UTE BRUGGEMANN,' HANS-DIETER KLENK,2 AND WALTER DOERFLER1*. Institute ofGenetics, University ofCologne, Cologne,' and Institute of Virology, ...
Vol. 55, No. 1
JOURNAL OF VIROLOGY, JUIY 1985, p. 117-125
0022-538X/85/070117-09$02.00/0 Copyright C) 1985, American Society for Microbiology
Increased Infectivity of Extracellular Adenovirus Type 12 UTE BRUGGEMANN,' HANS-DIETER KLENK,2 AND WALTER DOERFLER1* Institute of Genetics, University of Cologne, Cologne,' and Institute of Virology, University of Giessen, Giessen,2 Federal Republic of Germany Received 20 November 1984/Accepted 18 February 1985
Human adenovirus type 12 was propagated on human embryonic kidney cells, and the specific infectivities of intra- and extracellular virus particles were compared between 48 and 104 h after infection. Released virions exhibited a specific infectivity of up to 10 times higher than that of intracellular particles. The increased infectivity was apparently not due to enhanced rates of adsorption or penetration of extracellular virus. There may be a delay in the onset of viral DNA replication in intracellular virus-infected cells. Differences in the composition of intra- and extracellular virions were not recognized. Differences might also be sought in late expression or assembly of progeny virions or both. The data indicated that the virions released from the infected cells differed from those retained in the nucleus with respect to their specific infectivities. Active mechanisms of virus release have not yet been investigated.
particles seemed to contain the same DNA molecules. The polypeptide composition of intra- and extracellular virions was indistinguishable as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of [35S]methionine-labeled particles. Intra- and extracellular virions appeared to adsorb at comparable rates to HeLa cells, and viral DNA synthesis began at about the same time after inoculation but seemed to be less efficient after infection with intracellular virus. Virion polypeptides, in particular the virion fiber, were glucosylated to about the same extents in intra- and extracellular virions.
In the course of a systematic investigation on the infectivity of human adenovirus type 12 (Adl2), we discovered that extracellular virions liberated from human embryonic kidney (HEK) cells exhibited considerably higher specific infectivity than intracellular particles. In the past, most experiments conventionally carried out with adenoviruses were performed with intracellular virions extracted from infected cells. The extracellular virus comprising only a fraction of the total virus yield was often discarded. The high specific infectivity of extracellular virions raised interesting questions with respect to late steps of maturation and about the release of adenovirions which were assembled in the nucleus of the cell. Extracellular virions were not previously investigated for differences in structure, composition, or infectivity in comparison to intracellular virions. It was conceivable that adenovirions were further modified after the assembly stage in the nucleus and that one of these modifying events might be required for the efficient release of the virions. The mechanism of release of infectious adenovirions from the cell nucleus was not yet studied in detail. In principle there are two alternatives: (i) virions could be liberated passively, as it were, as a consequence of the destruction of cells in late stages of infection, or (ii) there might be an active process of virion release. If extracellular virions differed significantly from intracellular particles, it was conceivable that the mechanisms of infection and uncoating, perhaps also the late events in viral gene expression or assembly and release, exhibited striking differences depending on whether intracellular or extracellular virions were used for inoculation. In this communication we will demonstrate that the total extracellular infectivity of Adl2 represents about 25 to 30% of the infectivity of the intracellular virions, but only 2.5 to 3% of the mass of intracellular virions. The specific infectivity of extracellular adenovirions of type 2 (Ad2) or type 12, as determined by focus-forming assay (12), was approximately 10 times higher than that of intracellular virions. Increased infectivity of extracellular virions was observed between 48 and 104 h after infection. The buoyant densities of intracellular and extracellular virions as determined by equilibrium sedimentation in neutral CsCl density gradients were found to be identical. Extra- and intracellular Adl2 *
MATERIALS AND METHODS of Inoculation HEK, HeLa, or KB cells with extracellular Adl2. Semiconfluent monolayers of HEK or HeLa cells were inoculated with 2 ml of extracellular Adl2 per 75-cm2 cell sheet. Extracellular virus represented untreated medium derived from HEK cells previously infected with Adl2. For virus adsorption, 2 h were allowed. Subsequently, the inoculum was removed and replaced by fresh Dulbecco modified Eagle medium (1) supplemented with 10% fetal bovine serum. Intra- and extracellular virus was harvested 48, 72, 96, or 104 h after inoculation. For most experiments, virus was harvested at 72 h after inoculation. Extracellular virus from Adl2-infected HEK cells could also be used for the inoculation of KB cells growing in suspension culture. KB cells were collected by low-speed centrifugation, and the equivalent of 1,000 ml of KB cells was resuspended in 50 ml of medium from Adl2-infected HEK cells. After a 2-h adsorption period, the cells were pelleted and resuspended in fresh spinner medium. In the experiments reported here, suspension cultures were not routinely used. For practical purposes, however, it may be useful to mention that Adl2 can be more reliably produced by inoculating suspension cultures with extracellular Adl2. In a few exploratory experiments, extracellular virus from KB or HeLa cells was also tested as inoculum, but did not prove to be very effective. Purification of extra- and intracellular Adl2. Intracellular virus from Adl2-infected HEK cells was released from the cells by conventional techniques and purified as described earlier (3, 18). Additional purification steps are described below. For the purification of extracellular Adl2, the medium was freed of cells and cellular debris and was subse-
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quently concentrated about 40-fold by vacuum dialysis against Dulbecco modified Eagle medium. This concentrate or cell extracts containing intracellular virus were treated with equal volumes of trichlorotrifluoroethane (Freon 113). The aqueous phase was then layered on top of a composite gradient system (18) in SW41 tubes of the Beckman ultracentrifuge. The gradient system consisted of a preformed CsCl density gradient (5 ml) in 0.02 M Tris-hydrochloride, pH 8.0, ranging from 1.4 to 1.2 g/ml. On top of this gradient a 5 to 20% sucrose gradient (3 ml) in 0.02 M Tris-hydrochloride, pH 8.0, was layered. These gradients were centrifuged in a Beckman ultracentrifuge at 35,000 rpm for 2.5 h at 4°C and contained intra- or extracellular virus in parallel tubes. Virion bands could be detected in the isopycnic density stratum in the CsCl portion of the gradient system. Extracellular virion preparations yielded very faint bands. Intracellular or extracellular virions were removed by puncturing the nitrocellulose tubes from the sides and were rebanded after dilution in buoyant CsCl solution by equilibrium centrifugation. Either virus preparation was further purified by gel filtration over a Sephadex G150 column (17.5 by 0.8 cm in diameter) equilibrated with buoyant CsCl solution in 0.02 M Tris-hydrochloride, pH 8.0, to keep the salt concentration constant, because Adl2 virions have been shown to be unstable to changes in salt concentration (3). Intracellular or extracellular virus was then rebanded twice by equilibrium centrifugation in CsCl density gradients. Adl2-containing fractions were pooled separately for both the extra- and intracellular virus. Locations of virus bands were determined visually and ascertained by focus-forming assays or, for radioactively labeled preparations, by determining radioactivity in aliquot fractions. Immediately before use for infection, focus-forming assay, or biochemical analyses (see below), virus preparations were desalted by passage over a Sephadex G50 column (17.5 by 0.8 cm in diameter) equilibrated with 0.02 M Tris-hydrochloride, pH 7.5. Radioactive labeling of Adl2 preparations. Adl2inoculated HEK cells were labeled with [3H]thymidine (10 ,uCi/ml) (specific activity, 20 to 30 Ci/mmol), [14C]thymidine (about 1 ,uCi/ml) (specific activity, 50 to 60 mCi/mmol), or D-[6-3H]glucosamine (10 ,uCi/ml) (specific activity, 20 to 40 Ci/mmol) by adding the labeled compound directly to the medium 2 h after inoculation. When Ad12-infected cell cultures were labeled with [35S]methionine, the medium was changed 2 h after inoculation to medium devoid of unlabeled L-methionine, and 6.7 ,uCi of [35S]methionine (specific activity, >800 Ci/mmol) per ml was added at 12 or 24 h postinfection. In a few exploratory experiments, Ad12-infected HEK cells were labeled with L-[1-3H]fucose (specific activity, 70 to 90 Ci/mmol), D-[2-3H]mannose (specific activity, 30 to 60 Ci/mmol), or D-[6-3H]galactose (specific activity, 20 to 40 Ci/mmol) (10 ,uCi/ml) in each case. All radiolabeled compounds were purchased from Amersham Buchler, Braunschweig, Germany. Measuring infectious Adl2 by focus formation on HeLa cells and indirect immunofluorescence. The technique of Philipson (12) was used for measuring infectious Adl2 by focus formation on HeLa cells. Nearly confluent monolayers of HeLa cells growing on glass cover slips were inoculated with appropriate dilutions of intra- or extracellular Adl2 in phosphate-buffered saline (PBS) containing 2% fetal bovine serum. After 2 h of adsorption, the inoculum was removed and medium supplemented with 10% fetal bovine serum was added. Between 48 and 72 h after infection, the medium was removed and a 1:10 dilution in PBS of rabbit anti-Adl2 serum was applied to each cover slip for 30 min at room
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temperature. Subsequently, the cells were washed four times in PBS and a 1:10 dilution in PBS of fluoresceinconjugated caprine anti-rabbit immunoglobulin G (GIBCO Laboratories, Grand Island, N.Y.) was added. The cells were again incubated at room temperature for 30 min. The cells were then washed twice in PBS and embedded in glycerol. Fluorescent foci were counted over an ocular grid (1 cm2) in a Zeiss fluorescence microscope, using UV optics. For each sample, fluorescent foci were counted in 10 fields bounded by the ocular grid, and the average number of fluorescent foci per grid was used as a relative measure for Adl2 infectivity. Extraction of viral DNA and restriction analyses. DNA was extracted (5) from purified extra- or intracellular Adl2 after the final gel filtration step. DNA preparations were cleaved with the restriction endonuclease EcoRI, BamHI, HindIII, Mspl, or PstI. Fragments were separated by electrophoresis on 0.5 to 1.5% agarose gels, blotted to nitrocellulose (BA85) filters (14), and hybridized to 32P-labeled Adl2 DNA as described before (15). Adl2 DNA was labeled by nick translation (13), and DNA-DNA hybridization experiments were carried out as outlined elsewhere (19). Adl2-specific
fragments were eventually visualized by autoradiography and quantified by scanning spectrophotometry as described earlier (15). Cleavage patterns of DNA preparations ex-
tracted from intracellular and extracellular virions were compared. This procedure afforded a direct relative quantitation of the amounts of intracellular and extracellular viral DNA and thus of virions. SDS-polyacrylamide gel electrophoresis of Adl2 proteins.
D-[6-3H]glucosamine-
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extra- or
intracellular preparations of Adl2 virions were lyophilized and resuspended in 0.0625 M Tris-hydrochloride buffer, pH 6.8, containing 10% glycerol, 5% mercaptoethanol, and 3% SDS. The solutions were heated to 100°C for 5 min, and proteins were analyzed by electrophoresis on vertical 10% polyacrylamide gels (9), using a 5% stacking gel. The buffer was 0.025 M Tris-hydrochloride-0.19 M glycine-0.1% SDS. After electrophoresis at 250 V under cooling for 3 to 4 h, the gels were prepared for fluorography. Gels were autoradiographed on Kodak XAR-5 film for 1 to 2 weeks. As molecular size markers, [35S]-labeled Adl2 virions were coelectrophoresed in most experiments. In some experiments, virion polypeptides were analyzed on cylindrical polyacrylamide gels as described elsewhere (10). After electrophoresis, the gels were cut into slices, and the radioactivity in each slice was determined by scintillation counting
(10). Synthesis of Adl2-specific DNA. Southern blot and hybridization analyses are suitable methods to estimate the relative amounts of viral DNA produced in Adl2-infected cells. HEK cells were infected with extra- or intracellular Adl2 and, at various times after infection, the nuclear and cytoplasmic fractions were prepared. Cells were washed three
times in Tris-saline; subsequently, 2 ml of 0.01 M Trishydrochloride (pH 7.5)-0.005 M MgCl2-0.01 M NaCl-0.5% Nonidet P-40-O.05% deoxycholate per 75-cm2 cell surface was added. The cells were kept on ice for 10 min and scraped off the plastic surface, and nuclei and cytoplasm were separated by low-speed centrifugation. The total nuclear DNA was extracted by the SDS-proteinase K-phenol method described previously, omitting the CsCl equilibrium centrifugation step (16). The total nuclear DNA was cleaved with the EcoRI restriction endonuclease and analyzed for the presence of Adl2 DNA sequences by electrophoresis (1 ,ug of DNA per slot) on 0.5% agarose gels, by blotting, and
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FIG. 1. Quantitation of virus particles in intracellular and extracellular Adl2 preparations, HEK cells were inoculated with extracellular Adl2 derived from HEK cells, and at 72 h after infection the intracellular and extracellular Adl2 virions were harvested separately and purified as described in the text. Adl2 inocula were standardized by the fluorescent focus-forming assay. To assess the total relative amounts of virions in either compartment, the viral DNA was extracted from the total purified intracellular or extracellular Adl2. Amounts of DNA which corresponded to equivalent portions of intracellular or extracellular Adl2 particles were cut with EcoRI (a) or BamHI (b), the fragments were separated by electrophoresis on a 0.5% agarose gel and transferred to nitrocellulose filters (14), and Ad12 DNA was visualized by hybridization to 32P-labeled Adl2 DNA followed by autoradiography. The viral DNA bands, in particular those corresponding to EcoRI fragment B, C, or D or BamHI fragment A, B, or E, were quantitated by spectrophotometric scanning (15).
by hybridization to 32P-labeled Adl2 DNA. Amounts of viral DNA present in each DNA preparation were compared by scanning the intensity of viral DNA bands in autoradiograms (15). Comparison of buoyant densities of intra- and extracellular Adl2. Monolayers of HEK cells were infected with extracellular Adl2 and labeled with either [3H]thymidine or [14C]thymidine. Intra- and extracellular Adl2 were partly purified from 3H- or 14C-labeled cultures by one cycle of equilibrium centrifugation in CsCI density gradients. Subsequently, 3H-labeled extracellular Adl2 was mixed with 14Clabeled intracellular Ad12 and vice versa, and these mixtures were centrifuged to equilibrium in a conventional CsCl density gradient (3). The gradients were fractionated, and portions of each fraction were counted in a Packard 3395 Tri-Carb liquid scintillation spectrometer. Aqueous samples were dissolved in a toluene-methanol (50:50)-based scintillator as described previously (4). Adsorption of [35S]methionine- or [3H]thymidine-labeled virus to HeLa cells. Previously published techniques were used for adsorption (11, 17). HeLa cells growing in suspension culture were pelleted by centrifugation and resuspended
in 10 ml of PBS per liter of culture. Highly purified [35S]methionine- or [3H]thymidine-labeled intracellular or extracellular Ad12 was added to the cells, which were previously equilibrated for 5 min at 0 or 37°C, in a total volume of 10 ml of PBS. At 0, 5, 10, 15, 30, or 60 min after inoculation, 0.5-ml samples were removed, diluted 10-fold in ice-cold PBS, and immediately sedimented by low-speed centrifugation. The amount of radioactivity that was cell associated at various times after inoculation was determined by scintillation counting. RESULTS AND DISCUSSION The specific infectivity of extracellular Adl2 is about 10-fold higher than that of intracellular Adl2. Intracellular and extracellular Adl2 was extensively purified as described from Adl2-infected HEK cells growing in monolayers. Usually, 10 monolayers of 75 cm2 were inoculated with extracellular Adl2, and virus was harvested at different times between 48 to 104 h postinfection. Adl2 inocula were standardized by the fluorescent focus-forming assay. The total intracellular and extracellular virions from 10 mnonolayers of HEK cells were quantified on the basis of DNA
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FIG. 2. Glycosylation of the fiber polypeptides of intracelullar and extracellular Adl2 virions. Adl2 was replicated in HEK cells in medium containing D-[6-3H]-labeled glucosamine (10 ,uCi/ml). Ad12 inocula had been standardized by the fluorescent focus-forming assay. At 72 h after infection, the intracellular (a) or extracellular (b) Adl2 particles were separately purified. The polypeptide composition and the distribution of the D-[6-3H]-glucosamine label were determined by electrophoresis on cylindrical SDS-polyacrylamide gels (10). The positions of the major virion polypeptides were marked by electrophoresis of [35S]-methionine-labeled Adl2 virions on a separate gel (c). H, Hexon polypeptide; F, fiber polypeptide.
extractable from highly purified virions as described under Materials and Methods. The average of numerous independent experiments was determined. Aliquots of the total intracellular and extracellular Ad12 virion preparations were used to determine the focus-forming activity on HeLa cells. The indirect immunofluorescence technique was used as described elsewhere (12). The results of numerous independent determinations demonstrated that the total infectivity of the extracellular virus amounted to about 25 to 30% of that of the total intracellular virions, but the extracellular virus mass made up only 2.5 to 3.0% of the intracellular yield. Hence, the specific infectivity of the extracellular Adl2 had to be about 10 times higher than that of the intracellular virions. The relative intensities of the intracellular and extracellular Adl2 bands visible in CsCl density gradients had suggested a much lower relative infectivity of the extracellular virus. Similar results were obtained 48, 72, 96, or 104 h after infection, and the relative proportions of extracellular virus particles appeared to remain constant over that period of time. Similar observations were made with human Ad2. Upon inoculation of HeLa cells with intracellular or extracellular Adl2, fluorescent foci could be detected by the focus-forming assay starting at about 24 h after inoculation. At earlier times, the inoculated HeLa cell cultures were unstained as were uninfected HeLa cell control cultures. It proved essential to determine precisely the total amounts of
physical particles present in the intra- and extracellular compartments to evaluate the relative infectivity data on a quantitative basis. From aliquots of highly purified intra- or extracellular Adl2, the viral DNA was extracted and cleaved with restriction endonuclease EcoRI or BamHI, and the fragments were separated by electrophoresis on an agarose gel. After blotting and hybridization to 32P-labeled Adl2 DNA, the amounts of intra- and extracellular Adl2 DNA were compared by autoradiography and spectrophotometric scanning of the autoradiograms (15). The relative amounts of Adl2 DNA extracted from intra- or extracellular virions were taken as direct measures of the mass of Adl2 particles. The results (Fig. 1) demonstrated that the amount of extracellular Adl2 produced corresponded to only 2.5 to 3% of that of intracellular Adl2. This ratio was found between 48 to 104 h after infection. Thus, the specific infectivity of extracellular Adl2 had to be 10 times higher than that of intracellular Adl2 as judged by the focus-forming activities of these virus preparations. The objection could be raised that losses of viral DNA incurred during the extraction of DNA from unequal amounts of Adl2 virions might be disproportionate, and thus the quantitation presented might be invalidated. This possibility was ruled out by the following reconstruction experiment. The DNA in Adl2 virions was labeled with [3H]thymidine. Viral DNA was extracted from an undiluted purified Adl2 preparation and from 10- and 50-fold dilutions
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FIG. 3. Adsorption of intracellular and extracellular Adl2 to HeLa cells. [35S]methionine-labeled Adl2 virions purified from the intracellular (I) or extracellular (E) conlpartment of Adl2-infected HEK cells were adsorbed to HeLa cells at 0 or 37°C as described in the text. Adl2 inocula were standardized according to the same amount of [35S]methionine. At various times after adding virus to HeLa cells, the 3IS activity associated with HeLa cells was determined (see text).
of the same preparation, using standard extraction methods (5). Based on the proportion of [3H]thymidine recovered in DNA from each extraction experiment, it was concluded that recovery of DNA was not dependent on Adl2 concentration. It was, therefore, highly unlikely that DNA was selectively lost during the extraction of extracellular virions. Moreover, it was conceivable that the intracellular Adl2 virions were damaged by ultrasonic treatment and therefore exhibited a relatively lower infectivity. This possibility was ruled out by ultrasonic treatment of the medium from Adl2-infected HEK cells before purification of the extracellular virions. Even under these conditions, the same difference in infectivity between intra- and extracellular virions was observed. The additional possibility was considered that intracellular virions could have been aggregated in a form that would render intracellular particles relatively less infectious due to the agglomeration of many particles into one "infectious unit." The occurrence of such complexes could be ruled out by serially diluting [3H]thymidine-labeled adenovirus preparations in PBS plus 2% fetal bovine serum, or in 0.01 M Tris-hydrochloride-1 mM EDTA (pH 7.5), or in buoyant CsCI solution (5-, 10-, 50-, 100-, 500-, 103-, 104-, 105-fold) and by demonstrating that the decrease in radioactivity was perfectly proportional to the extent of dilution. This proportionality was independent of ionic strength. Thus, there was no evidence whatsoever for the formation of aggregated
complexes. It was concluded that, between 48 and 104 h postinfection,
extracellular Ad12 constituted about 2.5 to 3% by mass of the intracellular Adl2 yield in HEK cells. The specific infectivity of extracellular Ad12 was approximately 10-fold higher than that of intracellular Adl2. Similar experiments performed by infecting human KB or HeLa cells with extracellular Adl2 from HEK cells and by subsequently using extracellular Adl2 from KB or HeLa cells to inoculate KB or HeLa cells did not yield comparable amounts of infectious extracellular Adl2 in comparison to experiments in which HEK cells were used. The extracellular fluids from Adl2-inoculated KB or HeLa cells did cause cytopathic effects, but, for unknown reasons, Adl2 production did often not ensue. DNA and polypeptide composition of intra- and extracellular Adl2. The possibility existed that intra- and extracellular Adl2 preparations differed significantly in their chemical compositions. Thus, it was conceivable that intracellular Adl2 preparations contained an unexpectedly high proportion of young adenovirions (6, 8) and therefore exhibited 10-fold-lower relative specific infectivity. When the polypeptide compositions of [35S]methionine-labeled intracellular and extracellular Adl2 preparations were compared by disruption of the virions and standard SDS-polyacrylamide gel electrophoresis, significant differences in the standard autoradiographic patterns of either Adl2 preparation could not be observed (data not shown). Thus, there was no evidence for a preponderance of young virions in intracellular Adl2. Young virions would have contained high amounts of precursors of virion polypeptides, in particular of polypeptides VI, VII, and VIII (6), and would have caused
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significant changes in the polypeptide patterns of extracellular virions. Young virions of Adl2 have not yet been independently investigated. It has been assumed that differences in polypeptide composition would have been apparent upon SDS-polyacrylamide gel electrophoresis. It cannot be ruled out that some of the structural proteins differ in sequence between intra- and extracellular virions. This possibility will have to be investigated in the future. Since intracellular and extracellular Adl2 were purified by zone velocity sedimentation and by four cycles of equilibrium sedimentation in CsCl density gradients, it could also be ruled out that the Adl2 preparations were contaminated to any significant extent with noninfectious incomplete virions which had been shown to exhibit lower buoyant densities (2). Incomplete virions were removed by this purification procedure and, thus, could not account for differences in specific infectivities. The DNA preparations extracted from intra- or extracellular Adl2 particles were cleaved with restriction endonuclease EcoRI, HindIII, HpaI, MspI, or PstI, and cleavage patterns were compared by blotting and hybridization to 32P-labeled Adl2 DNA. Differences between intra- and extracellular Adl2 DNA could not be detected in this way (data not shown). This result also ruled out the possibility that intra- and extracellular Adl2 DNA might be methylated to different extents at 5'-CCGG-3' sites (7, 16). It was concluded that intra- and extracellular Adl2 particles contained the same viral DNA and had the same polypeptide composition as far as could be determined by standard technology. We have also compared the buoyant
densities of intracellular and extracellular Adl2 preparations in CsCl density gradients and found them to be identical. Do intra- and extracellular Adl2 particles contain different amounts of glycoproteins? There was evidence from previous work (8) that the fiber polypeptides of intracellular Ad2 were glycosylated. Adl2-infected HEK cells were labeled with [3H]glucosamine starting 2 h after infection. Intra- and extracellular virions were purified 72 h after infection. The distribution of D-[6-3H]-labeled glucosamine, the most suitable precursor of glycoproteins in eucaryotes, among the different Adl2 polypeptides in the intra- and extracellular virus preparations was determined by SDS-polyacrylamide gel electrophoresis on cylindrical gels. At the end of the electrophoresis experiment, gels were sliced, individual slices were dried on a filter, and the 3H activity was counted. As a size marker, [35S]methionine-labeled Adl2 was treated in the same way and electrophoresed on a separate gel (Fig. 2). The results demonstrated that a substantial portion of the D-[6-3H]glucosamine label in the intra- (Fig. 2a) and extracellular (Fig. 2b) Adl2 preparations coincided with the hexon and the fiber polypeptides, as already described for Ad2 virions (8). Figure 2b exhibits additional bands at fractions 15, 23, and 57 and a reduced band at fraction 82 in the extracellular virus. These differences were not observed reproducibly and were therefore not considered to be significant. Unresolved label on top of the gradient could not be accounted for. Significant differences in the patterns of labeling by D-[6-3H]glucosamine could not be detected for the polypeptides from intra- and extracellular virions. It was therefore concluded that intra- and extracellular Adl2 did
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not differ detectably with respect to their glycoprotein content. Similar results were obtained with Ad2. For comparison, we also used L-[1-3H]fucose, D-[2-3H]mannose, or D-[6-3H]galactose in attempts to label intraand extracellular Adl2. In comparison with D-[6-3H] glucosamine, both intra- and extracellular Adl2 incorporated by far less label of any of the former compounds. They were, therefore, not used as efficient precursors to label adenoviral glycoproteins. Rates of adsorption of intra- and extracellular Adl2 on HeLa cells. The rates of adsorption of intracellular and extracellular Ad12 were compared. HeLa cells in suspension in PBS were inoculated with [35S]methionine-labeled intracellular or extracellular Adl2. The rates of adsorption of Adl2 were determined at 37 and 0°C as described under Materials and Methods (17). The data presented in Fig. 3 demonstrated that intra- and extracellular Adl2 particles adsorbed to HeLa cells at comparable rates. Thus, the difference in specific infectivity could not have been due to a higher rate of adsorption of extracellular Ad12 particles. Does extracellular Adl2 penetrate or replicate more efficiently than intracellular Adl2? We examined the possibility that extracellular Adl2 was more infectious, because it might replicate more rapidly than intracellular virions. HEK cells growing in monolayers were inoculated with intra- or extracellular virions diluted to equal infectivity titers as determined by focus-forming assay. At various time periods after inoculation (3, 6, 10, 16, 18, 24, or 34 h after infection), total nuclear DNA was extracted as described under Materials and Methods. The DNA was cleaved with the EcoRI restriction endonuclease, fragments were separated by electrophoresis and transferred to nitrocellulose filters, and Adl2-specific DNA was detected by hybridization to 32p_ labeled Adl2 DNA as described above. In this way, the accumulation of Adl2 DNA in infected human cells was determined. The results (Fig. 4) demonstrated that both intra- and extracellular Ad12 initiated viral DNA synthesis between 10 and 16 h postinfection. Thus, there was no significant difference detectable in the time of initiation of viral DNA replication. At early times (3 to 16 h) after infection, somewhat lower amounts of parental Adl2 DNA were found in cells inoculated with extracellular virus (Fig. 4b) as compared with those inoculated with intracellular virus (Fig. 4a). This difference was expected since intra- and extracellular viral inocula had been used at equal titers; thus more intracellular particles had been added to the cells. Yet extracellular virus had replicated to levels comparable to those of intracellular virus by 24 to 34 h after infection. It therefore appeared that Adl2 DNA introduced into cells by extracellular virions had replicated more efficiently than DNA derived from intracellular virions. These findings rendered it unlikely that extracellular Adl2 virions had the capacity to penetrate the cytoplasmic membrane more efficiently than intracellular virions. These analyses thus revealed some differences in viral DNA replication between cells inoculated with intracellular and extracellular Adl2
virions. Conclusions. The data presented here demonstrate that extracellular Adl2 virions propagated on HEK cells had up to 10-fold-higher specific infectivities than intracellular virus particles. It was not readily apparent what had caused the increased infectivities of virions released into the medium. Since the proportion of virus particles excreted into the medium stayed approximately constant between 48 and 104 h after infection, it appeared unlikely that virus release was mainly caused by decay of infected cells. The presumptive
mechanism of an active release of adenovirions is unknown. The present data suggested that a subpopulation of adenovirions produced could be preferentially liberated, possibly due to special structural properties. With the standard analytical tools hitherto applied, we did not succeed in pinpointing significant differences in the composition of extracellular Adl2 as compared to intracellular Adl2 virions. Intra- and extracellular Adl2 seemed to adsorb and penetrate into host cells at comparable rates. Extracellular Adl2 might have an advantage in viral DNA replication. We have not yet investigated in detail the kinetics of late expression, assembly, or release of newly produced virus particles. It was peculiar that the increased infectivity of extracellular Adl2 virions was observed only after infection of human HEK cells. ACKNOWLEDGMENTS We are indebted to P. Gallimore, Birmingham, England, for gifts of primary HEK cells and to Petra Bohm and Gertrud Deutschlander for editorial work. We thank Hanna Mansi-Wothke for medium preparation. This research was supported by the Deutsche Forschungsgemeinschaft through SFB74-C1. LITERATURE CITED 1. Bablanian, R., H. J. Eggers, and I. Tamm. 1965. Studies on the mechanism of poliovirus-induced cell damage. I. The relation between poliovirus-induced metabolic and morphological alterations in cultured cells. Virology 26:100-113. 2. Burlingham, B. T., D. T. Brown, and W. Doerfler. 1974. Incomplete particles of adenovirus. I. Characteristics of the DNA associated with incomplete adenovirions of type 2 and 12. Virology 60:419-430. 3. Doerfler, W. 1969. Nonproductive infection of baby hamster kidney cells (BHK21) with adenovirus type 12. Virology
38:587-606. 4. Doerfler, W. 1970. Integration of the deoxyribonucleic acid of adenovirus type 12 into deoxyribonucleic acid of baby hamster kidney cells. J. Virol. 6:652-666. 5. Doerfler, W., U. Lundholm, and M. Hirsch-Kauffmann. 1972. Intracellular forms of adenovirus DNA. I. Evidence for a
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