JOURNAL OF VIROLO&Y, May, 1979, p. 462471 0022-538X/79/05-0462/10$02.00/0

Vol. 30, No. 2

Uncoating of Adenovirus Type 2 M. AMIN A. MIRZA AND JOSEPH WEBER de Departement Microbiologie, Centre Hospitalier Universitaire, Universite de Sherbrooke, Quebec, Canada JIH 5N4

Received for publication 17 October 1978

The uncoating of adenovirus type 2 and a temperature-sensitive mutant, tsl, was studied. HEp-2 cells were infected with 32P- or "251-labeled purified virions for various lengths of time, and the nuclear and cytoplasmic fractions were analyzed by sucrose gradient velocity sedimentation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Within 1 h of infection, virions were converted into three subviral structures: (i) subviral structures in the cytoplasm with a density greater than virions but which qualitatively still contained all virus polypeptides; (ii) corelike structures associated with both the nuclear and cytoplasmic fractions and composed of viral DNA and polypeptides VIa2, V, and PVII; and (iii) putative DNA-terminal protein complexes in the nuclei. The kinetic and compartmentalization studies suggested that the DNA-terminal protein complex is the end product of uncoating. The virions which were synthesized by tsl at the nonpermissive temperature and contained the precursor polypeptides PVI and PVII were found to be blocked in uncoating at the corelike stage. This block in uncoating provides the explanation for the lack of infectivity of these virions. A model for the uncoating of adenovirus is proposed.

The early literature on the attachment of viruses to cells and uncoating has been reviewed in general (3) and for adenoviruses in particular (9). Based largely on electron microscopic studies, adenovirus is thought to penetrate the cell membrane either directly or more likely by invagination. Upon lysis of the vacuoles, the virus is rapidly transported to the nuclear membrane, possibly along microtubules (4, 10, 16). Qualitatively intact particles are thought to associate with nuclear pore complexes and to deliver their genome through it into the nucleus via an aperture created by a missing penton (11). Very little biochemical information is available on intermediates in uncoating, their protein composition, and the role of individual viral polypeptides during uncoating (11). In view of recent advances concerning the core proteins and the availability of temperature-sensitive mutants specifically altered in these proteins (13, 19), information is particularly lacking on the possible role of these proteins and the nature of the final product of uncoating. In the present communication we have tried to fill this gap in our understanding of the uncoating process by making a comparative biochemical study using adenovirus type 2 (Ad2) and mutant tsl. This mutant was chosen because at the restrictive temperature it produces noninfectious virions which contain the precursor polypeptides PVI, PVII, and PVIII (13, 18).

MATERIALS AND METHODS Cells and virus. HEp-2 cells were grown in Dulbecco modified minimal essential medium (DMEM) prepared in the laboratory and supplemented with 10% heat-inactivated calf serum, penicillin (100 U/ml), and streptomycin (100 jig/ml). During the experiments, the medium contained 2.5% dialyzed calf serum and 0.4 mM arginine. The wild-type (WT) Ad2 was the parental strain of tsl used in all experiments. All experiments were performed with a single stock of passage 3, tsl-virus. Virus was purified by the following procedure. Cells were freeze-thawed three times, sonicated for 20 s, and clarified by low-speed centrifugation, and the supernatant was extracted twice with trichlorotrifluoroethane (Freon 113; Du Pont of Canada Ltd.). This material was gently pipetted on top of a preformed CsCl gradient (1.2 to 1.5 g/ml) and centrifuged for 1 h at 30,000 rpm in the SB283 rotor of an International B60 ultracentrifuge. Fractions were collected from the bottom of the tube, and the density was determined via the refractive index. The virions of density 1.34 g/ml were diluted with buffer (0.05 M Tris-hydrochloride, pH 8.1) and centrifuged to equilibrium in CsCl (1.4 g/ ml) in the same rotor at 24,000 rpm overnight. Highly purified virions were collected and dialyzed against 10 mM Tris-hydrochloride (pH 7.5) buffer containing 1 mM MgCl2 and 10% glycerol in the cold overnight to remove CsCl. The virions prepared in this manner remained structurally intact and retained ful biological activity as monitored by plaque assays. The ratio of particles to PFU of WT was 30, whereas that of tsl

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was 510.

Labeling with "P. HEp-2 cell monolayers in 32-

VOL. 30, 1979 ounce (960-ml) bottles were infected with WT or tsl at a multiplicity of 5 to 10 PFU/cell. At 1 h postadsorption at 330C, complete DMEM was added, and the infected cultures were shifted to 390C or maintained at 33°C as required. At 12 h postinfection the medium was removed, and monolayers were washed with phosphate-free medium and then incubated in the same medium for at least 1 h to exhaust residual phosphate. Next, each bottle received 1 to 2 uCi of carrier-free 32P (ICN) in 10 ml of fresh phosphate-free medium. The cell cultures were pulsed for 2 h under these conditions; then the complete DMEM was replaced, and cells were labeled continuously for the next 36 h. At 48 h postinfection infected cultures were collected, and virions were harvested as described above. lodination of purified virions. Viruses were iodinated under conditions shown to preserve biological activity (7). The purified unlabeled virus (0.05 ml) was added to a mixture containing 10 pl of each of the following: 1 M sodium phosphate buffer, pH 6.9; 0.1 N HCI; and carrier-free '"I (200 to 500 ,Ci) in 0.1 N NaOH. Iodination was initiated by the addition of 4 jig of chloramine T. After 30 to 45 s at room temperature, the reaction was stopped with 12.5 jig of K2S205. Labeled virus was separated from unreacted free iodine by velocity sedimentation in a sucrose solution buffered with 0.05 M Tris-hydrochloride, pH 8.1. Iodinated virus samples were suspended in 10 mM Trishydrochloride, pH 7.5, containing 1 mM MgCl2 and 10% glycerol and used for cell infection within 12 h

after labeling. Infection with labeled virus. Subconfluent monolayer cultures of HEp-2 cells in duplicate petri dishes were infected with 32p- or '25I-labeled virions at a multiplicity of infection of 1,000 physical particles per cell (determined spectrophotometrically, with one unit of absorbance at 260 nm equal to 1012 particles per ml). Equivalent amounts of radioactivity, contained in equivalent numbers of physical particles, were added in each case. The infected cells were incubated at 33 or 390C as required and harvested at various times postinfection. Cell fractionation. Infected cells were collected and washed with physiological Tris-saline to remove unadsorbed virus. The procedure used for the preparation of subcellular fractions was essentially the same as that described by Frost and Bourgaux (6), with some modifications. Washed cells were suspended in 1 ml of 0.1% Tween 80-0.01 M EDTA-0.01 M Trishydrochloride, pH 7.4, and transferred to a centrifuge tube. The suspension was shaken for 1 min on a Vortex junior mixer and then held in ice for 15 min. The suspension was centrifuged at 2,400 rpm for 5 min in a refrigerated Sorvall HG-4 centrifuge to sediment nuclei. The supernatant fluid, termed cytoplasmic extract, contained less than 5% of the newly synthesized DNA. The distribution of pulse-labeled protein and RNA indicated recovery of 90 to 95% of the cytoplasmic material in this fraction. These control experiments suggest that there could not be more than 10% cross-contamination. The nuclear pellet obtained as described above was washed a few times with Tris-saline and then suspended in 1 ml of buffer containing 0.25% deoxycholate

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or 0.5% Nonidet P-40, 0.01 M EDTA, and 0.01 M Trishydrochloride, pH 7.4. The nuclei were broken by 10 strokes in a tightly fitting Dounce glass homogenizer equipped with a Teflon plunger. The lysed nuclear suspension was centrifuged for 15 min at 4,500 rpm. The supernatant was separated and designated the nuclear fraction. The concentrations of Tween 80 and deoxycholate or Nonidet P-40 used to prepare subcellular fractions were considered safe, as the 3P-labeled purified virion used for infection remained physically stable when subjected to the detergent treatments. Lysis of nuclei with 0.25% deoxycholate or 0.5% Nonidet P-40 gave

identical results. Centrifugation. The analysis of cytoplasmic and nuclear fractions prepared from infected cells at various times postinfection was performed by sucrose gradient velocity sedimentation. A 0.2-ml sample was layered over 3.5 ml of a 5 to 25% linear sucrose gradient (1 M NaCl, 0.001 M EDTA, 0.01 M Trishydrochloride, pH 7.4) formed on top of a 0.3-ml cushion (56% CsCl, 25% sucrose, 0.001 M EDTA, 0.01 M Tris-hydrochloride, pH 7.4). Centrifugation was performed in an International B60 ultracentrifuge for 45 min at 35,000 rpm (SB405 rotor). Fractions (6 drops) were collected from the bottom of the tube, and either whole fractions or 20-pl samples were assayed for cold trichloroacetic acid-precipitable radioactivity. SDS-polyacrylamide gel electrophoresis. The polyacrylamide gel system was basically that described by Maizel (12). The sodium dodecyl sulfate (SDS) continuous system was used with a Hoeffer slab gel apparatus (Hoeffer Scientific, San Francisco, Calif.). The gels (0.75 mm thick, 10.5 cm high) consisted of a 12.5% resolving gel and a 5% stacking gel, with a ratio of acrylamide to bisacrylamide of 30:0.8. The gels were run at 30 mA/slab, stained with Coomassie brilliant blue, dried in vacuo, and autoradiographed with Kodak RP-oxomat medical X-ray film. Samples to be analyzed were collected in 0.1 ml of sample solution (0.05 M Tris, pH 6.8, 2% SDS, 1% mercaptoethanol, 10% glycerol, 0.001% phenol red), boiled for 2 min, and cooled. Either equal volumes or equal amounts of radioactivity were applied to the gels. Extraction of DNA-terminal protein complex and its characterization on sucrose gradients. Marker DNA-terminal protein complex was isolated from 32P-labeled virus essentially as described by Robinson et al. (15). One volume of 8 M guanidinium chloride in 0.01 M Tris (pH 8.0)-0.001 M EDTA was mixed with 1 volume of virus at 0°C. After 2 to 3 min about 10 volumes of ice-cold chloroform-isoamyl alcohol (24:1) was added, and the mixture was rotated slowly at 40C for 5 to 10 min. After centrifuging at 1,100 x g for 10 min at 4°C, the aqueous phase was dialyzed for about 1 h against 0.1 M NaCl-0.05 M Tris, pH 7.2-0001 M EDTA at 40C. Exactly the same procedure was used to extract DNA from nuclear fractions of WT-infected cells. The dialyzed samples were centrifuged through a 5 to 20% sucrose gradient containing 4 M guanidinium hydrochloride, 0.01 M Tris, pH 8.1, and 0.001 M EDTA. The gradients were centrifuged in an SB283 rotor (International Centrifuge) at 21,000 rpm and 40C

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for 16 h. Fractions were collected, and samples were assayed for cold trichlorocetic acid-precipitable radioactivity.

RESULTS

Kinetics of uptake of Ad2 and tsl virions by HEp-2 cells. To study the mechanisxm of in vivo uncoating, the kinetics of uptake of WT and tsl virions was investigated. Duplicate petri dishes containing equal numbers of HEp-2 cells were infected with 1,000 physical particles of 32P-labeled WT and tsl virions grown at 390C (subsequently referred to as ts1-39°C virions) per cell. At specified times after the addition of the viruses, the unadsorbed virus was removed, and cells were washed and lysed to prepare cytoplasmic and nuclear fractions as described above. Samples of subcellular fractions were assayed for cold trichloroacetic acid-precipitable radioactivity to measure the extent of uptake. Figure 1 shows the kinetics of uptake of WT and ts1-39°C virions by HEp-2 cells at 390C. The WT viral label entered the cytoplasm and the nucleus very rapidly and then leveled off within 15 min. There was a relatively greater amount of radioactivity in the nucleus than in the cytoplasm, suggesting extremely rapid transport from the cytoplasm to the nucleus. The uptake kinetics of ts1-39°C virions was similar to that of WT in that (i) it plateaued within 15 min postinfection WT

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FIG. 1. Kinetics of uptake of 32P-labeled Ad2 and tsl-39°C virions by HEp-2 cells at 39°C. HEp-2 cell monolayers in petri dishes were separately infected with 1,000 particles of 32P-labeled WT and ts1-39°C virions per cell. The total input was 90,000 cpm/petri dish for both viruses. The virus preparations had specific activities of 4 x 108 to 6 x 108 cpm/unit of absorbance at 260 nm. At 15, 30, and 60 min postinfection (P.I.) cells were harvested, washed, and lysed to prepare cytoplasmic and nuclear fractions as described in the text. Samples (20 ,l) were assayed for cold trichloroacetic acid-precipitable radioactivity. The total radioactivity which was cell bound after 60 min was 15 and 5% of the input for WT and ts1-390C virions, respectively.

and (ii) there was relatively more radioactivity in the nucleus than in the cytoplasmn. The net amount of radioactivity in tsl-39°C virion-infected cells was only 25 to 30% of that observed in WT. The uptake kinetics was also studied at 330C and was found to be similar to that seen at 390C (data not shown). tsl virions grown at 330C (tsl-33°C virions) behaved identically to WT at either 33 or 39°C (data not shown). These observations therefore indicate that ts1-390C virions are less efficient in uptake, compared with WT and ts1-330C virions at either 33 or 390C. Uncoating of 32P-labeled virions at 390C. The biochemical approach used to investigate the mechanism of uncoating of the virus was to monitor the metabolic fate of 32P-labeled virions in subcellular fractions at various times postinfection. The objective was to detect subviral structures with decreasing sedimentation coefficients in sucrose gradients generated as a result of uncoating. Based on this rationale, the following experiment was carried out. Equal numbers of HEp-2 cells were infected with 32P-labeled purified WT, tsl-390C, and tsl330C virions separately. The adsorption was allowed to occur at 390C for 15, 30, and 60 min, respectively. After these time intervals the cells were washed to remove unadsorbed virus and then lysed to prepare cytoplasmic and nuclear fractions. The subcellular fractions were analyzed by velocity sedimentation through sucrose gradients containing 1 M NaCl. Figure 2 shows the 32P radioactivity profiles of the subviral structures observed in the cytoplasm and nuclei of the infected cells. At 15 min postinfection the cytoplasm of the WT-infected cells contained 240S and heavier-sedimenting structures (Fig. 2J). The nuclei contained these structures in relatively greater amounts, but in addition they had a 31S peak running in the position of adenovirus marker DNA. The 31S product (which is DNase sensitive [data not shown]) accounted for over 50% of the total radioactivity recovered in the nuclear fraction, suggesting a rapid generation of DNA from heavier-sedimenting structures exclusively in the nuclei. ts1-330C virus uncoated in a manner similar to WT, generating 240S and heavier structures from both the cytoplasm and the nuclei (Fig. 2D through F). The nuclei contained a greater amount of these heavier-sedimenting structures; in addition they contained 31S DNA, the proportion of which increased with time. At 1 h postinfection 31S DNA represented about 30% of the total radioactivity recovered in the nuclei. The extent of generation of 31S DNA was lower than it was with WT. This means that tsl-330C virions uncoated slowly as compared with WT.

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accumulation of 150S complex in the nuclei relative to the cytoplasm, but no 31S DNA was generated. This result clearly shows that tsl6 v 240S 150S 31S 240S 390C virions underwent only partial uncoating, generating a 150S complex which failed to uncoat further to yield 31S DNA. It is evident, 2 therefore, that tsl-39°C virions were blocked at this intermediate stage of uncoating. That the ui subviral structures observed in the subcellular Q - ~~~~~~3400 fractions of WT-, tsl-33°C virion-, and tsl-39°C E 30MIN. 30MIN. virion-infected cells most probably represented 6 true products of in vivo uncoating was suggested d by the following control experiment. Purified 4; WT and tsl-39°C virions labeled with 32P or 2 [3S]methionine were subjected to Tween-de0l. C0 oxycholate treatment (the conditions used to lyse infected cells for preparation of subcellular °1600 ~ ~0~~~~~~~~~ ~ ~~.50 fractions) and then analyzed in sucrose gradients containing 1 M NaCl. Virions treated in this 6 manner sedimented as a single homogeneous CIA C peak at the bottom of the gradient (data not C 6.OM. . F 6.M.NI shown). This control, therefore, rules out the x) 2 possibility that the structures observed in the infected cells were artifactual. ciUncoating of 32P-labeled virions at 330C. r, 2400 40 20 30 In the previous section, the uncoating of WT, : ,J WT 1 5MIN .: ts1-330C, and tsl-390C virions was investigated 390C, the nonpermissive temperature for tsl. I~~~~~~ at It was of interest to study the mode of uncoating 1~ ~ 1, of these virions at 330C, the permissive temperature, to determine any differences. HEp-2 cells 2 were infected at 330C with 32P-labeled WT, tsl330C, and tsl-390C virions, and cytoplasmic and 1 0 20 30 40 nuclear fractions were analyzed on sucrose graFRACTION NUMBER FIG. 2. Sucrose gradient analysis of cytoplasmic dients exactly as described above. Figure 3 shows the radioactivity profiles of the and nuclear fractions of cells infected with 32P-labeled virions for various times at 39°C. Cytoplasmic subviral structures detected in the cytoplasmic and nuclear fractions were prepared from cells in- and nuclear fractions of infected cells. Uncoating fected with 32P-labeled WT, tsl-33°C, or tsl-39°C of WT at 330C generated subviral products simvirions as described in the legend to Fig. 1. The ilar to those observed at 390C (Fig. 3J). Qualisubcellular fractions were analyzed by velocity sedi- tatively, therefore, there were no differences, but mentation through sucrose gradients in the presence quantitatively 31S DNA represented only 30% of I M NaCl as described in the text. Fractions (6 of the total radioactivity in the nuclei. This is in drops) were collected and assayed for cold trichloro- contrast to the large amount of 31S DNA obacetic acid-precipitable radioactivity. Sedimentaion served at 390C, which suggests slower uncoating was to the left; 240S, 150S, and 31S indicate positions of marker polyoma virus, polyoma empty shell, and at 330C. tsl-330C virions (Fig. 3D through F) and ts1-390C virions (Fig. 3A through C) also adenovirus DNA, respectively. presented sucrose gradient profiles qualitatively The uncoating of tsl-39°C virions proceeded similar to those observed at 390C, but in keeping quite differently (Fig. 2A through C). At 15 min with WT the level of uncoating was somewhat postinfection the cytoplasm contained 240S and reduced. Significantly, the continued generation heavier structures and a significant species at of the 150S complex and the failure to uncoat to 150S which constituted about 60% of the total 31S DNA by tsl-390C virions even at 330C radioactivity recovered in the cytoplasmic frac- suggests that its structural alterations are irretion. The nuclei also contained this 150S com- versible (13). Uncoating of "2I-labeled virions and the plex in addition to heavier structures, but there effect of salt concentration during analysis. was a conspicuous absence of 31S DNA. At 30 and 60 min postinfection there was a greater After we established that tsl-390C virions were ATS-39

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The 32P and 125I labels ran paralel for the heavier structures in the cytoplasm (Fig. 4A) and the nuclei (Fig. 4B) of WT-infected cells. 6 240S 150S 31S The 125I radioactivity found at the top of the 64 4 ff gradient in both the cytoplasmic and nuclear o fractions represented solubilized proteins result2 o 2 o 6k ing from uncoating. It is significant to note that the bulk of the DNA and protein label, both the 0 1- 2100 heavy and light structures and the solubilized 8 &B 30 MI N. E 3OMIN. proteins, were found in association with the nuclear fraction. 6 The tsl-33°C virion-infected cells revealed structures similar to those observed with WT 4 9o /\ , t1 v; (Fig. 4C and D). However, less 31S DNA and 2 v soluble protein were associated with the nuclei 7 ; 4D). This reduction in 31S DNA and the (Fig. decrease in solubilized protein proportional 'so5000 8 C F 60MIN. again indicates that 60 MI N. virions uncoat less WT. with compared efficiently 6 v ,l The tsl-39°C virion profiles (Fig. 4E and F) 4 § Xshow that, although no 31S DNA was generated, partial uncoating was realized, as evidenced by 2 Ithe soluble proteins near the top of the gradient b_ 60

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blocked at an intermediate stage of uncoating, it was important to determine the nature of the 150S complex observed in both the cytoplasm and the nuclei of infected cells. Most specifically, the objective was to determine whether this 150S structure represented some sort of deoxyribonucleoprotein complex and, second, to identify its constituent polypeptides. To approach the problem, WT, tsl-330C, and tsl-39°C virions were labeled with 32P and 1251 separately. HEp-2 cells were infected with 32p- and 125I-labeled carried out out at at 39°C virions, and adsorption adsorption was was carried virions, and for 1 h. Unadsorbed virus was washed off, and the cells were lysed to prepare cytoplasmic and nuclear fractions which were analyzed on sucrose gradients containing 1 M NaCl. The results of this experiment are shown in Fig. 4.

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and the 150S complex. Cosedimenting 1I indicated that the 150S species was indeed a deoxyribonucleoprotein complex and may represent the viral core. Again, as in the case of WT and ts1-33°C virions, the bulk of the infecting material became associated with the nuclei. In all of the experiments described so far, the analysis of subcellular fractions of infected cells was carried out in sucrose gradients which contained 1 M NaCl. The question arose as to whether the subviral structures seen in the cytoplasm and nuclei of WT- and tsl-infected cells could not perhaps be generated from heavier native structures due to the presence of the high salt concentration in the gradient. We therefore repeated these experiments with sucrose gradients containing 0.1 M NaCl. The results were qualitatively and quantitatively indistinguishable from those obtained in the presence of 1 M NaCl (data not shown). In particular, the appearance of the 150S complex is therefore independent of salt concentration and may represent an intermediate in uncoating. Stability of the 160S complex. We described above observations which strongly suggested that up to 1 h postinfection ts1-39°C virions uncoated only partially, producing a 150S complex found both in the cytoplasm and the nuclei of the infected cells. It was essential to determine whether the 150S complex would undergo further uncoating to generate 31S DNA when the infected cells were allowed to incubate for a longer time. Equal numbers of HEp-2 cells were infected with 32P-labeled WT and tsl-390C virions separately. Adsorption was allowed to occur for 1 h at 390C; then the cultures were washed of unadsorbed virus, and one set was lysed immediately to prepare subcellular fractions. The other set of washed cultures received complete DMEM and was incubated further for 3 h at 390C, after which they were lysed to prepare cytoplasmic and nuclear fractions. The subcellular fractions obtained from infected cells at 1 h and 4 h postinfection were analyzed by sucrose gradients as described previously. Figures 5C and D show that by increasing the time of incubation to 4 h a somewhat larger proportion of WT 31S DNA was generated. Essentially all of the 3P became nucleus associated. In the case of ts1-39°C virions (Fig. 5A and B), there was no substantial change beween 1 h and 4 h postinfection in the proportion of radioactivity which was nucleus associated. However, the proportion of 150S complex versus the fast-sedimenting component doubled. Significantly, even after 4 h, no 31S DNA was generated by tsl390C virions (Fig. 5B). We therefore consider the 150S complex to be an intermediate of un-

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coating rendered stable by the pleiotropic effects of the tsl mutation. Initial products of uncoating of WT and tsl are similar. The sucrose gradient conditions used to analyze subcellular fractions of infected cells could not possibly efficiently resolve structures heavier than 250S. To examine these structures further, we pooled fractions 1 through 10 (from both cytoplasm and nuclei) from sucrose gradients (Fig. 2J) and analyzed them on preformed CsCl gradients. Figure 6 shows that these fast-sedimenting structures were in fact heterogeneous in density. The cytoplasm contained structures with densities of 1.46 and 1.48 g/cm3, and the nuclei also had these structures but in relatively greater amounts. Significantly, no intact virions with the typical density of 1.34 g/cm3 were observed in either the cytoplasm or the nuclei; this suggests that the initial uncoating of the virus, which results in the generation of structures having densities of 1.46 and 1.48 g/cm3, is very rapid. The structures having densities of 1.46 and 1.48 g/cm3 are therefore partly uncoated virions. These could be envisaged to represent the products of the first stage of uncoating. It is these structures which undergo further sequen-

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10 20 30 40 FRACTION NUMBER FIG. 6. CsCI gradient analysis of heavier subviral structures isolated from sucrose gradients. Fractions 1 through 10 from the bottom of the sucrose gradient (Fig. 2 and 3J) were pooled and analyzed on a preformed CsCl gradient as described in the text. Fractions were collected and assayed for cold trichloroacetic acid-precipitable radioactivity after their densities were determined.

tial uncoating, resulting eventually in the generation of 31S DNA. Fractions 1 through 10 pooled from the tsl-33°C virion gradients (Fig. 2D) and those from tsl-39°C virions (Fig. 2A) gave similar results when analyzed on CsCl gradients (data not shown). These results show that the initial products of uncoating of WT and tsl39°C virions are probably very similar. Is WT virus uncoated to the DNA-terminal protein complex? We have shown above that the uncoating of WT virus results in DNasesensitive material cosedimenting with 31S adenovirus marker DNA. To examine the physical homogeneity of this material, the appropriate top fractions (Fig. 4B, fractions 30 through 40) were pooled, made 4 M with guanidinium hydrochloride, and after a brief incubation at 4°C extracted with an isoamyl alcohol-chloroform mixture (15). The aqueous phase containing nucleic acid was collected and dialyzed as described above. The dialyzed sample was subjected to centrifugation through a sucrose gradient containing 4 M guanidinium hydrochloride. The marker DNA-terminal protein complex was prepared from purified virus by the same procedures. Figure 7 shows the sedimentation profile of the material extracted from the pooled fractions. This DNA sedimented as a homogeneous peak, coinciding with the marker DNA-terminal protein complex. Further evidence in support of our contention that the final product of uncoating of WT virions might be the DNA-terminal protein complex was obtained by the following experiment. In vivo uncoated 31S DNA, obtained from pooled fractions as described above (Fig. 4B, fractions 30 through 40) and extracted exactly as described above, was mixed with [3H]thymidinelabeled, deproteinized marker DNA (SDS-Pro-

nase-phenol) and centrifuged in a sucrose gradient containing 0.5 M NaCl. The radioactive profiles showed that the in vivo uncoated DNA sedimented significantly faster than the deproteinized marker DNA (data not shown). This result indicates that the in vivo DNA may be aggregated because of the presence of the "sticky" terminal protein (14). The experiment to show that the terminal protein was still on the DNA by looking for retarded migration of the terminal fragments in agarose gel electrophoresis after EcoRI endonuclease digestion was inconclusive due to the lack of sufficient counts. Although the above results are consistent with the hypothesis that the final product of uncoating of WT virus is the DNA-terminal protein complex, they do not prove it. 150S intermediate is a core complex. Since the tsl-39°C virions appeared to be blocked at an intermediate stage of uncoating, as evidenced by the accumulation of 150S complex, it was of interest to determine the polypeptide composition of this structure. Fractions 20 through 30, corresponding to 125Ij labeled 150S complex, were pooled separately from both cytoplasm (Fig. 4E) and nuclei (Fig. 4F). The pooled fractions were concentrated, boiled with SDS, and subjected to electrophoretic analysis on an SDS-polyacrylamide slab gel. The gel was dried and autoradiographed. Figure 8a shows the polypeptide composition of the nuclear 150S complex. As expected, the 150S complex contained polypeptides V and PVII and traces of polypeptide VII. The cytoplasmic 150S complex contained the same polypeptides (data not shown). Judging from the polypeptide com9 6

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FIG. 7. Characterization of DNA generated in the nuclei of WT virus-infected cells. 32P viral label corresponding to top fractions 30 through 40 (Fig. 4B) was extracted for nucleic acid by using the chloroform-isoamyl alcoholprocedure described in the text. This material was centrifuged through a sucrose gradient containing 4 Mguanidinium hydrochloride. Purified DNA-terminalprotein complex labeled with 3p was centrifuged in a parallel tube. Symbols: 0, Ad2 marker complex; 0, DNA isolated from top fractions 30 through 40 (Fig. 4B).

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of "2I-labeled WT virus-infected cells were pooled and concentrated. The corresponding fractions from the tsl-39°C virion-infected cells were also pooled (Fig. 4F). The concentrated samples were denatured with SDS and mercaptoethanol and subjected to electrophoresis on SDS-polyacrylamide slab gels. The heaviestsedimenting structure found in the nuclei of tsl39°C virion-infected cells contained all of the viral structural proteins (Fig. 9, lane b). The major proteins that were removed and found in the soluble fractions during uncoating were polypeptides II, III, and IV and, to a limited extent, core proteins V and PVII (Fig. 9, lane c). On the other hand, the heaviest nuclear structure found £ CI in the WT-infected cells contained nearly all of the viral structural polypeptides (Fig. 9, lane e), I,1 ½ HI> I with the notable exceptions of polypeptides IVa2 10-I i and VII, which could be quantitatively acP-4 counted for in the soluble pool (Fig. 9, lane f). In ,,,> 0-4 r, addition to these proteins, core protein V was also largely solubilized. The presence of IVa2 in the soluble fractions was in agreement with the FIG. 8. SDS-polyacrylamide gel electrophoresis of notion that IVa2 is a core protein (unpublished 150S complex isolated from the nuclei of cells infected data). These observations clearly establish anwith '251-labeled tsl-39°C virus. Fractions 20 through other significant difference in the way tsl-39°C 30, corresponding to 150S complex (Fig. 4F), were pooled and analyzed electrophoretically on SDS- and WT virions shed their structural polypeppolyacrylamide slab gels. The sample order was: tides during uncoating. channel a, 150S complex; channel b, ts1-39°C virus; DISCUSSION and channel c, WT virus. The densitometric scanning At the nonpermissive temperature tsl, a temof the "SI-generated autoradiogram in channel a is perature-sensitive mutant of Ad2, synthesizes also shown.

position, the 150S complex is some sort of a core structure. The basic polypeptides V, PVII, and VII are the core proteins of tsl-39°C virus (13), and it is intriguing to find these in the in vivogenerated 150S core complex, an intermediate in the uncoating pathway of the virus. Since tsl39°C virions were blocked at the level of 150S complex containing PVII, it is possible that the presence of this precursor core protein renders tsl-39°C virions incapable of undergoing normal and complete uncoating. It is therefore possible that the lack of infectivity of tsl-39°C virions is due to a block in uncoating caused by the presence of precursor polypeptide PVII. Polypeptide composition of subviral structures found in the nucleus of WT- and tsl-infected cells. We have shown that the initial products of uncoating consist of fast-sedimenting, dense structures as well as soluble proteins. We now examine the polypeptide composition of these products of uncoating found in the nuclei of WT- and tsl-39°C virion-infected cells. Fractions corresponding to the heaviestsedimenting structures (Fig. 4B, fractions 1 through 10) and the solubilized proteins (Fig. 4B, fractions 30 through 40) found in the nuclei

ab c II

III

d e f

....

*-

IV I Ia_ V __

&.I PVII> VII_*..

.-

Vll-

-

i -_IVa2

I

FIG. 9. Polypeptide analysis of subviral structures found in the nuclei of WT- and tsl-39°C virus-infected cells. The autoradiogram was 1"I generated. Channel a, tsl-39°C virus; channel b, tsl nucleus heaviest structure (Fig. 4F, fractions 1 through 10); channel c, tsl soluble proteins (Fig. 4F, fractions 30 through 40); channel d, WT virus; channel e, WT nucleus heaviest structure (Fig. 4B, fractions 1 through 10); channel f, WT soluble proteins (Fig. 4B, fractions 30 through 40).

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noninfectious virions which contain the precursors to the internal and core polypeptides VI, VII, VIII, X, XI, and XII (17). For brevity we refer to these virions as tsl-39°C virions. Recently we reported significant differences between WT and tsl core structures derived in vitro by disintegration of virions with pyridine (13). Our present studies were aimed at investigating the biochemical significance and implications of such structural differences to seek an explanation for the noninfectious nature of tsl390C virions. Preliminary experiments showed that tsl390C virions failed to program viral DNA, late protein, or even early protein synthesis (unpublished data). In this paper we demonstrate that the block to the infectivity of ts1-39°C virions in fact lies at a late stage in uncoating. Based on a comparative analysis of WT, tsl330C, and tsl-39°C virions, in which sucrose gradient sedimentation and SDS-gel electrophoresis of the products of uncoating in cytoplasmic and nuclear fractions were used, we propose a possible sequence of adenovirus uncoating (Fig. 10). Virions rapidly penetrate the cytoplasm without the loss of any particular class of structural proteins. Most of these virions increase in density, possibly as a result of the loss of some portion of the major shell proteins (Fig. 10, C and C"). Although the bulk of dense virions become tightly associated with the nucleus (Fig. 10, C), the data do not rule out the possibility

A

C

FIG. 10. Possible sequence of events during the first hour of infection with Ad2. Because of technical problems in the separation of nuclei and cytoplasm, it has not been possible to rule out the existence of the pathway identified by C" and D'. C represents C after injection of the core D. The block in uncoating of tsl-39°C virion is between D or D' and E. The composition of the various structures is as follows: A, B, C, and C" (complete, or nearly so, virions), polypeptides II, III, IIIa, IV, IVa2, V, VI, VII, VIII, IX, and X-XII and DNA; C (shells), polypeptides II, III, IIIa, IV, VI, VIII, and IX; D and D' (cores), polypeptides IVa2, V, and VII and DNA; E (DNA-terminal protein complex), polypeptide IVa2 and DNA.

J. VIROL.

that some of them (Fig. 10, C") release the core which in turn associates with the nucleus (Fig. 10, D'). At this point the core penetrates the nucleus (Fig. 10, D), leaving behind the empty shell (Fig. 10, C') which is now devoid of DNA and proteins IVa2 (a putative core protein; unpublished data), V, and VII. The core sheds proteins V and VII, and the fate of VIa2 is not known, giving rise to the possible final product of uncoating, the DNA-terminal protein complex (Fig. 10, E). tsl-39°C virions appear to be blocked in the conversion of their PVII-containing cores to DNA-terminal protein complex. Furthermore, a large proportion of ts1-39°C virion cores (the 150S complex) fails to penetrate or become associated with the nucleus. This suggests a possible alternative pathway of uncoating (Fig. 10, C" and D'). Electron microscope studies currently in progress indeed- support such an alternative pathway in the uncoating of tsl-39°C virions (B. Miles, R. Luftig, and J. Weber, unpublished data). The 150S complex end product of the uncoating of ts1-39°C virions contained polypeptides IVa2, V, and PVII. This polypeptide composition was identical, whether the complex was isolated from the cytoplasm or the nucleus and, furthermore, resembles the in vitro-obtained pyridine core (13). Clearly, the lack of infectivity of tsl-39°C virions is due either to the failure of complete uncoating or, alternatively, to an altered pathway of uncoating, as postulated above (Fig. 10, C" and D'). The question arises as to whether this block to infectivity could be overcome by increasing the time scale to days. The answer is apparently negative, as evidenced by the absence of even miniplaque formation by ts1-39°C virions after prolonged incubation at 330C (unpublished data). Defects in the DNA were ruled out by transfection experiments; tsl390C virion DNA and WT DNA formed plaques with comparable efficiency at 330C (Mirza and Weber, manuscript in preparation). Although in the present experiments we failed to detect a WT 150S core complex, very likely because of its short half-life, we feel that its existence can be inferred from the presence of this complex during the uncoating of ts1-33°C virions which are fully infectious. The scheme of adenovirus uncoating presented here appears to be consistent with previous reports on the subject (2, 4). Based on partly biochemical but mainly electron microscopic studies, it has been established that Ad5 virions moved vectorially from the plasma membrane to the nuclear envelope along microtubules. The work of Chardonnet and Dales (2-4) supports the view that the final uncoating of the

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virus occurs at nuclear pores, resulting in rapid and efficient transfer of DNA into the nucleus. Using a biochemical approach, we also find that uncoating, as shown by solubilization of structural proteins of the virus and generation of DNA, is a nucleus-associated phenomenon. It is important to point out, however, that because of the obvious limitation of our analytical technique, we cannot distinguish whether the uncoating of the core occurs inside the nucleus or at a perinuclear site. Our conclusions are also largely consistent with the original findings of Lonberg-Holm and Philipson (8), who used a biochemical approach similar to ours and identified the above-described three subviral structures as products of uncoating. ACKNOWLEDGMENTS This investigation was supported by a grant from the National Cancer Institute of Canada to J.W., who is also a Research Scholar of the National Cancer Institute of Canada.

LITERATURE CITED 1. Begin, M., and J. Weber. 1975. Genetic analysis of adenovirus type 2. I. Isolation and genetic characterization of temperature-sensitive mutants. J. Virol. 15:17. 2. Chardonnet, Y., and S. Dales. 1972. Early events in the interaction of adenoviruses with HeLa cells. III. Relationship between ATPase activity in nuclear envelopes and transfer of core material: a hypothesis. Virology 48: 342-359. 3. Dales, S. 1973. The early events in cell animal virus interactions. Bacteriol. Rev. 37:103-135. 4. Dales, S., and Y. Chardonnet. 1973. Early events in the interaction of adenoviruses with HeLa cells. IV. Association with microtubules and the nuclear pore complex during vectorial movements of the inoculum. Virology 56:465-483. 5. Frost, E., and P. Bourgaux. 1973. Attempts to find a specific intranuclear location for replicating polyoma virus DNA. Can. J. Biochem. 51:1225-1228.

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6. Frost, E., and P. Bourgaux. 1975. Decapsidation of polyoma virus: identification of subviral species. Virology 68:245-255. 7. Frost, E. H. E. 1977. Radioactive labelling of viruses: an iodination technique preserving biological properties. J. Gen. Virol. 35:181-185. 8. Lonberg-Holm, K., and L. Philipson. 1969. Early events of virus-cell interaction in an adenovirus system. J. Virol. 4:323-338. 9. Lonberg-Holm, K., and L Philipson. 1974. Early interaction between animal viruses and cells. Monogr. Virol. 9:1-148. 10. Luftig, R. B., and R. R. Weihing. 1975. Adenovirus binds to rat brain microtubules in vitro. J. Virol. 16: 696-706. 11. Lyon, M., Y. Chardonnet, and S. Dales. 1978. Early events in the interaction of adenoviruses with HeLa cells. V. Polypeptides associated with the penetrating inoculum. Virology 87:81-88. 12. Maizel, J. V., Jr. 1969. Acrylamide gel electrophoresis of proteins and nucleic acids, p. 334-370. In K. Habel and N. P. Salzman (ed.), Fundamental techniques in virology. Academic Press Inc., New York. 13. Mirza, M. A. A., and J. Weber. 1977. Genetic analysis of adenovirus type 2. VII. Cleavage modified affinity for DNA of internal virion proteins. Virology 80:83-97. 14. Rekosh, D. M. K., W. C. Russell, A. J. D. Bellet, and A. J. Robinson. 1977. Identification of a protein linked to the ends of adenovirus DNA. Cell 11:283-295. 15. Robinson, A. J., H. B. Younghusband, and A. J. D. Bellet. 1973. A circular DNA-protein complex from adenoviruses. Virology 56:54-69. 16. Weatherbee, J. A., R. B. Luftig, and R. R. Weihing. 1977. Binding of adenoviruses to microtubules. II. Depletion of high-molecular-weight microtubule-associated protein content reduces specificity of in vitro binding. J. Virol. 21:732-742. 17. Weber, J. 1976. Genetic analysis of adenovirus type 2. III. Temperature sensitivity of processing of viral proteins. J. Virol. 17:462-471. 18. Weber, J., M. Begin, and E. Carstens. 1977. Genetic analysis of adenovirus type 2. IV. Coordinate regulation of polypeptides 80K, Illa and V. Virology 76:709-724. 19. Weber, J., M. Begin, and G. Khittoo. 1975. Genetic analysis of adenovirus type 2. II. Preliminary phenotypic characterization of temperature-sensitive mutants. J. Virol. 15:1049-1056.