VIROLOGY
82, 182-195 (1977)
Mouse Adenovirus:
Growth of Plaque-Purified FL Virus in Cell Lines and Characterization of Viral DNA
STEVEN H. LARSEN1 Department
of Microbiology,
Johns Hopkins
AND
University
DANIEL NATHANS School of Medicine,
Baltimore,
Maryland
Accepted May 24,1977 The FL strain of mouse adenovirus (AdFL) was plaque-purified and grown in mouse 3T6 cells. Puritied virions resemble human adenoviruses by electron microscopy, and viral DNA shares physical properties with human adenovirus DNA. AdFL-DNA is a linear duplex with a molecular weight of 20 x 106, shows evidence of protein covalently linked at each end, and forms single-strand circles after denaturation, indicating inverted repeat sequences at or near the ends of the molecule. However, less than 10% nucleotide sequence homology was found between AdFL-DNA and the DNAs of Ad2, 7, or 12. Also, the C + G content of AdFL-DNA (44%) is lower than that of human adenovirus DNAs. While there was reactivity between extracts of AdFL-infected cells and antiserum against human adenovirus, sera against the T antigens of human adenoviruses of groups A, B, and C did not react with AdFL-infected cell lysates. Six restriction endonucleases were used to cleave AdFL-DNA and to construct cleavage maps of the DNA. These maps differed from those of Ad2 or Ad5. INTRODUCTION
Adenoviruses, defined by morphological, biological, and serological criteria, have been isolated from mammals, amphibians, and birds (Rowe and Hartley, 1962; Pereira, 19751, and several human strains have been characterized biochemically and genetically (Ginsberg and Young, 1976; Wold et al., 1977). We became interested in the eventual possibility of using murine adenovirus as a transducing virus for mouse cells and have therefore begun a molecular characterization of this virus. Two immunologically and pathogenically distinct strains of mouse adenovirus have been described: the FL strain (AdFL) isolated from Swiss mouse cells (Hartley and Rowe, 1960) and the K87 strain (AdK87) isolated from the feces of normal DKl mice (Hashimoto et al., 1966; van der Veen and Mes, 1974). In newborn mice AdFL produces a fatal generalized infection (Hartley and Rowe, 1960; Blailock et al., 19681, and in adult mice the 1 Author dressed.
to whom reprint
requests
should be ad-
virus causes a persistent, inapparent infection, with viruria lasting for months or years (van der Veen and Mes, 1973). In contrast, AdK87 produces only an intestinal infection in neonatal or older mice (DKl strain) without observable signs of disease (Sugiyama et al., 1967). Because of its wider tissue range we chose to work with AdFL rather than the K87 strain. Our initial experiments with AdFL, reported in the present communication, were designed to test its host range, to develop a procedure for preparing virions and viral DNA suitable for genetic and biochemical study, to characterize the viral DNA, and to establish cleavage maps of the viral genome. MATERIALS
AND
METHODS
Cells and virus. FL virus, originally isolated by Hartley and Rowe (19601, and anti-FL serum were obtained from Microbiological Associates, Bethesda, Maryland. The virus had been serially passed in Swiss mouse embryo cells and in Swiss mouse kidney cells without plaque purification. BALB/c 3T3 cells were generously
182 Copyright 0 1977 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
ISSN 0042-6822
MOUSE
ADENOVIRUS
provided by G. Todaro; Vero, KB, and HeLa cells, by T. J. Kelly, Jr.; Swiss 3T6 cells, by W. Gibson and W. Eckhart; L929 (hereafter referred to as L cells) and WI38 cells, by D. H. Carver; and HEp2 cells, by L. Aurelian. Human embryonic kidney cells were purchased from Microbiological Associates. Cells were grown and maintained in Eagle’s minimal essential medium (MEM) with Earle’s salts and the following serum concentrations: BALB/c 3T3, 20/10; L, 10/3; HeLa, 10/2; KB, lOC/ 2C; Vero, 10/2C; WI38, 10/2; HEp2, 10/2; CHL, 10/2; Swiss 3T6, lOC/lOC; where numbers refer to the percentage (v/v) of fetal calf serum or calf serum (C) for growth/maintenance. The cells were fed two or three times per week depending on their individual stability. Mouse cells used for growth of virus were cloned by plating various dilutions in 6-mm microwells, and colonies that arose from single cells were selected. An attempt was made to select clones that gave stable, flat monolayers in the case of BALB/c 3T3 and L cells. Swiss 3T6 clones were tested for high titer yields of FL virus and the best of five clones was used. Plaque assays. Appropriately diluted AdFL in 0.2 ml of MEM supplemented with 10% fetal calf serum was absorbed on confluent monolayers of BALB/c 3T3 or L cells. After 2 hr at 37” the cells were overlaid with 5 ml of the same medium plus 0.9% agar. The cells were fed 2.5 ml of agar-containing medium each 3 days thereafter and were stained with neutral red 6 to 9 days postinfection. Recently, it was found that the BALB/c 3T3 monolayer used for assay is more stable if the agar medium is supplemented with twice the normal amino acid concentration of MEM. Preparation of virus stocks. For preparation of a primary stock, the original stock from Microbiological Associates (found to be about lo4 PFU/ml) was diluted and plated on BALB/c 3T3 cells. A wellseparated plaque was selected at 10 days postinfection and replated. A second plaque was selected to infect 10 75-cm2 flasks of BALB/c 3T3 cells at an estimated m.0.i. of 10p4. CPE appeared maximal
183
DNA
after 10 days at 37”. The lysate was frozen and thawed three times and stored at -20”. The primary stock titer was 1.7 x 10’ PFU/ml. To prepare high titer secondary stocks, Swiss 3T6 confluent monolayers were infected with the primary stock at 0.1 to 0.3 PFU/cell and the lysate was frozen after about 6 days, yielding secondary stocks of 3 to 10 x lo* PFU/ml. Immunological assays. Immunological assays for AdFL antigenic determinants corresponding to human Ad virion antigens or T antigens were kindly performed by R. J. Toni and R. V. Gilden of the Frederick Cancer Research Center. To test for cross reaction between AdFL and human adenovirus, an antiserum active against the human adenovirus group (Hexon) antigen was reacted in complement fixation tests with lysates of BALB/c 3T3 cells that had been infected with AdFL 40 hrs previously. Control reactions were carried out with either uninfected 3T3 cell lysates or with lysates of Ad2-infected KB cells. Complement fixation reactions were also carried out between the same AdFL cell lysates and anti-T antigen sera from hamsters bearing tumors induced by representatives of each of the major groups of human adenoviruses: Ad2-SV40 hybrid virus (Group C), Ad7 and 21 (Group B), or Ad 12 (Group A) (Gilden et al., 1968). In each case positive and negative controls were run concurrently. Preparation
of virus
and viral
DNA.
Monolayers of Swiss 3T6 cells in 490-cm* plastic roller bottles (Corning) were infected with secondary virus stock at a multiplicity of 2 to 3 PFU/cell in fresh MEM plus 10% fetal calf serum. Carrier-free 13*Plphosphate (50 to 500 &i) was added at 24 hr postinfection when [32PlDNA was desired. The medium was replaced with phosphate-free MEM with 5% fetal calf serum just prior to 32P-labeling. At 4 days postinfection the cultures were frozen and thawed and virions were sedimented from the clarified lysate in an SW 27 rotor at 24,000 rpm for 80 min into a layer of 5 ml of CsCl, 1.2 g/ml, which was on top of a layer of 3 ml of CsCl, 1.5 g/ml, both solutions in TE buffer 110 mM Tris, pH 8.6; 1
184
LARSEN
AND
mM ethylenediaminetetraacetic acid (EDTA)]. Two bands appeared and were collected together from the bottom of the tube. This viral suspension was brought to a density of about 1.6 g/ml of CsCl by addition of solid CsCl, and 7.5 ml was distributed into each of several SW 41 Spinco tubes. Onto the virus suspension were layered 2.5 ml of CsCl of density 1.42 g/ml and then 2.5 ml of CsCl of density 1.26 g/ml, both in TE buffer. The tubes were centrifuged at 36,000 rpm for 1 hr in an SW 41 rotor. Two to five bands appeared near the density of 1.34 g/ml. The total viral bands were collected by use of a Pasteur pipet from the top of the tube and dialyzed for at least 2 hr vs TE buffer. For preparation of viral DNA, sodium dodecyl sulfate (SDS) was added to 0.5% and preincubated Pronase to 1 mg/ml, and the mixture was incubated for 2 hr at 37”. This DNA-containing solution was extracted three times with an equal volume of phenol saturated with 0.1 x SSC (Na-chloride, Na-citrate) and dialyzed for 48 hr at 4” against at least three changes of 0.1 x SSC. Alternatively, residual phenol was removed by two extractions with equal volumes of ether prior to dialysis. DNA-protein complexes. These were isolated by chloroform-isoamyl alcohol extraction and guanidine hydrochloride-sucrose gradient centrifugation as described by Robinson et al. (1973). Electron microscopy. Virus particles and viral DNA were prepared for microscopy by standard methods, using nitrocellulose-coated grids and, in the case of virions, negative staining with 1% uranyl acetate. DNA was spread in 40% formamide as described by Davis et al. (1971). For a length standard, SV40 form II DNA was mixed with AdFL-DNA before spreading and the two DNAs compared within a given field. Measurements were made with an electronic graphics calculator, Numonies Corp., North Wales, Pa. Restriction endonucleases. Reactions were carried out at 37” in the following solutions: EcoRI, 100 mM Tris-HCl (pH 7.21, 50 nib2 NaCI, 5 m&f MgC&, 2 mM 2mercaptoethanol; BamHI, 20 mM TrisHCl (PH 7.41, 7 mM MgCI,, _ _. 2 mM 2-mer-
NATHANS
captoethanol; HindIII, BglII, HpaI, and SalI, 20 mM Tris-HCl (pH 7.41, 7 mM MgCl*, 7 m&f 2-mercaptoethanol. BgZII was a gift of D. R. Shortle. Sal1 was purchased from New England Biolabs, Beverly, Mass., and the other restriction enzymes, from Bethesda Laboratories, Bethesda, Md. Agarose-gel electrophoresis. Agarose slab gels were made as previously described (Brockman and Nathans, 1974). When indicated, SDS was added just before pouring the gels. Five microliters of a solution containing 50% sucrose, 100 m&f EDTA (pH 7),0.1% bromophenol blue, and usually 1% SDS was added to each sample before applying it to the gel. DNA-DNA hybridization. DNAs in 0.1 x SSC were denatured in 0.2 N NaOH, boiled 20 min and quickly chilled on ice. The solutions were adjusted to a final concentration of 0.3 M NaCl, 10 n&f Tris-HCl (pH 7.51, 1 mM EDTA and incubated at 68”. At various times samples were assayed by hydroxyapatite as described by Brockman et al., (1973). An aliquot was counted directly to determine total DNA [3H]Thymidine-labeled concentration. Ad2-DNA (labeled in viuo) was a gift of R. L. Lechner. Unlabeled and nick-translated (6 to 8 x lo6 cpm/Fg) Ad7- and Adl2-DNAs were gifts of J. K. Mackey and M. Green. RESULTS
Growth
of Plaque-Purified
Virus
Seed FL virus, propagated by passage in mouse kidney cells, was plated on BALB/c 3T3 and on L cell monolayers (Fig. 1). In each case plaque formation occurred by single-hit kinetics. On 3T3 cells plaques developed earlier and grew to larger size, but the efficiency of plating was one-third to one-half that on L cells. For preparation of stocks, virus was first purified by serial plaque formation on BALB/c 3T3 cells and subsequently grown on 3T3 monolayers, as detailed in Materials and Methods. The resulting virus was neutralizable by antiFL antibody. Growth of virus was compared on monolayers of BALB/c 3T3 cells, L cells, and Swiss 3T6 cells. As seen in Fig. 2, in growth experiments BALB/c 3T3 cells and Swiss 3T6 cells gave maximal or near
MOUSE ADENOVIRUS
185
DNA
FL Plaques
BALB/c
ST3
L
FIG. 1. Differential plaquing of AdFL on BALBlc 3T3 and L cells. Each monolayer was infected with equal amounts of an FL suspension, stained with neutral red, and photographed on the tenth day.
FIG. 2. One-step growth curve of AdFL. Confluent monolayers of BALB/c 3T3, Swiss 3T6, and L cells in 1.6-cm wells were infected in duplicate with 1.8 x lo5 PFU of virus, yielding multiplicites of 1.2, 0:41, and 0.58 respectively. The virus titers of the cell-free medium alone (0) or of the total contents of each microwell (0) Hfter freeze-thawing was determined by plaquing on BALBlc 3T3 cells.
186
LARSEN AND NATHANS
maximal yields in about 3 days, and the total virus yield regularly exceeded that from L cells. For routine preparation of a working stock, 3T6 cells were used as described in Materials and Methods. Titers of virus were generally 3 to 10 x lo8 PFU/ ml as assayed on BALB/c 3T3 cells. Stability
of Virus
Infected BALB/c 3T3 cell lysate stored at -20” retained full viral infectivity for at least 12 months. Virus present in such lysates could be inactivated by incubation for 30 min at 56, but was resistant to ether (Hartley and Rowe, 1960), to 1.5% (v/v) Triton X-100, and to 1% sodium deoxycholate. In contrast, chloroform reproducibly reduced the virus titer about threefold, and 50% ethanol (v/v) rapidly inactivated the virus. Host Range
adenoviruses reacted with AdFL-infected cell lysates, we observed that the antiserum against human Ad strains also reacted with AdFL. However, none of the anti-T sera reacted with lysates of AdFLinfected cells. Preparation
of AdFL
Virions
and DNA
Mouse adenovirus purified as detailed in Materials and Methods had a buoyant density in CsCl solution of 1.34 g/ml. In addition to the main virus band, empty particles and several minor species of intermediate densities were also present, as reported earlier for human adenoviruses (Smith, 1965; Burlingham et al., 1974; Daniell, 1976). By electron microscopy, purified AdFL particles resembled human adenoviruses (Horne, 1962) in size, icosahedral structure, and apparent number of subunits per face (Fig. 3). Viral DNA was extracted from purified virions by Pronase digestion of SDS-disrupted particles followed by phenol extraction, as described in Materials and Methods. In a typical preparation, 100 ml of Swiss 3T6 cell lysate containing 5 x 1O’O PFU of virus yielded 110 pg of viral DNA. Taking the molecular weight of the DNA as 20 x lo6 (see below) we estimate the minimal ratio of complete virions to plaque-forming units in the lysate at about
Hartley and Rowe (1960) reported that FL virus showed no cytopathic effect on rat embryo, monkey kidney, KB, human embryonic skin, or muscle cells. We tested human embryonic kidney, KB, HEp2, W138, HeLa, Vero, and BSC-1 cells for their ability to support virus growth by infecting at multiplicities of 0.1 to 1 PFU per cell and assaying cell lysates for virus 6 to 15 days postinfection. Virus titers decreased during the 6- to 1Bday period from 60. lo- to lOOO-foldbelow the input level, and of in no case did FL virus cause detectable Molecular Weight and Configuration AdFL-DNA cytopathic changes, in contrast to its effect on control BALB/c 3T3 and L cells. Even at DNA extracted from purified AdFL viria multiplicity of 100, WI38 cells showed no ons and further fractionated by sedimentamorphological change. In all cases the hu- tion through a sucrose gradient as deman and monkey cells used supported the scribed in Materials and Methods was exgrowth of Ad2 or SV40. amined by electron microscopy, and its length distribution was measured relative Serologic Cross-Reaction with Human to form II SV40 DNA on the same grid. As Adenoviruses shown in Figs. 4A and 5, virion DNA is a Lysates of AdFL-infected BALB/c 3T3 linear duplex with a mean length 5.9 2 cells were assayed by complement fixation 0.32 (SD) times that of SV40. From these for antigens reactive with antiserum measurements and the published value of against human adenovirus virions or 3.3 x lo6 for the molecular weight of SV40 against the T antigens of Ad2, Ad7, Ad12, DNA (Gerry et al., 19731,we estimate that or Ad21, as described in Materials and AdFL-DNA has a molecular weight of 19.5 Methods. In agreement with the finding of k 1.1 x 106. However, as shown in Fig. 5, Hartley and Rowe (1960), who found that the length distribution is skewed toward antisera against the C group of human shorter molecules, probably because of the
MOUSE
ADENOVIRUS
DNA
187
presence of DNA with small deletions de- from which we infer that protein is bound rived from defective particles (see minor to its ends (also see below). species noted above). Taking the peak Another property shared by mouse and length shown in Fig. 5 as the length of human adenovirus DNAs is the presence complete AdFL genomes, a value of 20 x of terminal, inverted repeat sequences. As lo6 is obtained. seen in Fig. 4C, when SDS- and PronaseWhen human adenovirus DNA is ex- treated AdFL-DNA was denatured by 50% tracted from virions with guanidine hydro- formamide at 60” and the solution held at 4” chloride in the absence of protease and for 24 hr, many molecules appeared as sinSDS, circular DNA molecules are obtained gle-stranded circles, as reported for human due to the presence of terminally bound adenovirus DNAs by Garon et al. (1972) and Wolfson and Dressler (1972). protein (Robinson et al., 1973). Similarly, mouse adenovirus DNA extracted in this way also yielded circular duplex forms Cleavage Maps of AdFL-DNA For future use of AdFL it would be helpseen by electron microscopy (Fig. 4B), ful to have a detailed cleavage map of its DNA. For this purpose the sites cleaved by several restriction endonucleases were determined. The strategy for mapping the viral DNA was as follows: first, to determine molecular weights of DNA fragments produced by individual enzymes by comparing their electrophoretic mobilities in agarose to a set of standards; second, to identify end fragments using DNA with terminal proteins; and third, to determine the fragment patterns resulting from siFIG. 3. Electron microscopic appearance of negatively stained FL virions. Note both full-appearing multaneous cleavage by pairs of restric(leti) and empty-appearing (right) virus particles. tion enzymes. Since the enzymes employed
FIG. 4. Electron micrographs of FL-DNA. (A) FL-DNA isolated with Pronase treatment, as described in Materials and Methods. (B) FL-DNA isolated with guanidine-HCl, but without Pronase treatment, as described in Materials and Methods. In different preparations, DNA appearing as circles varied from about 30 to 60% of the total. (C) FL-DNA denatured and reannealed at low concentrations according to Wolfson and Dressler (1972). Arrows indicate SV40 DNA used as internal length standard.
188
LARSEN AND NATHANS
Ol 0
7 n:
n l-l: i-l l&l-l I
2
3
4
Length of FL DNA relative
5
6
7
to SV4O
FIG. 5. Histogram of FL-DNA length relative to SV40. The DNA used in this experiment was prepared as described in Materials and Methods and further purified by sucrose gradient centrifugation.
cleaved FL-DNA at relatively few sites, the above data were generally sufficient to deduce the sites of cleavage by each enzyme. Examples of this approach are illustrated in Figs. 6-8, and all of the data are presented in Tables 1 and 2. As shown in Fig. 6, EcoRI, BarnHI, and Hind111 produced three, six, and six fragments, respectively, the molecular weights of which were estimated by comparison with coliphage A and SV40 DNA fragments or, in the case of large fragments, by subtraction of the sum of the molecular weights of smaller fragments from the molecular weight of the starting DNA (Table 1). Figure 6 and Table 2 also show the analysis of double enzyme digests. To determine which fragments are at the ends of the viral DNA we applied the findings of Brown et al. (1975) and Sharp et al. (1976) that when DNA-protein complexes of Ad2- and Ad&DNA were digested with. a restriction enzyme, the end fragments did not migrate during electrophoresis in agarose; prior phenol extraction of such a digest resulted in loss of end fragments from the aqueous phase. Figure 7 shows the digest patterns of AdFL-DNA isolated with and without Pronase treatment and the effect of subseouentlv - - ~~~-~~.-~treat.- _-..
ing the restricted DNA-protein complexes with Pronase or phenol. As seen in the figure, EcoRI-B and -C fragments and HindIII-A and -F fragments are missing from the tracks that received digests of untreated DNA-protein complexes, and there is a smear of radioactivity near the origin. However, prior treatment of these digests with Pronase led to the appearance of the missing fragments. Also shown in Fig. 7 is the effect of phenol extraction of endonuclease-cleaved DNA-protein complex; againEcoRI-B and -C, andHindIII-A and -F are missing, but in this case no smear of readioactivity is found near the origin. (It should be noted that phenol extraction removes little full length DNAprotein complex.) Finally, we found that when the gel contained 0.5% SDS (and the sample had been treated with 2% SDS), the putative end fragments entered the agarose but migrated more slowly than the Pronase-treated DNA fragments (Fig. 8). All of these results are consistent with the presence of protein bound to each end of AdFL-DNA, resulting in altered electrophoretic mobility of end fragments in agarose gel. In addition to the analysis of EcoRI, HindIII, and BamHI fragments of AdFLDNA, similar analyses were carried out
MOUSE
ADENOVIRUS
189
DNA
I1 I
TABLE ENDONUCLEASE
Fragment
-Full hgth IS.7
-
4.3
-
3.3
-
t.3
-
FL DNA
l.It
FIG. 6. Autoradiogram of 3ZP-labeled FL-DNA restriction fragments following electrophoresis in agarose. 3ZP-labeled FL-DNA was isolated after Pronase treatment and digested singly withEcoR1 (Rll, BamHl (Hl), or Hind111 (dII1) or with pairs of these enzymes simultaneously. The position of some EcoRl products of A DNA and Hind111 products of SV40 DNA used as molecular weight markers in the same gel are indicated at the right in millions of . _ daltons.
EcoRl
1
R DNA
BW7lH YindIII 34 30.5 24.5 5.56 4.6 1.20
44b 23 12 10 7.6 3.5”
FRAGMENTS”
EglII
HpaI Sal1
57 28 136 2.6b
37 22.5 12.5b 11 9.5 6b 1.5
53b 19.5 12.5 11.5 3.5b
o Data are expressed in percentages of AdFLDNA. Fragment lengths 30% or less were estimated by electrophoretic mobility compared to EcoRI fragmerits (Allet et al., 19731 of coliphage h DNA or HindIII fragments (Danna et al., 1973; Carroll and Brown, 1976) of SV40 DNA. Fragment lengths greater than 30% were estimated by subtracting the sum of the lengths of all other fragments from 100%. b Indicates end fragments.
with DNA restricted by SalI, BgZII, and HpaI, and the results are given in Tables 1 and 2. Each enzyme produced two and only two putative end fragments judged by migration with and without SDS in the gels. Having identified terminal fragments for each enzyme and determined which end fragments were cut during double enzyme digestions, we could align the left and right ends of all the enzyme cleavage maps. Then by analysis of double-digest patterns, unique fragment orders could be deduced. In the case of HindHI, and BamHI, the fragment orders were confirmed by separate Hind111 or BumHI cleavage of each isolated EcoRI fragment. The resulting maps are shown in Fig. 9, which includes the coordinates of all fragments listed in Table 1, except fragment HpuI-G, whose position has not been determined. Moreover, by the methods used, fragments less than about 0.5% of the length of AdFL-DNA could have been missed. Homology between AdFLAd-DNAs
and
Human
A comparison of AdFL cleavage maps with those of human adenoviruses (Sambrook et al., 1975) was the first indication that FL-DNA was markedly different from human Ad-DNAs. Second, the buoyant
190
LARSEN
AND
NATHANS
IL TABLE
ENDONUCLEA~E
EcoRI
37 23 10 8.6 7.6 7.4 3.5 2.8
I &II
51 22 13 7.2 4.6 2.6
a Data are expressed
38 16.7 12.5 10 9.6 6.2 6.0 1.5
33 19.5 19.5 11.5 9.2 3.6 3.5
25 13.3 12 12 10 9.0 6.0 4.5 3.5 1.6 1.1
in percentages
38 23 20 6.7 5.0 3.5 2.0 1.8
I
&I
34 20 10.8 9.3 6.7 5.1 4.2 3.5 2.0 1.5 1.2
of AdFl-DNA.
density of 32P-labeled FL-DNA in CsCl (1.702 g/ml) compared to that of 3H-labeled Ad2-DNA in the same tube (p = 1.716 g/ ml; Pina and Green, 1965) indicated that FL-DNA had a low C + G content (44%) compared to AdB-DNA (57%). Third, we estimated the degree of nucleotide sequence homology between FL-DNA and the DNA of representatives of three groups of human adenoviruses. For this purpose 32P-labeled FL-DNA was mixed with a lOO- to 300-fold excess of 13HlDNA from Ad2, 7, or 12, the mixed DNAs were then fragmented and denatured, and the percentage of duplex [32PlDNA and 13HlDNA formed was determined during renaturation, as detailed in Materials and Methods. As shown in Fig. 10, whereas 85 to 90% of each of the human Ad-DNAs reannealed, less than 10% of the FL-DNA was converted to duplex form. Thus by this test, FL-DNA has little or no nucleotide sequence homology with Ad2-, 7-, or 12DNAs. DISCUSSION
Based on virion morphology and general properties of its DNA, AdFL is a typical adenovirus. It produces small plaques on L cells and large plaques on BALB/c 3T3 cell monolayers, and it grows sufficiently well in Swiss 3T6 cells or BALB/c 3T3 cells to allow preparation of purified virions and viral DNA for biochemical studies. AdFL
DIGESTION”
11‘lindI1: IE iindII1 :E:lindII1
lamHI [ 13amHI
EcoRI 6koR1
BaiLa [I iin+dIIl [ 1&I 35 22.5 14.3 14.3 5.5 4.6 1.7 1.3
2
R FRAGMENTS PRODUCED BY DOUBLE Endonuclease pairs
30 17.4 16 11.8 10.6 5.3 3.6 2.5 1.9 1.2
See footnote
3glII
13glII
&II
H&I
&I
&I
&I
28.5 23.5 10.2 10 9.6 7.6 3.5 2.6 1.2
14 13 11 11 9.2 9.2 9.0 7.6 6.3 3.4 1.5 0.9
23.5 12.9 12.2 11.5 11.4 8.0 7.6 3.5 3.5 1.9
36 11.7 12.2 11 9.3 3.4 3.4 2.6
39 17.5 13 12.5 11.4 2.6 2.5 1.2
WI &I 22.5 20 13.5 12.8 9.7 9.7 3.5 2.6 1.7
a to Table 1.
virions prepared from plaque-purified seed virus by equilibrium sedimentation in CsCl consisted predominantly of “complete” virus particles (p = 1.34 g/ml) with a lesser amount of empty particles and minor species of intermediate buoyant density. The minor species are presumably particles with incomplete genomes, as described for human adenoviruses (Smith, 1965; Burlingham et al., 1974; Daniell, 1976). The limited host range of AdFL is also characteristic of adenoviruses. As reported by Hartley and Rowe (19601, AdFL does not cause cytopathic effect in rat embryo, monkey kidney, human embryonic skin or muscle cells, or in KB cells. In our experiments neither cytopathic effect nor virus multiplication measured by plaque assay was detected in three different human cell lines, in human embryonic kidney cells, in WI38, or in two monkey kidney lines. Thus, of the cells tested, only mouse cells supported the growth of AdFL. Whether cells of other species, and particularly human cells, are abortively infected is now being investigated. The properties of AdFL-DNA extracted from purified virions are very similar to those of human adenovirus genomes. It is a linear duplex, the molecular weight of which is 20 x lo6 as estimated by electron microscopic length measurements relative to SV40 DNA. When the molecular weight
MOUSE
A
B
C
ADENOVIRUS
DNA
191
D A
B
C
D
FIG. 7. The effect of Pronase digestion or phenol extraction on the mobility of restriction fragments. (A) DNA isolated with Pronase digestion and phenol extraction. (B) DNA-protein complexes isolated without Pronase or phenol by the method of Robinson et al. (1973). (C) As in (B) except that the DNA was treated with Pronase just prior to electrophoresis. (D) As in (B) except that the DNA was extracted with phenol once just prior to electrophoresis.
was estimated by summing the electrophoretically determined molecular weights of all restriction fragments in appropriate digests, a value of 19.2 to 19.6 x lo6 was obtained. The length distribution of DNA
extracted from virions showed a skewing toward shorter molecules, again suggesting that some virions contain incomplete genomes. Similar to other adenovirus DNAs, AdFL-DNA also appears to have
LARSEN
192
AND
NATHANS
-SDS
+SDS ABCDEFGH
ABCDEFGH
FIG. 8. The effect of SDS on electrophoretic mobility of FL-DNA-protein complexes and their restriction products. (+SDS) Gel and running buffer contained 0.5% SDS. (-SDS) No SDS in gel or buffer, but 1% SDS in applied sample. (A) EcoRl digestion followed by Pronase. (B) EcoRl digestion, without Pronase. (C) Undigested DNA-protein complex, with Pronase treatment. (D) Undigested DNA-protein complex, without Pronase treatment. (E)BamHl digestion, followed by Pronase. (F)BamHl digestion, without Pronase. (G) Hind111 digestion followed by Pronase. (H) Hind111 digestion, without Pronase. At the exposure density shown, the BamHI-F fragment was not evident; however, it appeared after longer exposure, and the effect of SDS on its relative mobility was similar to that observed for the HindIII-F fragment.
protein linked to its ends, as judged by the presence of circular duplexes in material prepared without protease treatment (Robinson et al., 19731, by the Pronase-dependent mobility (Brown et al., 1975; Sharp et al., 1976; R. J. Roberts, personal communication), and by the SDS-dependent mobility of terminal restriction fragments. AdFL differs from human Ad strains, however, in the base composition of its DNA. Its C + G content (44%, estimated from buoyant density) is significantly lower than that reported for any human adenovirus DNA. AdFL-DNA has little or
no (~10%) nucleotide sequence homology with representative strains from three major groups of human adenoviruses (Ad12 of group A, Ad7 of group B, and Ad2 of group C). Also, there was no detectable reaction between human adenovirus anti-T antigen sera and extracts of AdFL-infected cells. Since the antisera used react with T antigens from all A group or several B group adenoviruses, or with Ad2 T antigen (C group), human adenovirus T antigens appear to differ from the analogous AdFL antigen(s). Nonetheless, since antiserum against human adenoviruses reacted with AdFL lysates by complement fixation as-
MOUSE B
Eco RI
BomHt
ffindm
ADENOVIRUS
193
DNA A
C 92.6
19.5
D
B
A
5.5
FE 3.5
JI
C II
B
D
23
A
D 97.4
TO B
12.5
B
A
13 C
EF 94.2SSB
56
46
C
C TO
A
E 73
35
MAP
analysis
and double enzyme digestion
(Ad21
30 Min.
F 94
UNITS
FIG. 9. Cleavage maps of AdFL-DNA deduced from end fragment patterns for six endonucleases. See text for details.
Hours
D a3
45 (Ad?,
60
A.6121
FIG. 10. Hybridization of FL-DNA in the presence of human adenovirus DNAs. Mixture 1: 32P-labeled FL-DNA (0) (85 rig/ml, 1.3 x lo5 cpm/gg) and 3H-labeled AdB-DNA (0) (11.5 pg/ml, 8100 cpmlpg). Mixture 2: 32P-labeled FL-DNA (@) (85 rig/ml, 1.3 x lo5 cpm/pg) and unlabeled salmon sperm DNA (11.5 pg/ml). Mixture 3: 3ZP-FL-DNA (B) (90 rig/ml, 6.6 x lo4 cpmlpg) and 3H-labeled Ad7-DNA (0) (28 pgglml, 890 cpm/ pg). Mixture 4: 32P-labeled FL-DNA (A) (90 rig/ml, 6.6 x lo4 cpm/pg) and 3H-labeled AdlB-DNA (A) (19 pg/ ml, 1460 cpmlpg). Mixture 5: 32P-labeled FL-DNA (x) (90 rig/ml, 6.6 x lo4 cpmlpg) and unlabeled salmon sperm DNA (20 fig/ml). The curves have all been normalized to 100% at zero time; the measured zero-time points (compared to a sample of the total reaction mixture) varied between 94 and 107%. At higher C$ values, 95% of 32P-labeled FL-DNA became hydroxyapatite-adsorbable.
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say (Hartley and Rowe, 1960; and present study), there is probably some sequence homology not detected by our experiments. A cleavage map of the AdFL genome was generated by the use of restriction enzymes that made between two and six scissions in the DNA molecule. For each enzyme, terminal fragments were identified by their shift in electrophoretic mobility due to pretreatment of the digest with Pronase or with SDS. Other fragments were positioned by analysis of digests produced by pairs of enzymes. Some uncertainties remain. First, the exact map positions of some cleavage sites are uncertain, due to the inaccuracy of molecular weight estimates of fragments greater than about 5 x lo6 daltons. Based on the consistency of map coordinates deduced from different double-digest patterns, we estimate that the map positions of large fragments may be in error by as much as 1 to 2 map units. Second, the position of the 1.5% HpaI-G fragment has not yet been determined. Third, in some experiments fragments less than 0.5% of the length of AdFL-DNA would have been missed owing to the inadequate specific activity of the DNA used. Detection of small fragments will require 32P-end-labeling of restricted DNA. As mentioned in the Introduction, our motive for investigating the molecular biology of still another adenovirus is the possibility that the mouse virus may eventually prove useful as a transducing vehicle for transferring rather large DNA molecules between different mouse strains or mouse cells in culture. The present study is a first step in the prerequisite characterization of the viral genome. ACKNOWLEDGMENTS We thank R. J. Toni, R. V. Gilden, R. J. Roberts, T. J. Kelly, Jr., and Gary Ketner for advice and materials; and we thank Jane Suthers, Janet Meyers, and Thomas Zeller for able technical assistance. This research was supported by grants from the Whitehall Foundation and the National Cancer Institute (CA 16519). S. L. is a postdoctoral trainee of the National Cancer Institute (CA 09139). REFERENCES ALLET, B., JEPPESEN, P. G. N., KATAGIRI, K. J., and DELIUS, H. (1973). Mapping the DNA fragments
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