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Structural proteins of adenoviruses
VIROLOGY
49,
745-757 (1972)
Structural VIII. Characterization LENNART
PRAGE,
Department of Microbiology, Institute
Proteins
of Adenoviruses
of Incomplete STEFAN
The Wallenberg of Biochemistry,
Particles
HOGLUND, Laboratory,
AND
Uppsala
Uppsala University,
of
Adenovirus
LENNART University, Uppsala,
Type 3’ PHILIPSON
Uppsala,
Sweden,
and
The
Sweden
Accepted June 20, 19Y.9
Incomplete virus particles were isolated from cells infected with adenovirus type 3. More than 30% of the particles recovered at 72 hr after infection were found to be incomplete. Polyacrylamide electrophoresis revealed that incomplete particles were deficient in the two major core polypeptides, but contained enhanced levels of a polypeptide present in complete virions in only minute amounts. The amino acid composition of incomplete particles showed reduced levels of both arginine and alanine compared to virions. These two amino acids are enriched in the core proteins. The main fraction of the incomplete particles contained little, or no, DNA. A small fraction, however, contained DNA which was smaller (about 16 S) than viral DNA, as determined by neutral and alkaline sucrose gradient centrifugation. DNADNA hybridizations revealed that the DNA of the incomplete particles was mainly of viral origin. The incomplete particle preparations contained reduced levels of infectivity compared to complete particles. Infectivity may either be caused by aggregation of complete and incomplete particles or by complementation. INTRODUCTION
Virus particles with incomplete genomes have been found in animal viruses containing RNA (Rhim et al., 1961; Halperen ed al., 1964; Smith et al., 1969; Nonoyama et al., 1970; Maize1 et al’., 1967; Huang et al., 1966; Kingsbury et al., 1970) as well as DNA (Watson et al., 1963; Breedis et al., 1962; Crawford et al., 1962; Blackstein et al., 1969; Yoshiike, 1968 a, b). Virus particles with a density considerably lower than that of complete virions were described in preparations of adenovirus type 12 (Smith, 1965; 1 This work was supported by grants from the Swedish Medical and Natural Science Research Council, the Swedish Cancer Society and the Knut and Alice Wallenbergs’ Foundation. Part of the work was performed at The Department of Microbiology, Rutgers Medical School, New Brunswick, New Jersey 08903, U.S.A., and was supported by grant CA-08851 of the National Cancer Institute.
Schiiojo et al., 1967; Maize1 et al., 1968) and were shown to display a morphology similar to complete virions by electron microscopy (Smith, 1965; Schimojo et al., 1967). “Top components” have also been recovered from adenovirus type 2 preparations (Kiihler, 1962; Maize1 et al., 1968), and they were found to be deficient in some of the core polypeptides present in complete virus particles (Maize1 et al., 1968). The adenoviruses have an icosahedral structure (Horne et aZ., 1959; Wilcox et al., 1963; Valentine and Pereira, 1965; Norrby, 1966) and the three major structural components of their capsids have been characterized in this laboratory (Pettersson et al., 1967, 1968; Petterson and Hoglund, 1969). Additional viral polypeptides have been demonstrated by polyacrylamide gel electrophoresis (Maize1 et al., 1968; Russell et al., 1968; Laver et al., 1967; Prage et al., 1968) 745
PRAGE,
746
H&LUND,
and an arginine-alanine rich protein (AAP) associated with the viral core has been characterized (Laver, 1970; Prage and Pettersson, 1971). The DNA in complete adenovirus particles is a linear, double-stranded molecule of about 23 X lo6 daltons (van der Eb and van Kesteren, 1966; Green et al., 1967b). In this report we describe a population of low density particles from adenovirus type 3. They contain a reduced amount of viral DNA and lack the major core polypeptides, but are enriched in another viral polypeptide only present in minute amounts in complete particles. The infectivity associated with these particles may be due to contamination with complete particles, but it cannot be excluded that it is caused by complementation. MATERIALS
AND
METHODS
Cell cultures and virus production. The prototype of adenovirus type 3 (Ad3) was originally obtained from Dr. Svedmyr (The Municipal Bacteriological Laboratory of Stockholm, Sweden), Spinner cultures of KB cells were infected at a multiplicity of 20-40 fluorescent focus units (FFU) per cell according to an earlier report (Pettersson et al., 1967). Infected cells were harvested after 72 hr, and incomplete and complete virus particles were isolated as described below. Virus labeled with 3H-thymidine (20 Ci/mmole) or carrier-free 32P-phosphate was processed in spinner cultures with the addition of 1 mCi of isotope per liter of culture at 12 hr after infection and were harvested at 72 hr p.i. Isotopes were purchased from New England Nuclear Corporation, Boston, Massachusetts. Isolation of incomplete and complete virus particles. Infected cells were suspended in 0.01 M Tris.HCI pH 8.1 containing 0.5% Nonidet P-40 (Shell Chemical Company) and sonicated at maximal effect for 4 r& in a Raytheon 10 kc sonic oscillator. The sonicate was spun for 15 min at 2000 g, and the supernatant was layered on a cushion of 1 ml CsCl with a density of 1.4 g/ml and centrifuged at 25,000 rpm for 45 min in a Beckman SW 50 rotor. The material on the top of the cushion was collected dropwise from the
AND
PHILIPSON
bottom of the punctured tube, diluted with 0.01 1M Tris .HCl, pH 8.1 and layered on the top of a performed CsCl gradient (2 ml of both 1.4 and 1.25 g/ml CsCl in 0.02 M Tris.HCI, pH 7.4) which was centrifuged at 40,000 rpm for 3.5 hr in the SW 50 centrifuge head. The major opalescent bands were collected separately from the bottom of the tube. The lower of these bands consisted of complete virus particles and was dialyzed against 0.25 1Msucrose, 0.02 M Tris, 0.001 IM MgClz HCl, pH 7.4 with 0.5 % n-butanol at +4”C overnight. A second CsCl gradient centrifugation was included in some experiments. Complete virus particles prepared as described above were considered to be sufficiently purified for the experiments to be presented. The upper band was diluted with 0.01 M TriseHCl, pH 8.1, and layered on 0.5 ml CsCl 1.4 g/ml + 0.5 ml 1.25 g/ml + 3 ml 30 % sucrose in 0.02 M Tris. HCI, pH 7.4 and centrifuged for 2.5 hr at 25,000 rpm in the SW 50 rotor. The visible band between the two CsCl solutions was collected and diluted as described above, and then layered on a preformed CsCl gradient (1.4-1.25 g/ml) and spun at 40,000 rpm for 3.5 hr. The single low density opalescent band was collected and dialyzed agaonst 0.25 M sucrose, 0.02 il!f Tris, 0.001 1M MgClz HCl, pH 7.4, with 0.5 % n-butanol. The dialyzed preparation was considered to be purified incomplete virus particles. Infectivity assays. Infectivity was assayed by fluorescent focus formation in monolayers of KB cells, using an antiserum directed toward purified adenovirus type 2 hexons, or by plaque formation in human embryo kidney (HEK) cells (Flow Laboratories). The fluorescent focus formation method (Philipson, 1961; Thiel and Smith, 1967) was performed as described previously (Philipson et al., 1968), and the results are expressed in fluorescent focus units (FFU). The plaque formation on HEK monolayers was done according to Strohl (1969), and the results are expressed in plaque-forming units (PFU) . Electron microscopy. The preparation of incomplete particles, obtained after CsCl gradient centrifugation, was placed on the specimen support of platinum apertures,
INCOMPLETE
ADENOVIRUS
which were covered by a thin carbon film. Most of the CsCl was removed by touching the solution of the specimen on a surface of a volatile buffer (ammonium acetate) containing 2.5 % glutaraldehyde. Excess material was removed by a filter paper, and the material was negatively contrasted by uranyl oxalate at pH 7.0 or potassium phosphotungstate (KPT) at pH 7.1. The specimens were examined in a Philips 300 electron microscope at 60 kV accelerating voltage. Isolation of viral and cellular DNA. DNA from virus particles was isolated according to Doerfler (1969) with minor modifications. The virus suspensions were dialyzed against 0.01 M TrisaHCl, pH 7.4, 0.001 M EDTA (ethylenediaminetetraacetic acid) and kept in this buffer for 5-10 days at +4’. A tenth volume of Pronase B (Calbiochem) in 0.1 M Tris.HCl, 0.005 M EDTA, 0.002 M mercaptoethanol, pH 7.4 was added to a final concentration of 1 mg/m.l, and the sample was incubated for 30 min at +37”. Sodium dodecyl sulfate (SDS) was then added to a final concentration of 0.3% whereupon the sample was left at room temperature for 1 hr. The sample was shaken with an equal volume of redistilled phenol, saturated with 0.01 M TrisaHCl, pH 7.4, for 15 min at +4” whereafter it was centrifuged for 15 min at 6000 g. The phenol extraction was repeated twice, and the water phase was then shaken twice with an equal volume of ether. The ether was evaporated with nitrogen gas, and the sample was dialyzed against 0.1 X SSC (SSC = 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0). The cellular DNA was prepared as described by Marmur (1961) from 5 X 10’ KB cells suspended in 2 ml of saline EDTA (0.15 M sodium chloride, 0.1 M EDTA, pH 8.0). The isolated DNA was taken up in 0.1 x ssc. DNA-DNA hybridization. A procedure described by Denhardt (1966) was used with slight modifications. Schleicher and Schuell type B-6 filters were soaked in 6 X SSC and thereupon washed with 5 ml of 6 X SSC. 50 or 5 pg of cellular or viral DNA in 10 ml 6 X SSC was adsorbed to the filters, followed by washing with 10 ml of 2 X SSC. The filters were
PARTICLES
747
dried overnight in room temperature before being baked for 3 hr at +80” in vacuum. The DNA adsorbed to the filters had been denatured in 6 X SSC for 7 min at + 100” and thereafter rapidly cooled in Dry Ice and ethanol. The baked filters were preincubated in glass scintillation vials for 6 hr at f65’ in 1 ml 0.02 % Ficoll (Pharmacia), 0.02 % polyvinylpyrrolidone and 0.02% bovine serum albumin in 3 X SSC. After the preincubation the labeled DNA was added in a volume of 0.1 ml, and the filters were incubated for 16 hr at +65”. The labeled DNA had been sonicated with the microtip for 20 set at setting 3 in a Sonifier Cell Disruptor Model W 185D (Heat Systems-Utlrasonic, Plainview, Long Island, New York), heated for 7 min at +lOO” and rapidly cooled in Dry Ice and ethanol. After the incubation the filters were washed five times with 20 ml of 0.01 M TriseHCl, pH 7.4, and dried. The radioactivity was measured in 5 ml of toluenePermafluor (Packard Instrument Company) scintillation fluid. Zonal sedimentation in sucrose density gradients was performed mainly as described by Burlingham and Doerller (1971) on linear 5-20 % (w/v) sucrose gradients on which the samples were layered in 0.1 ml 0.1 X SSC. The neutral sucrose solution was made in 1 M NaCI, 0.01 M TrisaHCI, pH 8.0 with 2 X lo-* M EDTA. The alkaline solution was made in 0.8 M NaOH, 0.2 M NaCl with 2 X lo-* M EDTA. The gradients were centrifuged in a Beckman SW 50 rotor at 39,000 rpm for 5 hr at +4”. They were collected dropwise from the bottom of the tubes (20 or 30 drops per fraction). Degraded herring DNA, 1 mg (Sigma) in PBS was added to each fraction, then trichloroacetic acid (TCA) was added to a final concentration of 10%. The precipitate was pelleted and dissolved in 0.5 ml 0.2 M NaOH and then counted in 5 ml toluene-Permafluor (Packard)-Bio-Solv Solubilizer Formula BBS-3 (Beckman) in proportions 96 : 4: 20 (v/v). Analytical polyacrylamide electrophoresis on 5 % gels in SDS were performed according to Maize1 (1966) and has been described in a previous report (Prage and Pettersson,
748
PRAGE,
HOGLUND,
1971). The gels were stained with 0.01% Coomassie Blue in 7 % acetic acid overnight. Destaining was performed by repeated washings with 7 % acetic acid. Densitometric tracings were recorded at 550 nm with a Gilford spectrophotometer Model 2400 with a linear transporter. Amino acid analysis. Samples of 0.3-0.5 mg were hydrolyzed in 6 M HCl (Pettersson et al., 1967) and analyzed according to Moore et al. (1958) with t,he “Biochrom” amino acid analyzer. Protein detemzination was made by the method of Lowry el al. (1951) with bovine serum albumin (Sigma) as a standard. DNA determination was made according to Burbon (1956) with degraded herring DNA (Sigma) as a standard. The samples in 5 % perchloric acid were heated for 20 min at +70”, and the precipitate was spun down. Both the supernatant and the precipitate was tested for DNA. The color was developed at +28” for 18-20 hr.
AND
PHILIPSON RESULTS
Characterization of Iwomplete Particles Incomplete virus particles were purified from AdS-infected KB cells as described in Materials and Methods. To avoid cont’amination with soluble proteins, a centrifugation t,hrough 30% sucrose on to a cushion of CsCl was introduced before the last CsClgradient centrifugation. The purified incomplete particles labeled with 3H-thymidine was mixed with 32P-labeled complete virions and run on a CsCl gradient (Fig. 1). The incomplete particles banded at a density of 1.310 g/ml, whereas the complete virions were recovered at 1.355 g/ml. The incomplete particle band in the CsCIgradients appeared as two very close bands which could not be satisfactorily separated. Optical density at 280 nm and radioactivity was measured in 2-drop fractions of a CsClcentrifugation of a purified preparation of incomplete particles labeled with 3H-thymi,J/CC
CPL I-
1200
1000
600
FRACTION
NUMBER
FIG. 1. CsCl gradient of a mixture of complete and incomplete Ad3 virus particles. The complete particles were labeled with 3*P (-) and the incomplete with 3H-thymidine (--). The inset shows a section of a CsCl gradient of purified incomplete Ad3 particles labeled with aH-thymidine ( -1 where the optical density at 280 nm (-) was determined.
INCOMPLETE
ADENOVIRUS
dine. The peak of the radioactivity was recovered at a density of 1.307 g/ml whereas the maximum of optical density was found at about 1.298 g/ml (Fig. 1, inset). Thus, it appears that only a minor fraction of the incomplete particles contain DNA, whereas the majority of the particles contain very little, or no, DNA. The lower of the two bands seen in CsCl gradients of incomplete Ad3 particles probably represents the DNA containing particles. In this report the combined bands will be treated as a single population. Complete and incomplete particles were TABLE
1
Armum OF INCOMPLETE AND COMPLETE Ad3 VIRUS PARTICLES ISOLATED FROM INFECTED KB-CELLS AT COMPLETION OF THE INFECTIOUS CYCLE
Preparation
Ib II
Protein bg)(l Ad3, complete
Ad3, incomplete
5040 (67) 9000 (63)
2510 (33) 5220 (37)
a The percentage distribution of protein in each type of particle is given within the brackets. b The preparations were derived from 5 X lo* t,o 109 infected KB cells.
PARTICLES
749
isolated from the same infected cultures, and the amount of protein in each population was determined. Table 1 shows that the incomplete particles represent about 30 % of the yield at 72 hr after infection as measured by protein determination. It should be noted, however, that the incomplete particles have been subjected to two more centrifugations than the complete particles. The relative amounts of the incomplete particles presented in Table 1 should therefore represent minimum values. The incomplete particles from the CsCl gradients were examined in the electron microscope (Fig. 2). Particles were observed where the contrast agent appeared to have penetrated into the capsids, however, about 20% of the particles appeared as morphologically intact without any contrast agent in the interior. The rim of the empty particle appeared occasionally wider than anticipated from a single row of capsomers (Fig. 2, inset), indicating that the capsids might have collapsed from one side and accumulated the contrasting agent in a sac at the top, rather than t’hat the contrast agent had penetrated into the empty capsid. Goniometer analysis has shown that fragile particles,
such as incomplete
virus particles,
FIG. 2. Electron micrograph of purified incomplete Ad3 particles contrasted by 2% KPT at pH 71. as described in Materials and Methods. The inset (X 220,000) shows a projection with an approximately hexagonal outline of the rim of the particle (contrasted by 1% uranyl oxalate at pH 7.0).
750
PRAGE, HOGLUND,
AND PHILIPSON
are flattened during drying of the specimen (Hiiglund and Blomqvist, 1972) although the contrast agent and the glutaraldehyde fixation should stabilize them. Composition of the Incomplete Particles
The amounts of protein and DNA in complete and incomplete particles are shown in Table 2. The incomplete particles contained only 0.5-0.6% DNA whereas the complete virions contained 14-16 %. The differencesin polypeptide composition between complete and incomplete particles were analyzed by SDS polyacrylamide gel electrophoresis. Three gels were run in parallel, one with complete, a second with incomplete particles and a third with a mixture of the two samples. All samples contained the same amount of hexons to allow semiquantitative as well as qualitative comparison (Fig. 3). The densitometric tracings of t,he stained gels show that the incomplete particles lack the two major core polypeptides (peak V, and VI-VII, according t’o the nomenclature of Maize1 et al., 1968) as well as a fast migrating polypeptide (polypeptide IX), but contain normal amounts of hexon and penton (peak II and III-IV). The incomplete particles contain a third major polypeptide (polypeptide A) which is represented in complete particles only in minute amounts. The electrophoresis of mixed samples suggeststhat this polypeptide corresponds to a polypeptide in complete virions, since no new peaks appeared, but increase in absorbance is seen at this position of polypepOideA. The polyacrylamide gel electrophoresis
I
d 2
3
4 CM
FIG. 3. Densitometric tracings of polyacrylamide gels run in parallel at 5 V/cm for 3.5 hr in neutral SDS. The anode is to the right. SDStreated complete particles (A), incomplete particles (B), and a mixture of complete and incomplete particles (C) are shown. The absorbance is given in arbitrary units. The peaks are numbered according to the nomenclature of Maize1 et al. (1968).
thus indicated that the incomplete particles lacked the arginine-alanine-rich protein TABLE 2 (AAP) which is the major core protein COMPOSITION OF~NCOMPLETEAND COMPLETE Ad3 (polypeptide VI-VII; Prage and Pettersson, VIRUS PARTICLES 1971). The amino acid composition of the incomplete particles presented in Table 3, Preparation Protein DNAa together with the composition of complete ells) old --Ad3 particles and the Ad3 AAP, agreeswith 1 Complete 168 28 (14.3) this interpretation. Incomplete 684 3.4 (0.5) Incomplete particles contain relatively II Complete 300 56 (15.7) less arginine and alanine, and more glutamic Incomplete 360 2.2 (0.6) and aspartic acid as well as leucine, than 0 The percentage of DNA is given within par- complete virions. The AAP is rich in entheses. arginine and alanine and poor in leucine and
INCOMPLETE
ADENOVIRUS
glutamic and aspartic acid as compared to complete particles.
TABLE 3 AMINO ACID COMPOSITION” Amino acid
Residues per 100 residues Ad3 virion
-
Characterization of the DNA from Incomplete Particles
-
T
Ad3 incomplete particle
Ad3 AAP”
_-
3.7 f 0.3 3.8 & 0.1 3.9 zk 0 1.8 i 0.6 1.4 z!z 0.1 1.6 f 0.1 8.5 f 0.2 5.6 i 0.2 213.2 i 2.0 7.2 i 0.8 12.3 f 0.2 13.2 f 0.2 8.0 f 0.1 7.5 l 0 8.5 i 0.5 7.2 f 0.1 5.4 i 0.2 6.6 f 0 2.4 i 0.1 7.7 f 0.2 9.3 f 0.2 7.4 i 0.5 6.5 f 0.1 6.4 f 0 8.0 f 2.0 7.8 f 0.1 7.0 f 0 7.7 f 0.1 19.4 f 0.2 8.7 i 0 ).05 f 0.05 Trace N.D.” 7.4 f 0.8 6.3 f 0.3 6.1 f 0.3 ND 2.9 f 0.2 3.0 f 0.2 3.6 f 0 2.5 f 0 3.5 f 0 8.5 f 0.1 2.2 f 0.4 7.2 f 0 5.0 z!2 0.1 0.8 f 0.4 4.9 i 0 4.3 rk 0 ND 3.8 h 0 * The figures given are the mean values of two different preparations. b Not detectable. c From Prage and Pettersson (1971).
LYS His Ax ASP Thr Ser Glu Pro GUY Ala w-cys Val Meth Ile Leu Tyr Phe
2000
751
PARTICLES
3H-thymidine labeled DNA was isolated from incomplete and complete Ad3 particles and subjected to sedimentation analysis in neutral and alkaline sucrose gradients. The samples were run in parallel at each pH, but in separate tubes. Figure 4 shows that the DNA derived from incomplete particles is smaller (around 16 S) than the complete viral genome, which has a sedimentation coefficient of 32 S at neutral and 34 S at alkaline pH (Burlingham et al., 1971). The neutral sucrose gradient with incomplete DNA showed a shoulder on the heavy side which was not seen in the alkaline gradients. The nature of this fast sedimenting DNA is unclear at present. To determine whether the DNA in the incomplete particles was of viral or cellular hybridizations were origin DNA-DNA performed with cellular or complete Ad3 viral DNA on the filters. The results in Table 4 show that almost all the DNA C
* i
i
I
2
3
4 EFFLUENT
I VOLUME
2
3
4
(ML)
FIG. 4. Sedimentation of DNA labeled with 3H-thymidine. The two left panels (A and B) show the sedimentation at neutral pH and the two right panels (C and D) at alkaline pH. DNA was isolated from complete (A and C) and incomplete (B and D) Ad3 particles.
752
PRAGE, TABLE
HOGLUND,
AND PHILIPSON
4
from incomplete particles hybridized with viral DATA, and only a small fraction with the cellular DXA. The incomplete Ad3 viral particles thus seem to contain small pieces of the viral genome.
DNA-DNA
HYBRIDIZATION OF DNA FROM COMPLETE AND INCOMPLETE Ad3
VIRUS PARTICLESO Source of radioactiw DNAb
:I
Radioactivity
bound to filters (cpm)
Infectivity
lontrol
DNA on fillterse
fdtersd
-Expt. I Ad3, complete particles (0.4 rg;6004wm) Ad3, incomplete particles (0.2 pg; 1498 cpm) Expt. II Ad3, complete
Ad3, complete
-I-
The infectivity of preparations of complete and incomplete Ad3 particles was determined with both the fluorescent focusforming and the plaque-forming methods. In Table 5 the titers of different preparatlons are listed. The infectivity of the incomplete particles is about 4 log units lower than that of a corresponding amount of complete particles when the quantitation is based on the amounts of protein. Since the fluorescent focus method is more convenient than the plaque method, the former was used for most experiments. The FFU, however, only measures accumulations of hexon in the cells, and it must not necessarily reflect a productive infection. The results in Table 5, however, show that the FFU and PFU data correspond, thereby validating the former as a measure of infectivity. It was feasible that t.he infectivity in preparations of incomplete particles was due to contamination with complete virions trailing behind the bulk of virions in the CsCl gradients, especially as these were harvested dropwise from the bottom of the tubes. Incomplete particles were mixed in excess with complete virus particles and subjected to a CsCl gradient centrifugation. The
KB
____
4092, 4012
294, 174
101
1218, 1093
134, 92
19
6880, 6970
590, 472
690
particles (0.4 pg; 6464 cpm) Ad3, incomplete 2373, 2509 346 particles (0.2 pg; 2417cpm) 899, 1222 KB (2.5 rg; 2350, 86, 73 cpm) I -i a All results are corrected for background
142
ra-
dioactivity. b The amount and the radioactivity added to each filter are given within brackets. The added
radioactivity was determined by spreading the radioactive DNA on a filter, drying it under a heating lamp, and counting it in scintillation liquid as described in Materials and Methods. c In Expt. I all filters had 5 rg DNA. In Expt. II the Ad3 filters were made with 5 yg DNA and the KB filters with 50 pg DNA.
d Control filters were made without DNA. TABLE INFECTIVITY
Preparation I Complete Incomplete II Complete Incomplete III Complete Incomplete IV Complete Incomplete V Complete Incomplete
IN PREPARATION
5
OF INCOMPLETE AND COMPLETE A&
FFU/ml
FFU/e
1.1 X 10’0 2.6 X lo6
3.5 x 7.5 x 4x 2.8 x
of Incomplete Particles
10’0 106 10’0 106
PFU/ml 2.4 6.0 3.0 1.9 6 5.6
5.1 8.3 1.8 1.2
X x x x
107 102 10’ 103
VIRUS PAk~~rc~~s
X X x X X X
109 1Oj 10’0 10” 10”’ lo6
2 x 106
PFu/,p
1.8 1.7 2 3.1
x x x x
107 103 10’ 103
2.2 x 103
INCOMPLETE
ADENOVIRUS
infectivity was scored throughout the gradient and the result is seen in Fig. 5. A distinct peak of infectivity is seen at the density of incomplete particles. Thus, the infectivity cannot be explained only by trailing from the complete virion peak, but these results do not exclude that complete particles remained trapped in the band of the incomplete particles. Because the amount of DNA in incomplete particles is reduced, it would be expected that several incomplete particles are needed to obtain a productive infection, which infers that the infectivity should display a multihit curve when plotted against particle concentration. Figure 6 shows the hit-curves obtained with the incomplete as well as the complete particles. The slope of the infectivity versus particle dilution of complete particles is consistent with a one-hit event leading to productive infection. The corresponding curves for the incomplete particles display slopes which are less steep than that for the complete particles. Such slopes may FFWML IO6
tc
PARTICLES
753
T
FIG. 6. The infectivity of different dilutions of preparations of incomplete (X---X) and (a- - -0) and complete (O--O) Ad3 particles. The outer scale applies to the complete, and the inner scale to the incomplete particles.
be observed if the infective particles contained interfering particles which were not, or less, infectious. Dilution of such mixtures should give proportionally higher infectivity as the probability of interference ‘between the two types of particles decreases with dilution. DISCUSSION
“\
I37
134
‘!30
4
i FRACTION
NUMBER
FIG. 5. CsCl gradient centrifugation of a mixture of purified incomplete and complete Ad3 particles. The infectivity of the indicated fractions was measured by the fluorescent focus-forming method. The incomplete particles were present in excess (about loo-fold) in the centrifuged mixture.
A population of virus particles with a density 0.05 g/ml lower than compIete virions was found in cells infected with adenovirus type 3. Such a low density implies a low content of DNA and suggests that the particles are more deficient than the defective virions described by Rlak (1971). The population of incomplete particles described in this report was, however, not homogeneous, since two very close bands could be seen in CsCl gradients when it was subjected to equilibrium density centrifugation. As a biochemically satisfactory separation of these two bands was difficult to achieve, the whole population was characterized as if it were homogeneous. Adenovirus type 3 was chosen for this study because it gives only two major bands
754
PRAGE, HOGLUND,
in CsCl gradients, the incomplete and the complete virion bands, which are present in a ratio of 1: 2 as calculated from the amount of protein in each population. There are only traces of two or three bands in between these. The yield of incomplete and complete Ad3 particles is furthermore very high. Several low density bands are seen in CsCl gradient runs of Ad2, but these are all faint (Maize1 et al., 1968; Prage, unpublished observations) and therefore not suited for biochemical characterization. The band patterns of Ad12 preparations is, interestingly, similar to the Ad3 pattern, with a single major top component of low density (Smith, 1965; Prage, unpublished observations). The yield of Ad12 is, however, low (Green et al., 1967a). The polypeptide composition of the incomplete particles revealed a pronounced deficiency of the major core proteins, which was also found by Maize1 et al. (1968) in DNA-lacking top components of Ad2 and Ad12. The lacking core proteins (polypeptides V and VI-VII according to Maizel’s nomenclature) amounts to about 20 % of the proteins in the virion (Maize1 et al., 1968; Prage and Pettersson, 1971). Interestingly, the incomplete particles contained an enhanced amount of a polypeptide that is present in complete virions only in minute amounts. It should be noted that any quantitative interpretations based on densitometric tracings of stained gels should be regarded with caution, since various polypeptides might bind the stain with different efficiencies. The amino acid composition confirmed that the incomplete Ad3 particles are deficient with regard to the arginine-alaninerich protein (AAP), which is the major core protein. These findings are correspondent to results obtained with naturally occurring empty polio capsids (Maize1 et al., 1967). These lack two polypeptides (VP2 and VP4), but are enriched in another polypeptide which is present in small amounts in complete virions (NCVPG or VPO). Although these two viral systems are unrelated, it might well be that they both mirror similar assembly mechanisms. Experiments are cur-
AND PHILIPSON
rently performed to elucidate the relation of the enriched protein in incomplete particles to the other viral polypeptides and to the capsid assembly process. The incomplete particles contained about 0.5 % DNA, which means that if the DNA is equally distributed among all the incomplete particles, each particle will contain approximately 3% of the amount present in complete Ad3 virions, assuming that they have only 80 % of the protein amount in complete particles. If the DNA is represented by a single piece, this should have a molecular weight of 7 X lo5 daltons, assuming that the complete Ad3 genome has a molecular weight of 23 X lo6 daltons (M. Green, quoted by Schlesinger, 1969). A rough estimate of the size of the incomplete DNA could be calculated from the sucrose gradients shown in Pig. 4. Assuming a sedimentation coefficient of 32 S at neutral and 34 S at alkaline pH (Burlingham et al., 1971) for the complete Ad3 genome, the incomplete DNA has a coefficient of about 16 S under both conditions. According to Studier (1965) this corresponds to a molecular weight of about 3.5 X lo6 ddtons which is 5-fold higher than the 7 X lo5 daltons calculated above. This discrepancy can be explained if only a fraction of the incomplete particles contained the bulk of DNA, which was suggested from the distribution of labeled DNA in the CsCl gradients. The electron micrograph also favored this interpretation since about 20 % of the incomplete particles was stained as complete particles, and may represent the DNA-containing incomplete particles. The DNA in the incomplete particles is probably not of cellular origin since almost all of it hybridized with viral DNA, although the presence of a small fraction of cellular DNA cannot be excluded. The small size speaks against that it is contaminating complete viral DNA. If this was the case, the contaminating virus particles should give an infectivity titer which is not less than 2 % of the infectivity in a corresponding amount of complete particles. The results show that the infectivity is more than lOOfold lower than the assumed 2%, thus indicating that the DNA in the incomplete
INCOMPLETE
ADENOVIRUS
PARTICLES
755
plete particles, as well as the explanation of particle preparations is not degraded DNA the different patterns of incomplete popuobtained from complete Ad3 virions during lations in CsCl gradients among different the preparation. The discrete size of the DNA in the in- types of adenoviruses is unknown. It complete particles, as suggested from the should be noted that the particles described distinct band in CsCl gradients as well as in this report are harvested at one single time point, after infection, and it is not from the sucrose gradient analysis indicates that the DNA pieces are produced by a improbable that the ratios between incomspecific mechanism. Whether they have any plete and complete virus particles differ relation to the endonucleolytic activity through the infectious cycle, which also may described by Burlingham et al. (1971) apply to the pattern in CsCl gradients. A proper study of the kinetics of the synthesis remains to be seen. It should also be noted that the same authors detected slowly sedi- of incomplete particles might give some information on the assembly mechanism for menting viral DNA in cells infected with adenoviruses. Ad2 or Ad12 (Burlingham and Doerfler, 1971). ACKNOWLEDGMENTS The incomplete particle preparations contained infectivity, although it was 4 log We are indebted to Dr. D. Eaker and Dr. R. Thorzelius for aid with the amino acid analysis. units lower than in corresponding amounts The skillful technical assistance provided by Ms. of complete particles. A contamination with complete virions is, however, very diflicult to I. Htibinette is also acknowledged. rule out at this low level. CsCl gradient REFERENCES centrifugations of incomplete particles have been observed to give a small amount of BLACKSTEIN, M. E., STANNERS, C. P., and FARMILO, A. J. (1969). Heterogeneity of polyoma infectivity at the density of complete virus DNA: Isolation and characterization of particles (unpublished observations). non-infectious small supercoiled molecules. J. The infectivity in the incomplete particle Mol. Biol. 42,301-313. preparations displayed a hit curve which BREEDIS,C.,BERWICK, L.,and ANDERSON, T.F. was not compatible with a viral genome (1962). Fractionation of Shope papilloma virus piece, which is about 15% of the complete in cesium chloride density gradients. v+oZogy genome. In such a case, the slope should be 17,84-94. BURLINCHAM, B. T., and DOERFLER, W. (1971). steeper than the one hit curve observed with Three size-classes of intracellular adenovirus complete particles. The results showed a hit deoxyribonucleic acid. J. Virol. 7,707-719. curve with a slope below unity, indicating that the observed infectivity was subject to &JRLINGHBM, B. T., DOERFLER, W., PETTERSSON, U., and PHILIPSON, L. (1971). Adenovirus endointerference. Since noninfectious particles nuclease : Association with the penton of adenomight interfere with infectivity caused by virus type 2. J. Mol. Biol. 60, 4S64. complete particles as well as by compleBURTON, K. (1956). A study of the conditions and menting DNA-containing incomplete parmechanism of the diphenylamine reaction for ticles, this analysis will not allow us to sepathe calorimetric estimation of deoxyribonucleic rate between the two alternatives, conacid. Biochem. J. 62,315323. CRAWFORD,L. V., CRAWFORD,E. M., and WATtamination or complementation. SON, D. H. (1962). The physical characteristics From this evidence we suggest that the of polyoma virus. I. Two types of particles. infectivity present in preparations of inVirology 18, 179-176. complete particles is due to contamination with complete Ad3 virions, although a DENHARDT, D. T. (1966). A membrane filter technique for the detection of complementary DNA. complementation mechanism obscured by Biochem. Biophys. Res. Commun. 23,641646. interference cannot be excluded at present. DOERFLER, W. (1969). Nonproductive infection of The contamination is probably caused by baby hamster kidney cells (BHK21) with adenotrapping of a few complete virus particles virus type 12. Virology 38, 587-606. in an excess of incomplete particles. GREEN, M., PIRA, M., and KIMES, R. C. (1967a). Biochemical studies on adenovirus multiplicaThe biological significance of the incom-
756
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H&LUND,
tion. XII. Plaquing efficiencies of purified human adenoviruses. Virology 31,562-565. GREEN, M., PrR.4, M., KIMES, R., WENSINK, P., MACHATTIE, L., and THOMAS, C. A. (196713). Adenovirus DNA. I. Molecular weight and conformation. Proc. :\:at. Acad. Sci. U.S.A. 57, 1302-1309. HALPF,REN, S., EGGERS, H. J., and TAMM, I. (1964). Complete and coreless hemagglutinating particles produced in ECHO 12 virus-infected cells. Virology 23, 81-89. H~GLUND, S., and BLOMQVIST, G. (1972). Some remarks on goniometer analysis of particulate objects. J. Ultrastruct. Res., in press. HORNE, R. W., BRF,NNI.;R, S., WATERSON, A. P., and WILDY, P. (1959). The icosahedral form of an adenovirus. J. Mol. Biol. 1, 84-86. HUANG, A. S., GRI
AND
PHILIPSON
fonated polystyrene resins-an improved system. Anal. Chem. 30, 1185-1190. NONOY~~MA, M., WATAS.~BIC, Y., and GRAH.AM, A. F. (1970). Defective virions of reovirus. J. Viral. 6,226-236. NORRBY, E. (1966). The relationship between the soluble antigens and the virion of adenovirus type 3. I. Morphological characteristics. ViroZogy28,236-248. PETTERSSON, U., and HOGLUND, S. (1969). Structural proteins of adenoviruses. III. Purification and characterization of adenovirus type 2 penton antigen. Virology 39,90-R%. PF;TTERSSON,U.,PHILIPSON, L.,and H~GLUND, S. (1967). Structural proteins of adenoviruses. I. Purification and characterization of adenovirus t,ype 2 hexon ant,igen. Virology 33,575~590. PETTERSSON, U.,PHILIPSON, L., and HBGLUND,S. (1968). Structural proteins of adenoviruses. II. Purification and characterization of adenovirus type 2 fiber antigen. Virology 35,204-215. PHTLIPSON, L. (1961). Adenovirus assay by the fluorescent cell-counting procedure. Virology 15, 263-268. PHILIPSON, L., LONBRRG-HOLM, K., and PE:TTISRSSON, U. (1968). Virus receptor interaction in an adenovirus system. J. ViroZ. 2, 1064-1075. PRAGE, L., and PETTGRSSON, U. (1971). Structural proteins of adenoviruses. VII. Purification and properties of an arginine-rich core protein from adenovirus type 2 and t,ype 3. Virology 45, 364373. PR.~GE, L., PETTERSSON, U., and PHILIPSON, L. (1968). Internal basic proteins in adenovirus. Virology 36,508-511. RHIM, J., SMITH, K. O., and MELNICK, J. (1961). Complete and coreless forms of reovirus (ECHO 10). Ratio of number of particles to infective units in the one-step growth curve. Virology 15, 428435. RUSSELL, W. C., LAVER, W. G., and SANDERSON, P. J. (1968). Internal components of adenovirus. Nature 219, 1127-1130. SCHIMOJO, H., YAM.ISHITA, T., YAMAMOTO, H., SUZUKI, E., and ABE, C. (1967). Empty particles of adenovirus 12. Jap. J. Med. Sci. Biol. 19,1-8. SCHLESINGER, R. W. (1969). Adenoviruses: The nature of the virion and of controlling factors in productive or abortive infection and tumorigenesis. Advan. Virus Res. 14, l-61. SMITH, K. 0. (1965). I. Quantitative correlations between some physical, antigenic and infectious properties. J. Immunol. 94,976-989. SMITH, R. E., ZWEERINK, H. J., and JOKLIK, W. K. (1969). Polypeptide components of virions, top components and cores of reovirus type 3. Virology 39,791-810. STROHL, W. A. (1969). The response of BHK21
INCOMPLETE
ADENOVIRUS
cells to infection with type 12 adenovirus. I. Cell killing and T antigen synthesis as correlated viral genome functions. Virology 39,642-652. STUDIER, F. W. (1965). Sedimentation studies of the size and shape of DNA. J. MoZ. Biol. 11. 373-390. THIEL, J. F., and SMITH, K. 0. (1967). Fluorescentfocus assay of viruses on monolayers in plastic petri dishes. Proc. Sot. Exp. Biol. Med. 125, 892495. V.LLENTINE, R. C., and PEREIRA, H. G. (1965). Antigens and structure of the adenovirus. J. Mol. BioZ. 13,13-20. VAN DER EB, A. J., and VAN KESTEREN, L. W. (1966). Structure and molecular weight of the DNA of adenovirus type 5. Biochim. Biophys. Acta 129,441~444. WATSON, D. H., RUSSELL, W. C., and WILDY, P.
PARTICLES
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(1963). Electron microscopic particle counts on herpes virus using the phosphotungstate negative staining technique. Virology 19, 250-260. WILCOX, W. C., GINSBERG, H. S., and ANDERSON, T. F. (1963). Structure of type 5 adenovirus. II. Fine structure of virus subunits. Morphological relationship of structural subunits to virus-specific soluble antigens from infected cells. J. Exp. Med. 118,307314. YOSHIIKE, K. (1968a). Studies on DNA from lowdensity particles of SV40. I. Heterogenous defective virions produced by successive undiluted passages. Virology 34, 391401. YOSHIIKE, K. (1968b). Studies on DNA from lowdensity particles of SV40. II. Noninfectious virions associated with a large plaque variant. Virology
34,402-409.
49,
745-757 (1972)
Structural VIII. Characterization LENNART
PRAGE,
Department of Microbiology, Institute
Proteins
of Adenoviruses
of Incomplete STEFAN
The Wallenberg of Biochemistry,
Particles
HOGLUND, Laboratory,
AND
Uppsala
Uppsala University,
of
Adenovirus
LENNART University, Uppsala,
Type 3’ PHILIPSON
Uppsala,
Sweden,
and
The
Sweden
Accepted June 20, 19Y.9
Incomplete virus particles were isolated from cells infected with adenovirus type 3. More than 30% of the particles recovered at 72 hr after infection were found to be incomplete. Polyacrylamide electrophoresis revealed that incomplete particles were deficient in the two major core polypeptides, but contained enhanced levels of a polypeptide present in complete virions in only minute amounts. The amino acid composition of incomplete particles showed reduced levels of both arginine and alanine compared to virions. These two amino acids are enriched in the core proteins. The main fraction of the incomplete particles contained little, or no, DNA. A small fraction, however, contained DNA which was smaller (about 16 S) than viral DNA, as determined by neutral and alkaline sucrose gradient centrifugation. DNADNA hybridizations revealed that the DNA of the incomplete particles was mainly of viral origin. The incomplete particle preparations contained reduced levels of infectivity compared to complete particles. Infectivity may either be caused by aggregation of complete and incomplete particles or by complementation. INTRODUCTION
Virus particles with incomplete genomes have been found in animal viruses containing RNA (Rhim et al., 1961; Halperen ed al., 1964; Smith et al., 1969; Nonoyama et al., 1970; Maize1 et al’., 1967; Huang et al., 1966; Kingsbury et al., 1970) as well as DNA (Watson et al., 1963; Breedis et al., 1962; Crawford et al., 1962; Blackstein et al., 1969; Yoshiike, 1968 a, b). Virus particles with a density considerably lower than that of complete virions were described in preparations of adenovirus type 12 (Smith, 1965; 1 This work was supported by grants from the Swedish Medical and Natural Science Research Council, the Swedish Cancer Society and the Knut and Alice Wallenbergs’ Foundation. Part of the work was performed at The Department of Microbiology, Rutgers Medical School, New Brunswick, New Jersey 08903, U.S.A., and was supported by grant CA-08851 of the National Cancer Institute.
Schiiojo et al., 1967; Maize1 et al., 1968) and were shown to display a morphology similar to complete virions by electron microscopy (Smith, 1965; Schimojo et al., 1967). “Top components” have also been recovered from adenovirus type 2 preparations (Kiihler, 1962; Maize1 et al., 1968), and they were found to be deficient in some of the core polypeptides present in complete virus particles (Maize1 et al., 1968). The adenoviruses have an icosahedral structure (Horne et aZ., 1959; Wilcox et al., 1963; Valentine and Pereira, 1965; Norrby, 1966) and the three major structural components of their capsids have been characterized in this laboratory (Pettersson et al., 1967, 1968; Petterson and Hoglund, 1969). Additional viral polypeptides have been demonstrated by polyacrylamide gel electrophoresis (Maize1 et al., 1968; Russell et al., 1968; Laver et al., 1967; Prage et al., 1968) 745
PRAGE,
746
H&LUND,
and an arginine-alanine rich protein (AAP) associated with the viral core has been characterized (Laver, 1970; Prage and Pettersson, 1971). The DNA in complete adenovirus particles is a linear, double-stranded molecule of about 23 X lo6 daltons (van der Eb and van Kesteren, 1966; Green et al., 1967b). In this report we describe a population of low density particles from adenovirus type 3. They contain a reduced amount of viral DNA and lack the major core polypeptides, but are enriched in another viral polypeptide only present in minute amounts in complete particles. The infectivity associated with these particles may be due to contamination with complete particles, but it cannot be excluded that it is caused by complementation. MATERIALS
AND
METHODS
Cell cultures and virus production. The prototype of adenovirus type 3 (Ad3) was originally obtained from Dr. Svedmyr (The Municipal Bacteriological Laboratory of Stockholm, Sweden), Spinner cultures of KB cells were infected at a multiplicity of 20-40 fluorescent focus units (FFU) per cell according to an earlier report (Pettersson et al., 1967). Infected cells were harvested after 72 hr, and incomplete and complete virus particles were isolated as described below. Virus labeled with 3H-thymidine (20 Ci/mmole) or carrier-free 32P-phosphate was processed in spinner cultures with the addition of 1 mCi of isotope per liter of culture at 12 hr after infection and were harvested at 72 hr p.i. Isotopes were purchased from New England Nuclear Corporation, Boston, Massachusetts. Isolation of incomplete and complete virus particles. Infected cells were suspended in 0.01 M Tris.HCI pH 8.1 containing 0.5% Nonidet P-40 (Shell Chemical Company) and sonicated at maximal effect for 4 r& in a Raytheon 10 kc sonic oscillator. The sonicate was spun for 15 min at 2000 g, and the supernatant was layered on a cushion of 1 ml CsCl with a density of 1.4 g/ml and centrifuged at 25,000 rpm for 45 min in a Beckman SW 50 rotor. The material on the top of the cushion was collected dropwise from the
AND
PHILIPSON
bottom of the punctured tube, diluted with 0.01 1M Tris .HCl, pH 8.1 and layered on the top of a performed CsCl gradient (2 ml of both 1.4 and 1.25 g/ml CsCl in 0.02 M Tris.HCI, pH 7.4) which was centrifuged at 40,000 rpm for 3.5 hr in the SW 50 centrifuge head. The major opalescent bands were collected separately from the bottom of the tube. The lower of these bands consisted of complete virus particles and was dialyzed against 0.25 1Msucrose, 0.02 M Tris, 0.001 IM MgClz HCl, pH 7.4 with 0.5 % n-butanol at +4”C overnight. A second CsCl gradient centrifugation was included in some experiments. Complete virus particles prepared as described above were considered to be sufficiently purified for the experiments to be presented. The upper band was diluted with 0.01 M TriseHCl, pH 8.1, and layered on 0.5 ml CsCl 1.4 g/ml + 0.5 ml 1.25 g/ml + 3 ml 30 % sucrose in 0.02 M Tris. HCI, pH 7.4 and centrifuged for 2.5 hr at 25,000 rpm in the SW 50 rotor. The visible band between the two CsCl solutions was collected and diluted as described above, and then layered on a preformed CsCl gradient (1.4-1.25 g/ml) and spun at 40,000 rpm for 3.5 hr. The single low density opalescent band was collected and dialyzed agaonst 0.25 M sucrose, 0.02 il!f Tris, 0.001 1M MgClz HCl, pH 7.4, with 0.5 % n-butanol. The dialyzed preparation was considered to be purified incomplete virus particles. Infectivity assays. Infectivity was assayed by fluorescent focus formation in monolayers of KB cells, using an antiserum directed toward purified adenovirus type 2 hexons, or by plaque formation in human embryo kidney (HEK) cells (Flow Laboratories). The fluorescent focus formation method (Philipson, 1961; Thiel and Smith, 1967) was performed as described previously (Philipson et al., 1968), and the results are expressed in fluorescent focus units (FFU). The plaque formation on HEK monolayers was done according to Strohl (1969), and the results are expressed in plaque-forming units (PFU) . Electron microscopy. The preparation of incomplete particles, obtained after CsCl gradient centrifugation, was placed on the specimen support of platinum apertures,
INCOMPLETE
ADENOVIRUS
which were covered by a thin carbon film. Most of the CsCl was removed by touching the solution of the specimen on a surface of a volatile buffer (ammonium acetate) containing 2.5 % glutaraldehyde. Excess material was removed by a filter paper, and the material was negatively contrasted by uranyl oxalate at pH 7.0 or potassium phosphotungstate (KPT) at pH 7.1. The specimens were examined in a Philips 300 electron microscope at 60 kV accelerating voltage. Isolation of viral and cellular DNA. DNA from virus particles was isolated according to Doerfler (1969) with minor modifications. The virus suspensions were dialyzed against 0.01 M TrisaHCl, pH 7.4, 0.001 M EDTA (ethylenediaminetetraacetic acid) and kept in this buffer for 5-10 days at +4’. A tenth volume of Pronase B (Calbiochem) in 0.1 M Tris.HCl, 0.005 M EDTA, 0.002 M mercaptoethanol, pH 7.4 was added to a final concentration of 1 mg/m.l, and the sample was incubated for 30 min at +37”. Sodium dodecyl sulfate (SDS) was then added to a final concentration of 0.3% whereupon the sample was left at room temperature for 1 hr. The sample was shaken with an equal volume of redistilled phenol, saturated with 0.01 M TrisaHCl, pH 7.4, for 15 min at +4” whereafter it was centrifuged for 15 min at 6000 g. The phenol extraction was repeated twice, and the water phase was then shaken twice with an equal volume of ether. The ether was evaporated with nitrogen gas, and the sample was dialyzed against 0.1 X SSC (SSC = 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0). The cellular DNA was prepared as described by Marmur (1961) from 5 X 10’ KB cells suspended in 2 ml of saline EDTA (0.15 M sodium chloride, 0.1 M EDTA, pH 8.0). The isolated DNA was taken up in 0.1 x ssc. DNA-DNA hybridization. A procedure described by Denhardt (1966) was used with slight modifications. Schleicher and Schuell type B-6 filters were soaked in 6 X SSC and thereupon washed with 5 ml of 6 X SSC. 50 or 5 pg of cellular or viral DNA in 10 ml 6 X SSC was adsorbed to the filters, followed by washing with 10 ml of 2 X SSC. The filters were
PARTICLES
747
dried overnight in room temperature before being baked for 3 hr at +80” in vacuum. The DNA adsorbed to the filters had been denatured in 6 X SSC for 7 min at + 100” and thereafter rapidly cooled in Dry Ice and ethanol. The baked filters were preincubated in glass scintillation vials for 6 hr at f65’ in 1 ml 0.02 % Ficoll (Pharmacia), 0.02 % polyvinylpyrrolidone and 0.02% bovine serum albumin in 3 X SSC. After the preincubation the labeled DNA was added in a volume of 0.1 ml, and the filters were incubated for 16 hr at +65”. The labeled DNA had been sonicated with the microtip for 20 set at setting 3 in a Sonifier Cell Disruptor Model W 185D (Heat Systems-Utlrasonic, Plainview, Long Island, New York), heated for 7 min at +lOO” and rapidly cooled in Dry Ice and ethanol. After the incubation the filters were washed five times with 20 ml of 0.01 M TriseHCl, pH 7.4, and dried. The radioactivity was measured in 5 ml of toluenePermafluor (Packard Instrument Company) scintillation fluid. Zonal sedimentation in sucrose density gradients was performed mainly as described by Burlingham and Doerller (1971) on linear 5-20 % (w/v) sucrose gradients on which the samples were layered in 0.1 ml 0.1 X SSC. The neutral sucrose solution was made in 1 M NaCI, 0.01 M TrisaHCI, pH 8.0 with 2 X lo-* M EDTA. The alkaline solution was made in 0.8 M NaOH, 0.2 M NaCl with 2 X lo-* M EDTA. The gradients were centrifuged in a Beckman SW 50 rotor at 39,000 rpm for 5 hr at +4”. They were collected dropwise from the bottom of the tubes (20 or 30 drops per fraction). Degraded herring DNA, 1 mg (Sigma) in PBS was added to each fraction, then trichloroacetic acid (TCA) was added to a final concentration of 10%. The precipitate was pelleted and dissolved in 0.5 ml 0.2 M NaOH and then counted in 5 ml toluene-Permafluor (Packard)-Bio-Solv Solubilizer Formula BBS-3 (Beckman) in proportions 96 : 4: 20 (v/v). Analytical polyacrylamide electrophoresis on 5 % gels in SDS were performed according to Maize1 (1966) and has been described in a previous report (Prage and Pettersson,
748
PRAGE,
HOGLUND,
1971). The gels were stained with 0.01% Coomassie Blue in 7 % acetic acid overnight. Destaining was performed by repeated washings with 7 % acetic acid. Densitometric tracings were recorded at 550 nm with a Gilford spectrophotometer Model 2400 with a linear transporter. Amino acid analysis. Samples of 0.3-0.5 mg were hydrolyzed in 6 M HCl (Pettersson et al., 1967) and analyzed according to Moore et al. (1958) with t,he “Biochrom” amino acid analyzer. Protein detemzination was made by the method of Lowry el al. (1951) with bovine serum albumin (Sigma) as a standard. DNA determination was made according to Burbon (1956) with degraded herring DNA (Sigma) as a standard. The samples in 5 % perchloric acid were heated for 20 min at +70”, and the precipitate was spun down. Both the supernatant and the precipitate was tested for DNA. The color was developed at +28” for 18-20 hr.
AND
PHILIPSON RESULTS
Characterization of Iwomplete Particles Incomplete virus particles were purified from AdS-infected KB cells as described in Materials and Methods. To avoid cont’amination with soluble proteins, a centrifugation t,hrough 30% sucrose on to a cushion of CsCl was introduced before the last CsClgradient centrifugation. The purified incomplete particles labeled with 3H-thymidine was mixed with 32P-labeled complete virions and run on a CsCl gradient (Fig. 1). The incomplete particles banded at a density of 1.310 g/ml, whereas the complete virions were recovered at 1.355 g/ml. The incomplete particle band in the CsCIgradients appeared as two very close bands which could not be satisfactorily separated. Optical density at 280 nm and radioactivity was measured in 2-drop fractions of a CsClcentrifugation of a purified preparation of incomplete particles labeled with 3H-thymi,J/CC
CPL I-
1200
1000
600
FRACTION
NUMBER
FIG. 1. CsCl gradient of a mixture of complete and incomplete Ad3 virus particles. The complete particles were labeled with 3*P (-) and the incomplete with 3H-thymidine (--). The inset shows a section of a CsCl gradient of purified incomplete Ad3 particles labeled with aH-thymidine ( -1 where the optical density at 280 nm (-) was determined.
INCOMPLETE
ADENOVIRUS
dine. The peak of the radioactivity was recovered at a density of 1.307 g/ml whereas the maximum of optical density was found at about 1.298 g/ml (Fig. 1, inset). Thus, it appears that only a minor fraction of the incomplete particles contain DNA, whereas the majority of the particles contain very little, or no, DNA. The lower of the two bands seen in CsCl gradients of incomplete Ad3 particles probably represents the DNA containing particles. In this report the combined bands will be treated as a single population. Complete and incomplete particles were TABLE
1
Armum OF INCOMPLETE AND COMPLETE Ad3 VIRUS PARTICLES ISOLATED FROM INFECTED KB-CELLS AT COMPLETION OF THE INFECTIOUS CYCLE
Preparation
Ib II
Protein bg)(l Ad3, complete
Ad3, incomplete
5040 (67) 9000 (63)
2510 (33) 5220 (37)
a The percentage distribution of protein in each type of particle is given within the brackets. b The preparations were derived from 5 X lo* t,o 109 infected KB cells.
PARTICLES
749
isolated from the same infected cultures, and the amount of protein in each population was determined. Table 1 shows that the incomplete particles represent about 30 % of the yield at 72 hr after infection as measured by protein determination. It should be noted, however, that the incomplete particles have been subjected to two more centrifugations than the complete particles. The relative amounts of the incomplete particles presented in Table 1 should therefore represent minimum values. The incomplete particles from the CsCl gradients were examined in the electron microscope (Fig. 2). Particles were observed where the contrast agent appeared to have penetrated into the capsids, however, about 20% of the particles appeared as morphologically intact without any contrast agent in the interior. The rim of the empty particle appeared occasionally wider than anticipated from a single row of capsomers (Fig. 2, inset), indicating that the capsids might have collapsed from one side and accumulated the contrasting agent in a sac at the top, rather than t’hat the contrast agent had penetrated into the empty capsid. Goniometer analysis has shown that fragile particles,
such as incomplete
virus particles,
FIG. 2. Electron micrograph of purified incomplete Ad3 particles contrasted by 2% KPT at pH 71. as described in Materials and Methods. The inset (X 220,000) shows a projection with an approximately hexagonal outline of the rim of the particle (contrasted by 1% uranyl oxalate at pH 7.0).
750
PRAGE, HOGLUND,
AND PHILIPSON
are flattened during drying of the specimen (Hiiglund and Blomqvist, 1972) although the contrast agent and the glutaraldehyde fixation should stabilize them. Composition of the Incomplete Particles
The amounts of protein and DNA in complete and incomplete particles are shown in Table 2. The incomplete particles contained only 0.5-0.6% DNA whereas the complete virions contained 14-16 %. The differencesin polypeptide composition between complete and incomplete particles were analyzed by SDS polyacrylamide gel electrophoresis. Three gels were run in parallel, one with complete, a second with incomplete particles and a third with a mixture of the two samples. All samples contained the same amount of hexons to allow semiquantitative as well as qualitative comparison (Fig. 3). The densitometric tracings of t,he stained gels show that the incomplete particles lack the two major core polypeptides (peak V, and VI-VII, according t’o the nomenclature of Maize1 et al., 1968) as well as a fast migrating polypeptide (polypeptide IX), but contain normal amounts of hexon and penton (peak II and III-IV). The incomplete particles contain a third major polypeptide (polypeptide A) which is represented in complete particles only in minute amounts. The electrophoresis of mixed samples suggeststhat this polypeptide corresponds to a polypeptide in complete virions, since no new peaks appeared, but increase in absorbance is seen at this position of polypepOideA. The polyacrylamide gel electrophoresis
I
d 2
3
4 CM
FIG. 3. Densitometric tracings of polyacrylamide gels run in parallel at 5 V/cm for 3.5 hr in neutral SDS. The anode is to the right. SDStreated complete particles (A), incomplete particles (B), and a mixture of complete and incomplete particles (C) are shown. The absorbance is given in arbitrary units. The peaks are numbered according to the nomenclature of Maize1 et al. (1968).
thus indicated that the incomplete particles lacked the arginine-alanine-rich protein TABLE 2 (AAP) which is the major core protein COMPOSITION OF~NCOMPLETEAND COMPLETE Ad3 (polypeptide VI-VII; Prage and Pettersson, VIRUS PARTICLES 1971). The amino acid composition of the incomplete particles presented in Table 3, Preparation Protein DNAa together with the composition of complete ells) old --Ad3 particles and the Ad3 AAP, agreeswith 1 Complete 168 28 (14.3) this interpretation. Incomplete 684 3.4 (0.5) Incomplete particles contain relatively II Complete 300 56 (15.7) less arginine and alanine, and more glutamic Incomplete 360 2.2 (0.6) and aspartic acid as well as leucine, than 0 The percentage of DNA is given within par- complete virions. The AAP is rich in entheses. arginine and alanine and poor in leucine and
INCOMPLETE
ADENOVIRUS
glutamic and aspartic acid as compared to complete particles.
TABLE 3 AMINO ACID COMPOSITION” Amino acid
Residues per 100 residues Ad3 virion
-
Characterization of the DNA from Incomplete Particles
-
T
Ad3 incomplete particle
Ad3 AAP”
_-
3.7 f 0.3 3.8 & 0.1 3.9 zk 0 1.8 i 0.6 1.4 z!z 0.1 1.6 f 0.1 8.5 f 0.2 5.6 i 0.2 213.2 i 2.0 7.2 i 0.8 12.3 f 0.2 13.2 f 0.2 8.0 f 0.1 7.5 l 0 8.5 i 0.5 7.2 f 0.1 5.4 i 0.2 6.6 f 0 2.4 i 0.1 7.7 f 0.2 9.3 f 0.2 7.4 i 0.5 6.5 f 0.1 6.4 f 0 8.0 f 2.0 7.8 f 0.1 7.0 f 0 7.7 f 0.1 19.4 f 0.2 8.7 i 0 ).05 f 0.05 Trace N.D.” 7.4 f 0.8 6.3 f 0.3 6.1 f 0.3 ND 2.9 f 0.2 3.0 f 0.2 3.6 f 0 2.5 f 0 3.5 f 0 8.5 f 0.1 2.2 f 0.4 7.2 f 0 5.0 z!2 0.1 0.8 f 0.4 4.9 i 0 4.3 rk 0 ND 3.8 h 0 * The figures given are the mean values of two different preparations. b Not detectable. c From Prage and Pettersson (1971).
LYS His Ax ASP Thr Ser Glu Pro GUY Ala w-cys Val Meth Ile Leu Tyr Phe
2000
751
PARTICLES
3H-thymidine labeled DNA was isolated from incomplete and complete Ad3 particles and subjected to sedimentation analysis in neutral and alkaline sucrose gradients. The samples were run in parallel at each pH, but in separate tubes. Figure 4 shows that the DNA derived from incomplete particles is smaller (around 16 S) than the complete viral genome, which has a sedimentation coefficient of 32 S at neutral and 34 S at alkaline pH (Burlingham et al., 1971). The neutral sucrose gradient with incomplete DNA showed a shoulder on the heavy side which was not seen in the alkaline gradients. The nature of this fast sedimenting DNA is unclear at present. To determine whether the DNA in the incomplete particles was of viral or cellular hybridizations were origin DNA-DNA performed with cellular or complete Ad3 viral DNA on the filters. The results in Table 4 show that almost all the DNA C
* i
i
I
2
3
4 EFFLUENT
I VOLUME
2
3
4
(ML)
FIG. 4. Sedimentation of DNA labeled with 3H-thymidine. The two left panels (A and B) show the sedimentation at neutral pH and the two right panels (C and D) at alkaline pH. DNA was isolated from complete (A and C) and incomplete (B and D) Ad3 particles.
752
PRAGE, TABLE
HOGLUND,
AND PHILIPSON
4
from incomplete particles hybridized with viral DATA, and only a small fraction with the cellular DXA. The incomplete Ad3 viral particles thus seem to contain small pieces of the viral genome.
DNA-DNA
HYBRIDIZATION OF DNA FROM COMPLETE AND INCOMPLETE Ad3
VIRUS PARTICLESO Source of radioactiw DNAb
:I
Radioactivity
bound to filters (cpm)
Infectivity
lontrol
DNA on fillterse
fdtersd
-Expt. I Ad3, complete particles (0.4 rg;6004wm) Ad3, incomplete particles (0.2 pg; 1498 cpm) Expt. II Ad3, complete
Ad3, complete
-I-
The infectivity of preparations of complete and incomplete Ad3 particles was determined with both the fluorescent focusforming and the plaque-forming methods. In Table 5 the titers of different preparatlons are listed. The infectivity of the incomplete particles is about 4 log units lower than that of a corresponding amount of complete particles when the quantitation is based on the amounts of protein. Since the fluorescent focus method is more convenient than the plaque method, the former was used for most experiments. The FFU, however, only measures accumulations of hexon in the cells, and it must not necessarily reflect a productive infection. The results in Table 5, however, show that the FFU and PFU data correspond, thereby validating the former as a measure of infectivity. It was feasible that t.he infectivity in preparations of incomplete particles was due to contamination with complete virions trailing behind the bulk of virions in the CsCl gradients, especially as these were harvested dropwise from the bottom of the tubes. Incomplete particles were mixed in excess with complete virus particles and subjected to a CsCl gradient centrifugation. The
KB
____
4092, 4012
294, 174
101
1218, 1093
134, 92
19
6880, 6970
590, 472
690
particles (0.4 pg; 6464 cpm) Ad3, incomplete 2373, 2509 346 particles (0.2 pg; 2417cpm) 899, 1222 KB (2.5 rg; 2350, 86, 73 cpm) I -i a All results are corrected for background
142
ra-
dioactivity. b The amount and the radioactivity added to each filter are given within brackets. The added
radioactivity was determined by spreading the radioactive DNA on a filter, drying it under a heating lamp, and counting it in scintillation liquid as described in Materials and Methods. c In Expt. I all filters had 5 rg DNA. In Expt. II the Ad3 filters were made with 5 yg DNA and the KB filters with 50 pg DNA.
d Control filters were made without DNA. TABLE INFECTIVITY
Preparation I Complete Incomplete II Complete Incomplete III Complete Incomplete IV Complete Incomplete V Complete Incomplete
IN PREPARATION
5
OF INCOMPLETE AND COMPLETE A&
FFU/ml
FFU/e
1.1 X 10’0 2.6 X lo6
3.5 x 7.5 x 4x 2.8 x
of Incomplete Particles
10’0 106 10’0 106
PFU/ml 2.4 6.0 3.0 1.9 6 5.6
5.1 8.3 1.8 1.2
X x x x
107 102 10’ 103
VIRUS PAk~~rc~~s
X X x X X X
109 1Oj 10’0 10” 10”’ lo6
2 x 106
PFu/,p
1.8 1.7 2 3.1
x x x x
107 103 10’ 103
2.2 x 103
INCOMPLETE
ADENOVIRUS
infectivity was scored throughout the gradient and the result is seen in Fig. 5. A distinct peak of infectivity is seen at the density of incomplete particles. Thus, the infectivity cannot be explained only by trailing from the complete virion peak, but these results do not exclude that complete particles remained trapped in the band of the incomplete particles. Because the amount of DNA in incomplete particles is reduced, it would be expected that several incomplete particles are needed to obtain a productive infection, which infers that the infectivity should display a multihit curve when plotted against particle concentration. Figure 6 shows the hit-curves obtained with the incomplete as well as the complete particles. The slope of the infectivity versus particle dilution of complete particles is consistent with a one-hit event leading to productive infection. The corresponding curves for the incomplete particles display slopes which are less steep than that for the complete particles. Such slopes may FFWML IO6
tc
PARTICLES
753
T
FIG. 6. The infectivity of different dilutions of preparations of incomplete (X---X) and (a- - -0) and complete (O--O) Ad3 particles. The outer scale applies to the complete, and the inner scale to the incomplete particles.
be observed if the infective particles contained interfering particles which were not, or less, infectious. Dilution of such mixtures should give proportionally higher infectivity as the probability of interference ‘between the two types of particles decreases with dilution. DISCUSSION
“\
I37
134
‘!30
4
i FRACTION
NUMBER
FIG. 5. CsCl gradient centrifugation of a mixture of purified incomplete and complete Ad3 particles. The infectivity of the indicated fractions was measured by the fluorescent focus-forming method. The incomplete particles were present in excess (about loo-fold) in the centrifuged mixture.
A population of virus particles with a density 0.05 g/ml lower than compIete virions was found in cells infected with adenovirus type 3. Such a low density implies a low content of DNA and suggests that the particles are more deficient than the defective virions described by Rlak (1971). The population of incomplete particles described in this report was, however, not homogeneous, since two very close bands could be seen in CsCl gradients when it was subjected to equilibrium density centrifugation. As a biochemically satisfactory separation of these two bands was difficult to achieve, the whole population was characterized as if it were homogeneous. Adenovirus type 3 was chosen for this study because it gives only two major bands
754
PRAGE, HOGLUND,
in CsCl gradients, the incomplete and the complete virion bands, which are present in a ratio of 1: 2 as calculated from the amount of protein in each population. There are only traces of two or three bands in between these. The yield of incomplete and complete Ad3 particles is furthermore very high. Several low density bands are seen in CsCl gradient runs of Ad2, but these are all faint (Maize1 et al., 1968; Prage, unpublished observations) and therefore not suited for biochemical characterization. The band patterns of Ad12 preparations is, interestingly, similar to the Ad3 pattern, with a single major top component of low density (Smith, 1965; Prage, unpublished observations). The yield of Ad12 is, however, low (Green et al., 1967a). The polypeptide composition of the incomplete particles revealed a pronounced deficiency of the major core proteins, which was also found by Maize1 et al. (1968) in DNA-lacking top components of Ad2 and Ad12. The lacking core proteins (polypeptides V and VI-VII according to Maizel’s nomenclature) amounts to about 20 % of the proteins in the virion (Maize1 et al., 1968; Prage and Pettersson, 1971). Interestingly, the incomplete particles contained an enhanced amount of a polypeptide that is present in complete virions only in minute amounts. It should be noted that any quantitative interpretations based on densitometric tracings of stained gels should be regarded with caution, since various polypeptides might bind the stain with different efficiencies. The amino acid composition confirmed that the incomplete Ad3 particles are deficient with regard to the arginine-alaninerich protein (AAP), which is the major core protein. These findings are correspondent to results obtained with naturally occurring empty polio capsids (Maize1 et al., 1967). These lack two polypeptides (VP2 and VP4), but are enriched in another polypeptide which is present in small amounts in complete virions (NCVPG or VPO). Although these two viral systems are unrelated, it might well be that they both mirror similar assembly mechanisms. Experiments are cur-
AND PHILIPSON
rently performed to elucidate the relation of the enriched protein in incomplete particles to the other viral polypeptides and to the capsid assembly process. The incomplete particles contained about 0.5 % DNA, which means that if the DNA is equally distributed among all the incomplete particles, each particle will contain approximately 3% of the amount present in complete Ad3 virions, assuming that they have only 80 % of the protein amount in complete particles. If the DNA is represented by a single piece, this should have a molecular weight of 7 X lo5 daltons, assuming that the complete Ad3 genome has a molecular weight of 23 X lo6 daltons (M. Green, quoted by Schlesinger, 1969). A rough estimate of the size of the incomplete DNA could be calculated from the sucrose gradients shown in Pig. 4. Assuming a sedimentation coefficient of 32 S at neutral and 34 S at alkaline pH (Burlingham et al., 1971) for the complete Ad3 genome, the incomplete DNA has a coefficient of about 16 S under both conditions. According to Studier (1965) this corresponds to a molecular weight of about 3.5 X lo6 ddtons which is 5-fold higher than the 7 X lo5 daltons calculated above. This discrepancy can be explained if only a fraction of the incomplete particles contained the bulk of DNA, which was suggested from the distribution of labeled DNA in the CsCl gradients. The electron micrograph also favored this interpretation since about 20 % of the incomplete particles was stained as complete particles, and may represent the DNA-containing incomplete particles. The DNA in the incomplete particles is probably not of cellular origin since almost all of it hybridized with viral DNA, although the presence of a small fraction of cellular DNA cannot be excluded. The small size speaks against that it is contaminating complete viral DNA. If this was the case, the contaminating virus particles should give an infectivity titer which is not less than 2 % of the infectivity in a corresponding amount of complete particles. The results show that the infectivity is more than lOOfold lower than the assumed 2%, thus indicating that the DNA in the incomplete
INCOMPLETE
ADENOVIRUS
PARTICLES
755
plete particles, as well as the explanation of particle preparations is not degraded DNA the different patterns of incomplete popuobtained from complete Ad3 virions during lations in CsCl gradients among different the preparation. The discrete size of the DNA in the in- types of adenoviruses is unknown. It complete particles, as suggested from the should be noted that the particles described distinct band in CsCl gradients as well as in this report are harvested at one single time point, after infection, and it is not from the sucrose gradient analysis indicates that the DNA pieces are produced by a improbable that the ratios between incomspecific mechanism. Whether they have any plete and complete virus particles differ relation to the endonucleolytic activity through the infectious cycle, which also may described by Burlingham et al. (1971) apply to the pattern in CsCl gradients. A proper study of the kinetics of the synthesis remains to be seen. It should also be noted that the same authors detected slowly sedi- of incomplete particles might give some information on the assembly mechanism for menting viral DNA in cells infected with adenoviruses. Ad2 or Ad12 (Burlingham and Doerfler, 1971). ACKNOWLEDGMENTS The incomplete particle preparations contained infectivity, although it was 4 log We are indebted to Dr. D. Eaker and Dr. R. Thorzelius for aid with the amino acid analysis. units lower than in corresponding amounts The skillful technical assistance provided by Ms. of complete particles. A contamination with complete virions is, however, very diflicult to I. Htibinette is also acknowledged. rule out at this low level. CsCl gradient REFERENCES centrifugations of incomplete particles have been observed to give a small amount of BLACKSTEIN, M. E., STANNERS, C. P., and FARMILO, A. J. (1969). Heterogeneity of polyoma infectivity at the density of complete virus DNA: Isolation and characterization of particles (unpublished observations). non-infectious small supercoiled molecules. J. The infectivity in the incomplete particle Mol. Biol. 42,301-313. preparations displayed a hit curve which BREEDIS,C.,BERWICK, L.,and ANDERSON, T.F. was not compatible with a viral genome (1962). Fractionation of Shope papilloma virus piece, which is about 15% of the complete in cesium chloride density gradients. v+oZogy genome. In such a case, the slope should be 17,84-94. BURLINCHAM, B. T., and DOERFLER, W. (1971). steeper than the one hit curve observed with Three size-classes of intracellular adenovirus complete particles. The results showed a hit deoxyribonucleic acid. J. Virol. 7,707-719. curve with a slope below unity, indicating that the observed infectivity was subject to &JRLINGHBM, B. T., DOERFLER, W., PETTERSSON, U., and PHILIPSON, L. (1971). Adenovirus endointerference. Since noninfectious particles nuclease : Association with the penton of adenomight interfere with infectivity caused by virus type 2. J. Mol. Biol. 60, 4S64. complete particles as well as by compleBURTON, K. (1956). A study of the conditions and menting DNA-containing incomplete parmechanism of the diphenylamine reaction for ticles, this analysis will not allow us to sepathe calorimetric estimation of deoxyribonucleic rate between the two alternatives, conacid. Biochem. J. 62,315323. CRAWFORD,L. V., CRAWFORD,E. M., and WATtamination or complementation. SON, D. H. (1962). The physical characteristics From this evidence we suggest that the of polyoma virus. I. Two types of particles. infectivity present in preparations of inVirology 18, 179-176. complete particles is due to contamination with complete Ad3 virions, although a DENHARDT, D. T. (1966). A membrane filter technique for the detection of complementary DNA. complementation mechanism obscured by Biochem. Biophys. Res. Commun. 23,641646. interference cannot be excluded at present. DOERFLER, W. (1969). Nonproductive infection of The contamination is probably caused by baby hamster kidney cells (BHK21) with adenotrapping of a few complete virus particles virus type 12. Virology 38, 587-606. in an excess of incomplete particles. GREEN, M., PIRA, M., and KIMES, R. C. (1967a). Biochemical studies on adenovirus multiplicaThe biological significance of the incom-
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INCOMPLETE
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