Defective simian virus 40 genomes: Isolation and growth of individual clones

VIROLOGY

62,

112-124

(19’74)

Defective

Simian

Virus 40 Genomes: of Individual

JANET Department

of Biochemistry,

E. MERTZ Stanford

University

Isolation

and Growth

Clones AND

PAUL

Medical

BERG

Center, Stanford,

California,

94305

Accepted July 18, 1974 Eleven defective deletion mutants of simian virus 40 (SV40) have been isolated. The methodology developed for this purpose involves the use of temperature-sensitive mutants as helpers. Included are procedures for screening for the presence of mutants, plaque-purifying, propagating, and assaying the titer of stocks of these mutants, and assigning mutants to complementation groups. These same procedures may be adaptable to enable isolation of deletion mutants of other animal viruses as well. INTRODUCTION

Although conditional-lethal, temperature-sensitive (ts) mutants of simian virus 40 (SV40) have provided valuable information about the physiology and molecular biology of the viral life cycle (see Tooze, 1973; Eckhart, 1974, for reviews), their utility is limited to genes that code for proteins. Their usefulness is further restricted by any “leakiness” or ability of the mutant proteins to function at the restrictive temperature and by the difficulties encountered in trying to identify the altered polypeptides and to accurately map the mutant loci in the viral genome. On the other hand, mutations resulting from substantial alterations in the viral DNA structure (e.g., deletions, insertions, duplications, and substitutions) would be more likely to cause nonleaky alterations of the viral phenotype. In addition, their mutant functions could be more easily identified and mapped using gel electrophoresis and heteroduplex analysis, respectively (Maizel, 1969; Davis et al., 1971). One source of such altered SV40 genomes is the defective virus particles that accumulate when SV40 is serially propagated at high multiplicities of infection (Yoshiike, 1968; Tai et al., 1972). In this paper we present a general method for cloning, propagating, and assaying the infectivity of defective SV40 mu-

tants having gross alterations in the structure of their genomes. Subsequent papers (Mertz and Berg, 1974; Herzberg, Mertz, Berg, Cameron, and Davis and Carbon and Berg, manuscripts in preparation) will describe how this and other methods have been used to isolate individual clones of viable deletion mutants, restriction endonuclease-generated deletion mutants, and mutants containing small inserts of dA:dT in their genomes, respectively. MATERIALS

AND

METHODS

Cell lines. CV-1P cells obtained from S. Kit and MA-134 cells from J. S. Pagan0 are established lines of African green monkey kidney (AGMK) cells. They were grown in plastic petri dishes (Nunclon) using Dulbecco’s modification of Eagle’s medium (Gibco) supplemented with 10% calf serum (Microbiological Associates), 500 units/ml penicillin and 100 &ml streptomycin sulfate in a 37”, 5% CO, incubator. Primary AGMK cells (Flow Laboratories) were grown as described above with 10% fetal bovine serum replacing the calf serum. After viral infection, confluent monolayers of cells were maintained in medium containing 2% fetal bovine serum. Viruses. SV40 strain Rh911 (Girardi, 1965), from J. Vinograd, was serially plaque-purified four times on CV-IP cells,

CLONING

SV40 DELETION

grown two times for 72 hr from input multiplicities of infection (m.0.i.) of 0.05, and then grown on MA-134 cells from an initial m.o.i. of 0.01 until about 90% of the cells exhibited cytopathic effect. The infected cells and culture fluid were frozen and thawed twice, extracted with 0.1 vol chloroform, and centrifuged to remove cellular debris. After addition of 0.05 vol 1.0 M Tris-HCl (pH 7.5) and fetal bovine serum to 2%, the supernatant fluid (stored at -20”) served as the starting stock of wild-type (WT) SV40 virus (renamed RhSll-Stanford, WT800). Stocks of the temperature-sensitive (ts) viral mutants tsA30, tsB4, and tsB*ll (Robb et al., 1972; Tegtmeyer and Ozer, 1971; Tegtmeyer, 1972) (kindly supplied by P. Tegtmeyer) were prepared as described above after single cycles of growth at 32”; each infection was performed at a m.o.i. of
MUTANTS

113

I DNA was treated with EcoRI enzyme under conditions previously described (Mertz and Davis, 1972). (b) HpaII, a restriction endonuclease from Hemophilus parainfluenzae (Sack and Nathans, 1973; Sharp et al., 1973) was purified by a modification of the second method of Sharp et al. (1973). After phosphocellulose chromatography, the enzyme was dialyzed against buffer A (Sharp et al., 1973), applied to a 1 x 11-cm DEAE-cellulose (Whatman DE-52) column and eluted with a linear gradient (80 ml) of O-O.8 M KC1 in buffer A. The enzyme activity, eluting at 0.04-0.10 M KCl, was then applied to the carboxymethylcellulose column. The final enzyme preparation, free of any detectable exonuclease activity, still contained a trace though noninterfering amount of HpaI endonuclease. HpaII endonuclease reactions were performed for l-4 hr at 37’ in 10 mM Tris-HCl (pH 7.5), 5 mM MgCl,, 0.4 dithiothreitol, and 100 fig/ml gelatin (autoclaved, Difco-Bacto). Plaque assays. Monolayers of CV-1P cells were grown in 60-mm Falcon petri dishes. (a) Virus (0.2 ml per plate), diluted in Tris-buffered saline (TBS, Kimura and Dulbecco, 1972) containing 2% fetal bovine serum, was allowed to adsorb, with occasional rocking of the plates, for 1’12-2 hr at 37”. The cells were overlaid with 5 ml of agar medium (Auto-Pow Minimal Essential Medium (Flow Labs) containing 4% fetal bovine serum, 1.0% Agar (Difco-Bacto), penicillin, and streptomycin) and incubated at the appropriate temperature in a 5% CO, incubator. With temperaturesensitive mutants, 32-33” and 40.5-41.0” served as the nonrestrictive and restrictive temperatures, respectively. The cells were fed 5 days after infection with 3 ml per plate of agar medium (containing 1% instead of 4% fetal bovine serum). After an additional 4 days, agar medium containing 0.01% neutral red was added; cultures at 41” were placed at 39.5” to prevent cell killing by neutral red. Plaques were counted between the ninth and thirteenth day after infection or until the number of plaques became constant. Plaque assays performed at 32-33” were continued, with weekly additions of agar medium, for up to 30 days. (b) Plaque assays using DNA were

114

MERTZ

AND BERG

carried out using a modification of the sucrose, 0.2-0.8 N NaOH, 0.8-0.2 M NaCl, method of McCutchen and Pagan0 (1968). 5 mM EDTA). The total number of 32P counts that sedimented in the region of The monolayers were washed twice with TBS and then incubated for lo-20 min at SV40 Form I marker DNA and remained room temperature with 0.2 ml of TBS acid precipitable (in the presence of added carrier DNA) after a further treatment in containing the DNA and 500 kg/ml DEAE-Dextran (2 x lo6 daltons, Phar- alkali at 90” for 10 min served as the macia Fine Chemicals). After washing the measure of viral DNA replication. cell monolayers twice more with TBS, RESULTS they were overlaid with agar medium, and Starting material for isolation of defectreated subsequently as described above. Immunofluorescent staining. The in- tive SV40 genomes. SV40 virus particles direct immunofluorescent staining tech- with defective genomes accumulate when plaque-purified virus is serially passaged in nique was used for the detection of viralof induced antigens. After infection and a susceptible cells at high multiplicities suitable period of incubation, the cells were infection (Yoshiike, 1968). These defective rinsed twice with TBS, soaked for 30 set in viral DNAs are generally smaller than the virus absolute methanol, and then air dryed. To DNA obtained from plaque-purified stain the cells, they were incubated for 2 hr and have gross alterations (deletions, substitutions, additions) in their molecular at 37” with the appropriate SV40-specific structure (Tai et al., 1972). Since the antiserum diluted in phosphate-buffered saline (PBS) (Robb and Martin, 1970), molecular defects contained in these DNAs are distributed somewhat randomly washed thoroughly with PBS, and then throughout the molecule (Risser and Mulincubated with the appropriate fluoreder, 1974; J. E. Mertz and P. Berg, manustein-conjugated globulin (anti-globulin) these preparations (Robb, 1973). The sequential sera used script in preparation), were: SV40 anti-T hamster serum (Flow provide a rich source of mutants with substantial modifications in various porLabs) and rabbit anti-hamster-globulin globulin (Sylvana Co.) for T antigen; and tions of their genomes. Our starting material for the isolation of anti-SV40 bovine serum (Flow Labs) and rabbit anti-bovine-globulin globulin (Syl- such mutants was obtained by four serial vana Co.) for V antigen. A Leitz Ortholux passages of wild-type SV40 (WTSOO) on microscope fitted with a halogen lamp, dry primary AGMK cells using the undiluted darkfield condenser and FITC filter was lysate obtained from each passage to initiused to determine the fraction of fluorescate the subsequent infection. The SV40 ing cells in the population. DNA obtained from the fourth cycle of Assay for viral DNA replication. Monoinfection was judged to be grossly defective layers of CV-1P cells in 32-mm Falcon by the following criteria: the titer (plaquedishes were infected as described above forming units (PFU)/ml) was more than with 0.038 pg of DNA in 0.1 ml of TBS 200-fold lower than that of an analogous containing 500 pg/ml of DEAE-Dextran lysate produced by low m.o.i. infection; the and then incubated at 37” with medium supercoiled DNA from the infected cells containing 2% dialyzed fetal bovine serum was very heterogeneous in size with moleand 2% anti-SV40 horse serum (Flow Labs) cules ranging from less than 0.6 to someto minimize reinfection with progeny virus. what greater than 1.0 SV40 fractional Forty hours later, 180 PCi of [32P]inorganic length; the defective DNA yielded delephosphate (NEN) was added. Fifty hours tion, substitution, and addition loops when postinfection, the cells were lysed (Hirt, heteroduplexes formed between them and 1967). The DNA contained in the supernawild-type (WT) SV40 DNA were examined tant was incubated at 37” for 20 min in 0.2 by electron microscopy; and, although N NaOH to hydrolyze RNA. The amount about 99% of the molecules in WT DNA of viral DNA formed was determined on a preparations are cleaved once at map posilinear alkaline sucrose gradient (5-20% tions 0 and 0.735 by the EcoRI and HpaII

CLONING

SV40 DELETION

restriction endonucleases, respectively (Morrow and Berg, 1972; Mulder and Delius, 1972; Sharp et al., 1973), 55% of the DNA molecules in this stock was resistant to cleavage by EcoRI endonuclease, 47% was resistant to HpaII endonuclease, and 23% was resistant to both enzymes (J. E. Mertz and P. Berg, manuscript in preparation). The EcoRI endonuclease-resistant and HpaII endonuclease-resistant DNA molecules were each separated from the cleaved molecules by equilibrium centrifugation in cesium chloride-ethidium bromide gradients. These two heterogeneous DNA preparations served as the sources of defective SV40 DNA used in all subsequent experiments and from which cloned isolates of defective SV40 were recovered. SV40 DNAs resistant to cleavage by EcoRI or Hpa.II endonuclease are defective in expressing “late” functions. Preparations of EcoRI and HpaII endonucleaseresistant DNAs have markedly reduced infectivities in conventional plaque assays (Table 1). Consequently, to isolate and propagate such defective genomes, a procedure is needed which permits complementation of their defect(s). Temperature-sensitive (ts) mutants of SV40 can be used for this purpose. To decide which of the known ts mutants could complement these defective DNAs for growth, it was necessary to identify the defective function(s) in the EcoRI and HpaII endonuclease-resistant DNA populations. After infection, both the EcoRI and HpaII endonculease-resistant DNAs inducme the “early” SV40specific T antigen and replicate their DNAs at nearly normal levels (Table l), but they fail to produce the “late” virion capsid (V) antiIgen. Consequently, molecules lacking Ieither the EcoRI or HpaII restriction endonuclease-recognition site (map position 0 and 0.735, respectively) can express those ,functions preceding and including viral DNA replication (“early” functions) but they cannot express all of the “late” functions. This result agrees with previous reports that these two restriction sites are contained within the “late” region of the genome (Khoury et al., 1973; Sambrook et al., 1973; Kelly and Lewis, 1973; Morrow et

115

MUTANTS TABLE

1

INDUCTION OF SV40-SPECIFIC ANTIGENS, VIRAL DNA REPLICATION, AND PLAQUE FORMATION BY EcoRI AND HpaII ENDONUCLEASE-RESISTANTSV40 DNAs” DNA resistant to

Antigen-positive cells

endonuclease T

V

Viral DNA replication

PFU

(7%of WT) EcoRI

47

0.2

46

1.6”

HpaII

31

0.03

36

0.01’

u Monolayers of CV-1P cells (3 x 10’ cells per 32-mm dish) were infected with 0.038 pg (in 0.10 ml) of the specified D&A, incubated at 37” for 72 hr in medium containing 2% anti-SV40 horse serum, and then fixed, stained, and scored for the number of antigen-positive cells per dish as described in Methods. The values for antigen-positive cells are averages obtained from two dishes; wild-type (WT) DNA induced 1280 T antigen-positive cells per dish and 1490 V antigen-positive cells per dish. The ability of the DNA to replicate was assayed as described in Methods; the values for the endonuclease-resistant DNAs are the averages from two separate analyses, where the amount of SV40 DNA induced by WT DNA was 13,120 32P cpm in one of the assays. Plaque-forming units were measured as described in Methods; the value for WT DNA was 1.5 x 10’ PFUlrg. b This preparation of EcoRI endonuclease-resistant DNA was contaminated with l-2% EcoRI endonuclease-sensitive DNA as determined by further treatment of the DNA preparation with EcoRI endonuclease; a different preparation of EcoRI endonucleaseresistant DNA yielded only 1 x lo3 PFU/pg or 0.01% of the WT DNA value. ( Eighty percent of these plaques contained a viable, plaque-morphology mutant that lacks the HpaII endonuclease-cleavage site; these mutants have deletions surrounding the HpaII endonucleaserecognition site which vary in size from about 80 to 190 base pairs (Mertz and Berg, 1974).

al., 1973). On this basis tsA30, a nonleaky “early” mutant which can complement the “late” mutants tsB4 and tsB*Il for growth (Tegtmeyer, 1972), was selected as the “helper” virus for growing the restriction enzyme-resistant “late” mutants. Plaque assay for “late” mutants by complementation with an “early” mutant. Generally, propagation and plaque assays of defective animal viruses have been accomplished using helpers under conditions where the helper virus cannot grow by itself

116

MERTZ

(Boeye et al., 1966; Kawai and Hanafusa, 1972). In this instance we have chosen to use the nonleaky, temperature-sensitive mutants of SV40 as helper viruses. Our procedure for isolating individual clones of defective “late” SV40 mutants can be illustrated using the late ts mutant, tsB4. Coinfection of monolayers of CV-1P cells with tsB4 and tsA30 virus or DNA, as described in the legends to Fig. 1 and Table 2, respectively, followed by incubation of the cells at 41”, results in the appearance of plaques within 10 days after infection (see Fig. Id). Therefore, mutants capable of complementing tsA30 for growth can be isolated as “mixed-plaques” that appear at high temperature in a helper assay. Furthermore, the number of plaques seen when the amount of tsA30 helper virus per dish is kept constant is directly proportional to the amount of tsB4 virus used in the infection (Fig. 2A), and, as expected,

AND BERG

there is a direct linear relationship between the apparent titer of tsB4 and the amount of the complementing helper (tsA30) used in the assay (Fig. 2B). Assays of tsA30 with constant levels of tsB4 helper give similar results (data not shown). We, therefore, further conclude that this helper virus procedure can probably be used as a standard, relatively rapid method for titering stocks of any (defective as well as ts) mutant that is capable of growing well when complemented by a helper. It should be noted that at the highest levels of tsA30 helper used the apparent titer of tsB4 in the complementation assay approaches, and even surpasses, the titer obtained at 32” in the absence of helper virus (dashed line in Fig. 2B). (A simple explanation for these higher-than-expected apparent titers is that they are due to an increased efficiency of plaque formation at the higher temperature; for example, the

FIG. 1. Morphology of plaques formed after coinfection of tsA30 with WT(c), tsB4 (d), and the defective mutants, dl-818 (e) and dl-811 (f). Monolayers of CV-1P cells (1.4 x lO%O-mm dish) were mixedly infected by incubation at 37” for 2 hr with a small amount of the indicated mutant and 5.3 x 10’ PFU of tsA30 virus (titer determined by 32” plaque assay), overlaid with agar medium, and then incubated at 41’ as described in Methods. Nine days later, neutral red was added and dishes were put at 39.5’. The photograph was taken 10% days postinfection. (a) mock infected (no mutant or helper virus): (b) tsA30 helper virus only; (c) WT with tsA3O virus; (d-f) tsB4, dl-818, dl-811 mutants, respectively, with tsA30 virus.

CLONING TABLE

SV40 DELETION

117

MUTANTS

2

PLAQUE-FORMING ACTIVITY OF DEFECTIVE SV40 DNA IN THE PRESENCEAND ABSENCE OF tsA30 “HELPER” AT 41” a DNA resistant 10 endonuclease

z

Helper None

tsA3O DNA

tsA30 virus (PFU/ dishID 2 x IO’

2 x 105

EcoRI

6 x 105’

2 x lo6

ND”

ND

HpaII

5 x 103’

7 x 10”

2 x 10’

2 x 105

o The defective DNAs are the same as those described in Table 1. The ability (PFU/pg) of these defective DNAs to produce plaques on CV-1P monolayers (approximately 1 x lo6 cells/dish) was assayed as described in Methods. When helper DNA (0.01 rg/dish) was used, the two DNA samples were mixed together with 500 rg/ml DEAE-Dextran before being added to the cells (0.2 ml/dish). Where tsA30 virus was used as helper, infection with virus (as in Methods) was performed immediately after adsorption of the defective DNA; when the order of these two infections was reversed, the specific infectivity was roughly 3-fold lower. b Titer of tsA.30 virus was measured in a plaque assay at 32” (see Methods). c See footnotes to Table 1. d Not determined.

ratio of WT PFU obtained at 41” to that at 32” is about 3.) Since, even at 41”, high levels of tsA30 virus will kill cell monolayers, the quantity of tsA30 used as helper has been kept within the range of 0.01-0.1 PFU/cell. Consequently, to eliminate possible errors that could arise from an inaccurate estimate of the m.o.i. of the helper used for titering mutant virus stocks, a standardized stock of a ts mutant capable of complementing the same helper is usually assayed at the same time as the unknown stocks, thereby providing an appropriate correction factor. For example, if the complementation titer of the standard ts mutant, d.etermined in the parallel assay, is one-fourth of its titer determined at 32”, the measured (apparent) complementation titers of the unknown mutant virus stocks are multiplied by four. screening for, and plaque“Cloning,” purifying defective mutants. Having estab-

A

PFU OF tsB4

(320)

100

II 10-3

B

II

III

10-z

10-l

II 100

MO1 OF tsA30

FIG. 2. A. Proportionality in the coinfection plaque assay of tsB4 using tsA30 as helper. Varying quantities of tsB4 virus (in 0.1 ml) (titered at 32” in a standard plaque assay) were mixed with 0.1 ml of tsA30 helper virus (m.o.i. = 0.04) directly in 60-mm dishes containing monolayers of CV-1P cells and the cells were infected and incubated as described in the legend to Fig. 1. All points are the averages of the number of plaques produced at 41” in duplicate dishes; the horizontal bars indicate the actual values obtained for each dish. B. Relationship between the apparent titer of tsB4 and the m.o.i. of tsA30 helper virus. Mixed infections of tsB4 and tsA30 virus were performed as described in Fig. 2A. The apparent titer of tsB4 at each level of tsA30 helper was determined from the best-fit straight line obtained from plots similar to that shown in Fig. 2A. The relative titer is calculated by dividing the apparent tsB4 titer at each level of helper virus by the titer of the same tsB4 virus stock assayed in parallel at 32” in the absence of helper virus. The open triangle, representing the value obtained from Fig. 2A (rel. titer = 0.21; m.o.i. of helper = 0.04), demonstrates the efficiency of the assay with the standard amount of tsA30 virus used in helper plaque assays.

118

MERTZ

lished that the late mutant tsB4 can be detected and grown as a mixed-plaque with tsA30 as the helper, we coinfected CV-1P cells with the restriction endonucleaseresistant DNA preparations and tsA3O DNA. With 0.01 pg of tsA30 DNA per dish as helper, there was a 3- and 140-fold increase in the number of plaques produced by the EcoRI and HpaII endonuclease-resistant DNAs, respectively, as compared to infections without helper (see Table 2). Therefore, the helper DNA probably permits many of the defective DNAs to form plaques. Although coinfection with tsA30 virus also increased the apparent titer of defective DNA, it was not as effective as tsA30 DNA (Table 2). Probably 0.01 pg of helper DNA is enough DNA to infect almost all of the cells which are susceptible to infection at a particular time by the defective DNA (under the conditions used, only l-2% of the cells are susceptible to infection with SV40 DNA (Dubbs and Kit, 1971; DePamphilis and Berg, unpublished)) whereas, with the multiplicities of helper virus used, only a small fraction of the cells receiving defective DNA are also infected with virus. Because the apparent titer of defective DNA is proportional to the m.o.i. of the coinfecting helper virus (see Table 2), maximum efficiency of mixed-plaque formation can only be obtained when most of t,he cells in the monolayer are infected with helper; however, this is not achievable in practice because such high multiplicities of infection with the helper virus would destroy the cell monolayer. Quite possibly, higher efficiencies could be achieved if cells were infected with the mutant DNA and high levels of helper virus and then reseeded in an infectious center assay. Plaques appearing on plates which had been coinfected with tsA30 were picked with a Pasteur pipette, mixed with 1 ml TBS containing 2% fetal bovine serum, frozen and thawed twice, shaken briefly with chloroform (approximately 0.2 ml) to disrupt, viral aggregates, and then tested for the presence of helper and defective virus. Various dilutions of these virus suspensions were used to infect cells with and without additional tsA30 helper virus. Fig-

AND BERG

ure 3 shows that with the defective mutant dl-818 (dl = deletion (Robb et al., 1972)) many more plaques were formed in the presence of added tsA30 than in its absence: at a dilution of approximately 104fold, between 50 and 100 plaques were produced with the tsA30 helper, but no plaques without it. As expected, the doseresponse relationship with helper follows a single-hit curve. Most of the plaques formed in the absence of added tsA30 are undoubtedly due to coinfection by the defective virus and the low level of tsA30 contained in the sample; some of the plaques may also result from the presence of wild-type recombinants and revertants. The dose-response curve in the absence of added helper should be close to a two-hit curve; the fact that the curve is somewhat less than two-hit in this experiment is perhaps due to inadequate dispersion of the virus suspension. Quite clearly, this simple technique (plating f helper) can be used to determine if plaques consist predominantly of complementing mutants. Eight of eleven plaques picked from coinfections with EcoRI endonucleaseresistant and tsA30 DNAs and 11 of 12 picked from coinfections with HpaII endonuclease-resistant and tsA30 DNAs I

I

I

I

I

I

I

I

103 DILUTION

I

I

I

I

I

I

104 OF VIRUS

I

I 105

FIG. 3. Plaque assay of mutant virus with tsA30 helper virus. A virus stock containing the defective mutant dl-818 and the tsA30 helper used in growing it was assayed for plaque-forming activity with and without added &A30 helper virus. All points are averages of duplicate dishes. Filled circles (O), infection with added tsA30 helper (m.o.i. = 0.03 PFU/ cell); open circles (0), without added helper virus. The solid (-) and dashed (- -) lines are theoretical one-hit dose-response curves; the dotted line (----) is the theoretical curve for a two-hit process.

CLONING

SV40 DELETION

MUTANTS

119

showed O-10 plaques when 0.1 ml of a mutant DNA are needed than can be obtained by the method summarized above 40-fold dilution of the plaque suspension was assayed without additional tsA30 and in Table 3. For this purpose we have virus. However, the infections with added developed another protocol for propagating tsA30 helper virus gave dozens of plaques defective mutants. After titering the virus (at least five times more in all cases). preparations obtained as described above, Generally, the mixed-plaques, whether tsA30 virus is added to make its concentraproduced by tsB4 and tsA30 coinfections tion about equal to that of the late ts or (Fig. Id) or from coinfections by defective defective virus. Cells (MA-134) are inmutants and !-~A30 (Fig. le,f), were considfected with the virus mixture (m.o.i. of 3-7 erably smaller than those produced by PFU of each virus/cell) and, after incubawild-type (Fig. lc). The remaining four tion at 33” for 14-18 days, virus or viral plaque suspensions yielded roughly equal DNA is isolated from each dish as denumbers of plaques (> 100 PFU/O.l ml of a scribed previously (Table 4). In this in40-fold dilution) in the presence or absence stance, the recoveries of both helper and of added tsA30 virus and were presumed to defective virus and intracellular DNA are contain predominantly or exclusively wildnearly equivalent to those found with WT type virus. These plaques were not exam(or tsA30 at the nonrestrictive temperained further. ture) virus grown under the same condiThose plaques that were significantly tions. The reason for the reduced yields at complemented by added tsA30 were seri- 41”, but normal yields under otherwise ally plaque-purified by coinfections with a equivalent conditions at 33”, is not understood at the present time. mixture of an approximately 800-fold diluPreparation of purified homogeneous tion of each plaque suspension and added tsA30 helper virus. Generally, this proce- populations of defective mutant DNAs. To separate a defective mutant from its helper dure was repeated twice to obtain plaques consisting of a single defective virus and DNA, the closed circular DNA obtained the tsA30 helper. from the lower band of a CsCl-ethidium Propagation and production of large bromide gradient after equilibrium centrifquantities of defective mutants. Eleven ugation was incubated with EcoRI endonuindependent plaque isolates, five from the clease (if the mutant was derived from the DNA pool) EcoRI endonuclease-resistant DNA pool EcoRI endonuclease-resistant or with HpaII endonuclease (if it came and six from the HpaII endonucleaseresistant DNA pool were selected for furfrom the pool of HpaII endonuclease-resistant DNA) and then sedimented in a neuther study. To obtain DNA preparations and high titer virus stocks of these putative tral CsCl gradient as described in the legend to Fig. 4. Each of the 11 mixed DNA defective mutants, monolayers of CV-1P cells (4 x lo6 cells/lOO-mm dish) were preparations yielded two peaks of labeled DNA (see Fig. 4 for example). Each faster infected with 0.6 ml of a mixture containing 4 x 105 PFU of tsA30 virus and peak cosedimented with, or slightly behind, marker SV40 Form I DNA. DNA one-twentieth of the last mixed-plaque obtained from the faster-sedimenting peak isolate (approximately 5 x lo4 PFU) . After 7 days of incubation at 41”, viral DNA or was resistant ( >98%) to further cleavage virus was isolated from each culture as by the appropriate restriction endonuclease and was the mutant DNA used in described in Methods (data from equivasubsequent experiments. The slower sedilent infection conditions is shown in Table menting DNA corresponded to cleaved 3). Under these conditions, the combined yield of both complementing viruses is unit length linear tsA30 helper DNA. The about 5515% that of wild-type; the yield of amount of DNA in each preparation that was resistant to cleavage by the restriction viral DNA recovered from the Hirt extracts endonucleases varied from about 50 to 96% is also roughly 10% that of wild-type DNA. (Table 5); the reason for this variation may For many purposes higher titers of mutant virus stocks or larger quantities of be related to the genome structures of the

120

MERTZ

AND BERG

TABLE YIELD OF VIRUS AND VIRAL DNA Infecting Mutant

3

FROM CELLS CO-INFECTED AT Low MULTIPLICITIES OF INFECTION WITH LATE MUTANTS AND tsA30 VIRUS AT 41”” DNA

Virus

viruses PFU/plate

+ Helper Mutant

Individual

Total

Percent of total as late mutant

Total xdplate

Percent of total as defective DNA”

WT800

None

WT

1.0’

1.0

-

1.W

-

WT800

tsA30

WT A30

0.73 ND

ND

-

0.91

-

tsB4

tsA30

B4 A30

0.048 0.027

0.075

64

0.25

-

dl-811

tsA30

811 A30

0.073 0.067

0.14

52

0.11

55

dl-818

tsA30

818 A30

0.037 0.012

0.049

76

0.13

87

n Monolayers of MA-134 cells (7 x lo6 cells/lOO-mm dish) were coinfected with 0.6 ml of a virus suspension containing either 7 x 10’ PFU of WT or 7 x 10” PFU of each of the viruses indicated in the Table. After 17-18 days of incubation at 41”, virus and viral DNA were prepared as described in Methods. The yield of wild-type virus was determined by the standard plaque assay; for tsA30 and the late mutants tsB4, dl-811. and dl-828, virus yields were measured by complementation plaque assays using tsB4 and tsA30 virus as helper, respectively. DNA yields were determined from purified samples using the extinction coefficient 1 A,,, = 50 pg/ml. The values shown have been normalized to those obtained with WT 800 as the infecting virus. endonuclease (see b Percent of purified DNA preparation resistani to cleavage by EcoRI restriction Methods). e WT virus yield was 2.7 x IO”’ PFU/dish. * WT DNA yield was 79 pg/dish. e ND = not determined.

defective mutants (see Discussion). Electron microscopic examination of each of these restriction endonucleaseresistant DNAs revealed an essentially homogeneous population of circular molecules (Fig. 5 shows mutant cll-816); the mean sizes of the various mutants range from 0.71 to 0.96 SV40 fractional length (Table 5). Thus, our procedure permits isolation of individual clones of defective mutants of SV40. A subsequent publication (J. E. Mertz and P. Berg, in preparation) will report the results of heteroduplex analysis of these defective DNAs which show that: (1) most (>95%) of the molecules in each cloned defective DNA preparation have a large deletion encompassing a unique region of the SV40 genome, but the exact location and size of the deletion varies from one cloned defective genome to

the next; (2) these deleted regions include the EcoRI and/or HpaII endonucleasecleavage site; and (3) most of the mutants also have sizable duplications of that portion of the SV40 genome containing the origin of viral DNA replication. DISCUSSION

This paper describes a general method for cloning and propagating defective mutants of SV40 by complementation with SV40 temperature-sensitive mutants as helper. Defective SV40 and polyoma mutants have also been cloned by Brockman and Nathans (1974) and Fried (1974), respectively, using a somewhat similar approach. In principle, mutants containing deletions, or any type of defect, in a transcomplementable region of the SV40 genome can be cloned and propagated using

CLONING

SV40 DELETION TABLE

YIELD

OF VIRUS AND VIRAL

Infecting Mutant

121

MUTANTS

4

DNA

FROM CELLS CO-INFECTED AT HIGH MULTIPLICITIES OF INFECTION LATE MUTANTS AND tsA30 VIRUS AT 33’”

DNA

virus

viruses + Helper

Total rg/plate

Total

Percent of total as late mutant

Percent of total as defective DNA

PFU/plate Mutant

Individual

WITH

None

t.sA30

tsA30

1 .O”

1.0

-

1.0’

-

tsB4

b-A30

tsB4 tsA30

1.83 0.48

2.31

79

1.26

-

tsB4(41°)

tsA30

tsB4 tsA30

0.075 0.055

0.13

58

0.30

-

dl-811

tsA30

dl-811 tsA30

0.19 0.61

0.80

24

0.94

29

dl-818

tsA30

dl-818 tsA30

0.61 0.4’2

1.03

59

1.15

56

u Monolayers of MA-134 cells (8 x lo6 cells/lOO-mm dish) were infected as described in Table 3 with tsA30 virus alone (m.o.i. about 4), with tsB4 and tsA30 (m.o.i. about 6 and 4, respectively), with defective mutant dl-811 and tsA30 (m.o.i. about 3 and 7, respectively), or with defective mutant dl-818 and tsA30 (m.o.i. about 5 each). After infection at 37” and 14 to 18 days of incubation at 33” (except where indicated) or 11 days for the incubation at 41”, virus and viral DNA were isolated as previously described. Yields were determined as mentioned in Table 3 and have been normalized with respect to the values obtained when tsA30 alone was used as the infecting virus. ’ tsA30 virus yield was 7.5 x lo9 PFUidish. ’ tsA30 DNA yield was 45 wg/dish.

bottom

10

20. FRACTION

30 NUMBER

40

FIG. 4. Separation of defective mutant DNA (dl821) from tsA30 helper DNA by velocity sedimentation in a neutral CsCl gradient. A mixture of dl-821 and tsA30 DNA, obtained as described in Methods, was treated with HpaII restriction endonuclease (see Methods) and then sedimented in a CsCl gradient (preformed by centrifugation overnight at 40,000 rpm and 20°; average p = 1.50, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA in a SW 50.1 rotor at 40,000 rpm and 20” for about 6 hr. Eight-drop fractions were collected, spotted onto Whatman 3 MM filters, dried, and counted by liquid scintillation spectroscopy

the appropriate ts mutant as helper. (Of course, a deletion, insertion, or other defect which prevents encapsidation of the resulting viral mutant DNA cannot be cloned by this method). Indeed, Herzberg, Mertz, Berg, Cameron, and Davis (unpublished results) have resected portions of SV40 DNA with EcoRII restriction endonuclease (Yoshimori, 1971) and cloned the resulting “early” and “late” deletion mutants by complementation with appropriate ts virus helpers. In this instance, the restriction endonuclease was used to randomly “cut out” segments of viral DNA rather than to select for preexisting mutants lacking restriction sequences; because of their smaller size, the resulting mutants could then be separated from WT or helper DNA using agarose gel electrophoresis. Our approach has several limitations though each one may be surmountable. Restriction endonucleases have been used to select from a heterogeneous defective

122

MERTZ TABLE

AND BERG

5

ciable difference is size between the defective and helper DNA (e.g., electrophoretic or centrifugal separations), has been useful tsA30 VIRUS HELPER AT 41” n for preparing defective mutant DNAs for structural and physiological studies. Howsv40 fractional Mutants from Percent of EcoRl endonutotal DNA length (mean : ever, the method is inadequate if a particustandard error)’ clease-resistant as defective lar mutant DNA does not differ from the mutant” pool helper in its sensitivity to cleavage by a known restriction endonuclease or if it does dl-81P 52 0.706 I 0.004; not differ appreciably from the helper DNA 0.861 i 0.004’ dl-812 85 0.886 i 0.003 in size (i.e., those containing small deledl-813 85 0.866 2 0.003 tions or insertions). The procedure dedl-816 90 0.923 f 0.003 scribed here is also inadequate for prepardl-817 87 0.9’23 * 0.005 ing cloned stocks of defective virus since Mutants from normal and defective virions cannot presHpaII endonuently be separated from each other on the clease-resistant pool basis of their sensitivity or resistance to cleavage by restriction endonucleases. This dl-814 0.889 zt 0.004 87 latter problem is particularly troublesome dl-815 83 0.956 i 0.003 since many physiologic studies are easier to dl-818 0.820 i 0.006 96 dl-819 0.882 i 0.004 95 perform by infecting with virus rather than dl-820 94 0.926 zt 0.004 viral DNA. Quite possibly, both of these dl-821 0.888 * 0.008 79 difficulties can be dealt with by using appropriate helpers. For example, if a 0 Monolayers of CV-1P cells were coinfected with a sizable deletion defective mutant virus obtained from single plaques helper virus containing (that includes a restriction site) can be and tsA30 helper virus (m.o.i. = 0.11 and then mutants containing incubated at 41” as described in the text; the viral used to complement it should be DNAs were extracted and purified as described in small deletions or insertions, RELATIVE QUANTITIES AND SIZES OF THE DEFECTI\E MUTANT DNAs OBTAINED AFTER COINFECTION WITH

I

Methods. h These values represent the proportion of circular DNA obtained from each mixed infection that was resistant to cleavage by either the EcoRI or HpaII restriction endonuclease as determined by sedimentation in neutral CsCl gradients (see Fig. 4). c Determined as described in the legend to Fig. 5. The standard deviations were all less than 4% of their means. d The cloned defective mutants have been numbered 811 through 821 with “dl” = deletion and “-” meaning complementation group not yet determined, following the nomenclature suggested by Robb et al., 1972. e This mutant yielded two populations of EcoRI endonuclease-resistant DNA. Both have the same sequence deleted, but the smaller one lacks the duplicated segment found in the other (J. E. Mertz and P. Berg, manuscript in preparation).

DNA preparation for mutant molecules that lack the corresponding restriction sequence; the same property is used to separate the cloned defective mutants from their complementing helper DNA. This procedure, or one which relies on an appre-

*O-

0.6

1.0 0.8 0.9 0.7 SV40 FRACTIONAL LENGTH

1.1

FIG. 5. Histogram of measured contour lengths of dl-816 DNA. dl-816 DNA, separated from tsA30 helper DNA by the procedure described in Fig. 4, was mounted for electron microscopy by the formamide technique of Davis et al. (1972). The grid was shadowed, examined, and photographed and the molecules were measured as previously described (Me& and Davis, 1972). Excluding the two measurements falling outside the main distribution, the remaining 41 molecules were 0.923 f 0.003 (mean + SE; 0 = 2.2%) SV40 fractional length; the PM2 DNA used as an internal standard on the same photographs had a standard deviation of 1.8% of its mean length.

CLONING

SV40 DELETION

possible to discard the restriction endonuclease-resistant helper or to separate the two kinds of DNA by physical means. Moreover, if the deletion in the helper virus appreciably alters the virion’s buoyant density, isopycnic banding could be used to separate the helper from the desired viral isolate. The ability to plaque-purify the mutants and to propagate them at low multiplicities of infection reduces the possibility that new defectives will arise while maintaining the stock or that the helper will outgrow the mutant. The latter problem is a realistic concern when wild-type virus is used as helper (Brockman and Nathans, 1974; Fried, 1974), although, in our procedure, the formation of wild-type revertants or recombinants (Dubbs et al., 1974) can still lead to overgrowth of the mutant population by wild-type virus. Furthermore, deletion mutants, once isolated, may not maintain their original genomic structures; duplications of the portion of the SV40 genome containing the origin of viral DNA replication frequently appear, and DNA molecules containing such duplications quickly become the predominant species in the population, presumably due to their more rapid rate of DNA replication (see Table 5 for examples; J. E. Mertz and P. Berg, unpublished). The helper assay, besides permitting the cloning of certain types of mutants, is also useful for obtaining accurate titers of ts or deletion mutants. For example, the titer of any complementable ts mutant can be more rapidly determined at 41” in the presence of an appropriate helper than with the usual plaque assay performed at 32-33”. Moreover, use of the appropriate helper virus permits each member of a mixture of two mutant viruses to be titered separately, provided both are in different complementation groups (e.g., a mixture of tsB4 and tsA30 or of a deletion mutant and a ts mutant). Complem.entation group analysis of SV40 mutants can also be greatly simplified by using the helper assay procedure. If complementation is defined as the ability of two mutants to form “mixed-plaques” similar to those shown in Fig. 1, the com-

123

MUTANTS

plementation group of any mutant (deletion as well as tsj can be readily determined by asking which ts mutants (choosing one or more from each complementation group) can serve as helper for mixedplaque formation at 41”. In this manner, we have confirmed Tegtmeyer and Ozer’s observation (1971) that tsB*II can complement tsA30 but not tsB4 (J. E. Mertz and P. Berg, unpublished). ACKNOWLEDGMENTS We thank M. Thomas and J. Ferguson for EcoRI restriction endonuclease, P. Tegtmeyer for the ts mutant strains, J. Robb for his help. and hospitality in performing the antigen assays, and P. Sharp, B. Sugden, and J. Sambrook for making their results with the HpaII restriction endonuclease available to us prior to publication. This work was supported in part by research grants from the U.S. Public Health Service (GM13235-09, 5 TI GM 196-15) and the American Cancer Society (VC-23C). J.E.M. is a U.S. Public Health Service Trainee. REFERENCES BOEYE, A., MELNICK, J. L., and RAPP, F. (1966). SV40-adenovirus “hybrids”: Presence of two genotypes and the requirement of their complementation for viral replication. Virology 28, 56-70. BROCKMAN, W. W., and NATHANS, D. (1974). The Isolation of simian virus 40 variants with specifically altered genomes. Proc. Nat. Acad. Sci. USA 71, 942-946. DAW, R., SIMON, M., and DAVIDSON, N. (1971). Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. In “Methods in Enzymology” (L. Grossman, and K. Moldave, eds.) Vol. 21, pp. 413-428. Academic Press, New York. DUBBS, D. R., and KIT, S. (19711. Spontaneous virus production by clonal lines of simian virus 40-transformed cells and effects of superinfection by deoxyribonucleic acid from mutant simian virus 40 strains. J. Viral. 8, 430-436. DUBBS, D. R., RACHMELZR, M., and KIT, S. (1974). Recombination between temperature-sensitive mutants of simian virus 40. virology 57, 161-174. ECKHART, W. (1974). Genetics of DNA tumor viruses. Annu. Reo. Genet. (in press). FRIED, M. (1974). Isolation and partial characterization of different defective DNA molecules derived from polyoma virus. J. Viral., 13, 939-946. GIRARDI, A. J. (1965). Prevention of SV40 virus oncogenesis in hamsters, I. Tumor resistance induced by human cells transformed by SV40. Proc. Nat. Acad. Sci. USA 54, 445-451. GREENE, P. J., BETLACH, M. C., GOODMAN, H. M., and

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