeling

Analysis

It has been established that cleavage of certain precursor polypeptides occurs late in maturation (Ishibashi and Maize& 1974; Edvardson et al., 1976). Further, we have previously described (Weber, 1976) a mutant of Ad2 (tsl) in which cleavage products VI, VII, and VIII and X, XI, and XII are absent although DNA is encapsidated. This prompted us to verify whether cleavage of the precursor polypeptides occurred in ts4. Pulse-chase experiments were carried out as described by Weber (1976). Fig ure 9 shows the autoradiogram of a pulsechase experiment at 39”. The results indicate that, during the pulse, identical polypeptide bands are obtained in wt and ts4, with the following exceptions: (i) Polypeptide VII is already present in the pulse to a small extent; (ii) incorporation into the cellular polypeptides is less suppressed in the ts6infected cells compared with the wt-infected cells. During the chase, however, polypeptides VII, VIII, X, XI, and

ADENOVIRUS

5

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MUTANT

25

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35

FAILS

TO ENCAPSIDATE

40

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DNA

20

25

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40

FRACTIONS

FIG. 5. Temperature shift-down immediately after the pulse. Cells were labeled for 3 hr at 39” (20 &i of [“S]methionine/mll and then transferred to 33” and chased for different lengths of time. The cells were then lysed and centrifuged in a CsCl gradient, and the radioactivity was assayed as usual. (O-0) Pulse at 39”; (0-O) chase at 33” for 4 hr; (0-O) chase at 33” for 24 hr. The arrows indicate the density of the particular regions.

XII fail to appear in ts4. It is difficult to distinguish polypeptide VI on these gels on account of its comigration with polypeptide Vb. But it is clear that the cleavage of certain precursors does not occur in ts4 at 39”. This experiment was repeated at 33” and the results showed that the polypeptide patterns of ts4 and wt were indistinguishable. Moreover, very similar results were obtained when KB cells were used instead of HEp-2 cells (data not shown). Temperature-Shift Experiments The fact that cleavage of several precursor proteins in ts4 does not occur at the nonpermissive temperature raises the question whether the precursors are so altered at 39” that the cleavage mechanism can no longer proceed. The second alternative is that it may be the cleavage mechanism that becomes nonfunctional in ts4 at 39”. The experiments conducted to eluci-

date this problem were similar to the temperature-shift experiments described for tsl (Weber, 1976). Figure 10 shows the results of the temperature-shift experiments using HEp-2 cells. Because polypeptides VI and X to XII were poorly resolved we shall concentrate on the appearance of polypeptide VII. During the shift-down, processing of PVII appears to occur to the extent that after a long chase (40 hr) PVII and VII are present in about equal amounts, whereas only a negligible amount of PVII is cleaved during the shiftup experiment. This suggests that the ts4 mutation is immediately expressed upon shift-up, whereas, in terms of this polypeptide at least, the tsCmediated inhibition of cleavage is largely removed by shifting down. Viral Particles and TC To answer the question

of whether

TC

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-

110

-

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A

90 "b x

EO-

E

70-

t‘

-

0 ; E

60-

CJt

50-

: 40

-

30

-

20

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. 5

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IS

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FRACTIONS

FIG. 6. Temperature shift-up immediately after the pulse. Cells were labeled for 5 hr at 33” (20 pC!i of [35Slmethionine/ml) and then transferred to 39” and chased for different lengths of time. The cells were treated as described in Fig. 5. (0-O) Pulse at 33”; (O-O) 4-hr chase at 39”; (0-O) 20-hr chase at 39”. The arrows indicate the density in grams per cubic centimeter.

39c

33c WT

16 Jr4

I WT

24 ,ts4

FIG. 7. Photograph illustrating band patterns obtained during a temperature-shift-down experiment. Wild-type and ts4 virus were grown at 39”, labeled for 3 hr (at 20 hr postinfection) with [YSlmethionine and chased in nonradioactive medium for 13 hr at 39”. Some of the infected cells were lysed and centrifuged in a CsCl gradient, while the rest of the infected cells were transferred to 33” where chases of different lengths of time were conducted (8, 16, and 24 hr). After the different chases the virus was harvested and centrifuged in a CsCl gradient as usual.

synthesized by ts4 at 39” have the same polypeptide composition as the TC of the wt, the following experiment was done: HEp-2 cells were infected with wt and ts4 at 39” and labeled with [35S]methionine as described earlier for the production of TC

and virions. The TC and virion bands were pipetted out of the centrifuge tubes, dialyzed, lyophilized, dissolved in the SDSlysing buffer, and submitted to electrophoresis. The results are shown in Fig. 11. Polypeptides V, PVII, and VII are absent

ADENOVIRUS

MUTANT

FAILS

TO ENCAPSIDATE

DNA

133 7

40

35

30 “b x 25 P a 0 20 z F $ 0

15

10

5

FRACTIONS

8. Temperature shift-down after a chase at 39”. The experimental conditions are described in Fig. 7. The centrifuge tubes were fractionated and the radioactivity measured as usual. (0-U) Virus grown at 39”; (O-O) 8-hr chase at 33”; (0-O) 16-hr chase at 33”. The arrows indicate the density in g/cm”. FIG.

from the TC of both wt and ts4. Nevertheless, the following differences were observed in the TC of ts4: There seems to be a more prominent band in the 14,000-MW region; polypeptide VIII is more intensely labeled; polypeptides XI and XII are present in significant quantities, whereas polypeptide X is completely absent in both ts4 and wt. Occasionally we noticed that polypeptide IIIa of ts4 migrates slightly more slowly than that of the wt. It appears, therefore, that tsCTC contain some polypeptides (VIII, XI, and XII) which appear in the wt only after packaging of DNA and a series of maturation cleavages (Weber, 1976). These results argue against a common precursor for polypeptides VIII and X and, by the same token, open the possibility that polypeptides VIII, XI, and XII may be derived from a single event if not a single precursor. DISCUSSION

This investigation was aimed at understanding the effects of the ts4 mutation and thereby gaining more information on the maturation process of Ad2. We have

demonstrated that ts4 fails to synthesize mature virus particles but, instead, accumulates empty shells termed top components (TC). It is to be noted that ts4 shows some mutant effect even at the permissive temperature, by accumulating a larger amount of TC than the wt. Knowing that ts4 synthesizes DNA normally at 33 and 39 (Weber et al., 1975), we believe the ts block is expressed at the level of viral DNA packaging. Examination of TC produced by ts4 at 39” under the electron microscope does not reveal any morphological defect when compared with the wt. This observation does not rule out subtle differences, such as evidenced by the polypeptide composition of tsCTC which contain increased amounts of polypeptides VIII, XI, and XII, as compared with wt-TC. The defect could therefore reside in the DNA-protein complex or some component of the DNA encapsidation mechanism. It has been suggested that TC are likely precursors of virions (Ishibashi and Maizel, 1974; Rosenwirth et al., 1974). More recent experiments, using gentle extraction methods have identified at least three steps in adenovirus assembly; intermedi-

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Pulse 1

I

3

Chase , 10

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Mock ,

26

WEBER

Pulse 1

3

Chase , 10

,

26

95K 72K

VII VIII

FIG. 9. SDS-polyacrylamide-gel autoradiogram of wt-, ts4-, and mock-infected infected cells were pulse-labeled with [YS]methionine for 1 hr (20 hr postinfection) active medium for 3, 10, and 26 hr. We note here that during the chase polypeptides fail to appear in ts4.

ates, young virions, and mature virions (Edvardson et al., 1976). Nevertheless, we showed that once ts4 produces TC at 39” it is not possible to “cure” them by a shiftdown to 33”. The de nouo synthesis of virions proceeds normally, however. It was surprising to find that these virions synthesized after the shift to 33” did not incorporate the radioactive virus polypeptides from the excess pool. The reasons for the dichotomy of these pools is presently under investigation. If the TC are precursors of virions, then, in tsCinfected cells following a shift-down, a new pool of competent TC would arise and, indeed, one observes an increase in the light-scattering TC band concomitant with the appearance of mature virions. The long periods of chase at both 33 and 39” indicates that TC are very stable structures and it is unlikely that they are broken down and reused in the synthesis of virions. Nevertheless, it would be interesting to investigate the origin of the small amount of radioactivity in

HEp-2 cells at 39”. The and chased in nonradioVII, VIII, X, XI, and XII

the virions obtained after the shift-down experiments with ts4 (Fig. 8). One possibility would be that these counts represent the processed core protein VII which was packaged into the newly synthesized virions along with the DNA. SDS-polyacrylamide-gel electrophoresis of pulse-chase experiments showed that ts4 fails to accumulate certain polypeptides believed to be cleavage products (Anderson et al., 1973; Weber, 1976). All the mutants studied in this laboratory up to now have been found to be defective in at least the cleavage of PVII into VII at 39”. This strengthens evidence that cleavage of PVII is a late stage in maturation of Ad2. The electrophoretic patterns obtained after the temperature-shift experiments indicate that the ts4 block can be removed to a large extent during a shift-down (Fig. 10). This brings about the processing of at least PVII into VII. That cleavage could account for the radioactivity found in the virion region during the “curing” experi-

ADENOVIRUS

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PCCC” 1 11 19 40

FAILS

WT 33 -39

TO ENCAPSIDATE

ts 4 I 39,->33

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ts 4 I 33+

39

Is I

lOOK95K-

.I I I

52K-, 50K-

,Illa .IV V

PVll

IX

FIG. 10. Autoradiogram of temperature-shift experiments. HEp-2 cells infected with wt and ts4 were grown at 33 and 39”. At 20 hr postinfection the cells at 39” were pulse-labeled (P) with 13Yllmethionine for 1 hr, whereas the cells at 33” received the same treatment at 40 hr postinfection. After the pulse, the cells at 33 were shifted up to 39” while the cells at 39” were shifted down to 33”. Chases (C) in nonradioactive medium (100x methionine concentration) were then conducted for different lengths of time (11,19, and 40 hr at 33”; 1, 8, and 23 hr at 39”).

ments of ts4 (Fig. 8). Moreover, the electrophoretic patterns after the shift-up experiments show that ts4 blocks the cleavage mechanism. This is in agreement with the results obtained by looking for DNA encapsidation after a similar shift-up experiment . Upon comparison of the polypeptides of TC produced by both ts4 and wt at 39” (Fig. 11) we observe that polypeptides VIII, XI, and XII are present in larger quantities in ts4. This could mean that, in ts4, cleavage of TC is carried to a further extent than in the wt-TC. The absence of polypeptide X from both wt- and tsCTC designates this polypeptide as a separate entity and invites speculations as to its origin. For instance this observation indicates that polypeptide X does not have the same origin or is not derived from the same event which produces polypeptide VIII. We have not been able to pinpoint exactly in what way ts4 interferes with en-

capsidation. In fact, recently published work might suggest that TC may be generated by the loss of DNA from intermediates in the maturation of virus particles (Edvardson et al., 1976). Further experiments are required to check this possibility. Mapping studies of ts4 (to be published elsewhere) places this mutation between 30 and 38 on the physical map. As the right strand of this region appears to code for polypeptides V, III, and IIIa (Lewis et al., 19751, the mutation must reside in one of these polypeptides. Since polypeptides III and IIIa are located on the outer capsid of the virion (Everitt et al., 19751, whereas V becomes associated with particles as DNA is encapsidated, this would suggest that the mutation may lie in polypeptide V. We are presently investigating several aspects of ts4 which we believe might throw some light on the mechanism of DNA encapsidation, and the role of polypeptide V in this process.

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A.

B,C

II1OOK Ill llla

,Va Vb/VI

VI-

-14K

Xl2 XII FIG. 11. Autoradiograms of wt virus, wt-TC, and ts4-TC produced at 39”. The virus was grown, labeled with [%]methionine, and centrifuged as described in text. The different viral bands obtained after centrifugation were dialyzed, lyophilized, dissolved in the SDS-sample solution, and used for electrophoresis. Since samples A, B, and C were cut out from different parts of the same slab gel, the alignment of X, XI, and XII is misleading: On the original gel the two fastest-migrating bands of sample C clearly comigrated with polypeptides XI and XII. ACKNOWLEDGMENTS We thank Pierre Magny for the electron microscopy. This work formed part of a thesis submitted by G.K. in partial fulfillment of the M.Sc. degree. J.W. is Research Scholar of the National Cancer Institute of Canada. This study was supported by a grant from the N.C.I.C. and Grant MT-4164 from the Medical Research Council of Canada. REFERENCES ANDERSON, C. W., BAUM, P. R., and GESTELAND, R. F. (1973). Processing of adenovirus a-induced proteins. J. Viral. 12, 241-251. B&IN, M., and WEBER, J. (1975). Genetic analysis of adenovirus type 2. I. Isolation and genetic characterisation of temperature-sensitive mutants. J. Virol. 15, l-7. BURLINGHAM, B. T., BROWN, D. T., and DOERFLER, W. (1974). Incomplete particles of adenovirus. I. Characteristics of the DNA associated with in-

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