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SV40 defectives selected during low multiplicity passage on A172 human glioblastoma cells
VIROLOGY
112.461-4’71 (1981)
SV40 Defectives Selected during Low Multiplicity A 172 Human Glioblastoma Cells
Passage
on
DANA CARROLL,**’ JOANNA L. HANSEN,* EDWARD B. MARYON,* AND FRANK J. O’NEILL**+ Departments of +CeUu.iar, Viral and Molecular Bidogy, and tPat*, University crf Utah Medial Center and *Research Service, Veterans Administration Hospital, Salt L&e Ci&, Utah 8blS2 Accepted Fekry
13, 1981
The gliobastoma line, A172, is unusual among human cell lines in being permissive for SV40 infection, and differs from many host cell types in allowing the accumulation of viral defectives on low multiplicity passage. We have characterized these Al72-derived defectives and find that most are reiteration mutants, containing multiple copies of both the viral replication origin (including the J3glI site at map position 0.669) and sequences from the opposite side of the standard genome (around the BamHI site at map position 0.143). This distinguishes them from defectives accumulated during high-multiplicity passage in monkey cells, which generally have only reiterated origins. When well characterized, monkey cell-derived defectives (ev-1101, d6, az, 4) were passed in Al72 cells, the characterized defectives were lost, and sometimes replaced by other defectives present in the same stock. Surprisingly, this was true even when the characterized defective carried sequences from the BamHI region of standard SV40. These findings are taken to support the notion that sequences in addition to the viral replication origin are positively selected during defective propagation, but that different requirements for such sequences are set by monkey cells and Al72 cells.
INTRODUCTION
Studies of simian virus 40 (SV40) defectives have served to help define and emphasize some functions of the standard virus (Kelly and Nathans, 1977; Fareed and Davoli, 1977). Since they are carried in the presence of wild-type helper, defectives need retain, and often are found to amplify, only those portions of the genome which cannot function in tram, but are required for propagation. In addition, the virus utilizes host cell machinery extensively for the replication of its DNA, so insights can be gained into cellular functions. Quite sensibly, all highly evolved SV40 defectives retain multiple copies of the viral replication origin, but have lost most other wild-type sequences. These reiterated origins confer a competitive replication advantage on the defectives, with i To whom reprint
requests should be addressed. 461
respect to standard virus, and account in large measure for their interference activity (Lee and Nathans, 1979). In earlier experiments, we found that Al72 human glioblastoma cells accumulate SV40 defectives, even on low multiplicity passage (O’Neill, 1976; O’Neill and Carroll, 1978). Analysis of several of these A172-derived defective genomes (Carroll and O’Neill, 1978) showed that they had retained and reiterated, in addition to the viral replication origin, viral sequences from the region opposite the origin on the wild-type genome which we call the termination region.2 Examination of characterized defectives from other sources revealed that, with a single exception, they ’ By this designation, we do not imply a function for this region, but merely acknowledge the fact that bidirectional replication of standard virus, and possibly early transcription, normally terminate in this vicinity. 0042-6822/81/100461-11$02.00/O Copyright All rights
0 1981 by Academic Press. Inc. of reproduction in any form reserved.
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CARROLL
carried reiterated copies of the viral termination region or of host cell-derived DNA sequences (Carroll and O’Neill, 1978). This led us to suggest that these sequences, like the replication origin, perform in cis an essential function for virus propagation, or at least contribute to the competitive advantage. These sequences are proposed to be selected during defective evolution and not simply carried along passively with the origins. The function served by the termination region is probably also essential to standard virus, and can be served by a wide variety of monkey chromosomal DNA sequences. In this study, we have examined several populations of SV40 defectives arising in Al72 cells to see how general the retention of the termination region is. We found that a majority of A172-derived defectives, but only a minority of monkey cellderived defectives, have equal reiterations of viral origin and termination regions. We have also tested the ability of wellcharacterized monkey cell-derived defectives to propagate in Al72 cells. The human cells selectively amplified different defectives from those preferred monkey cells, but this selection was not simply correlated with the presence of the viral termination region. MATERIALS
AND METHODS
Cells and viruses. General aspects of culturing Al72 (human glioblastoma) and TC-7 (African green monkey kidney) cells and their SV40 infections have been given previously (O’Neill and Carroll, 1978): Stocks containing the characterized defectives a3, dg, a; (Davoli et al., 1977) were obtained from Dr. George Fareed; the ev1101 strain (Lee et al., 1975) was provided a We have observed that when Al72 cells are allowed to grow to high densities on consecutive passages, they undergo a sort of transformation, following which they saturate at higher densities than the original line, no longer support efficient growth of SV40, and no longer amplify defectives on low multiplicity passage. We have not studied this phenomenon extensively, but find it can be avoided by passaging the Al72 cells more frequently and growing them in 5%, instead of 10%. serum.
ET AL.
by Dr. Daniel Nathans; and Repo (Shenk and Berg, 1976) by Dr. Thomas Shenk. Independent passage series were started from these stocks: (1) undiluted on TC-7; (2) undiluted on A172; (3) diluted lo3 at each passage on A172. Intracellular viral DNA was purified from Hirt supernates by banding in ethidium bromide-CsCl gradients (O’Neill and Carroll, 1978). Analysis of viral DNAs. Restriction enzymes were purchased from New England Biolabs (Beverly, Mass.) or Bethesda Research Laboratories (Rockville, Md.). Analytical digestions of 0.1-1.0 pg of viral DNA samples were done under conditions specified by the suppliers. Electrophoretic analyses were performed in 1.0% agarose, 1.5% agarose, or composite 2.0% polyacrylamide, 0.5% agarose gels, as previously described (O’Neill and Carroll, 1978; Carroll and O’Neill, 1978). For detailed restriction mapping, the intact defective b3 and the BglI fragments a, b, c, and d were recovered from 1.0% agarose gels by one of two techniques. Most commonly, the gel segments were dissolved in saturated NaI and the DNA bound to, then eluted from, glass powder (Vogelstein and Gillespie, 1979) or glass fiber filters (Chen and Thomas, 1980). Some samples were eluted electrophoretically and purified by passage over a small DEAE-cellulose column (Fedoroff and Brown, 1978). For Southern blot hybridizations, nicktranslated 32P probes were prepared as described by Rigby et al. (1977). DNA fragments were blotted from agarose gels onto Schleicher and Schuell BA83 nitrocellulose strips according to Southern (1975). Hybridizations were performed in 50% formamide, 5 X SSC, 10% dextran sulfate, as described by Wahl et al. (1979). The plasmid pCa1004 (R. Thayer, M. Singer, and T. McCutchan, in preparation) containing a 344-bp insert of African green monkey satellite DNA was provided by Dr. Maxine F. Singer. DNAs were spread for electron microscopy using the formamide procedure of Davis et al. (1971) and were examined in a JEOL-100s electron microscope. SV40 map positions are based on the published DNA sequence of strain 776
SV40 DEFECTIVES
(Reddy et al., 1978; Fiezs et al., 1978; Van Heuverswyn and Fiers, 1979) and are given as fractional distance from the EcoRI site, measured in the usual direction (Kelly and Nathans, 1977).
463
IN Al72 CELLS y------A112
,
-JC
J--,
RESULTS
The BgWBamHI Assay Digestion with the restriction
enzyme
BgtI provides a simple assay for the SV40 replication origin. There is a single BglI site in the nondefective genome (Fiers et al., 1978; Reddy et al., 1978), included in the T antigen binding region at map position 0.659 (Tjian, 1978; Fig. 1). Since the BgZI recognition site is (effectively) a hexanucleotide sequence (e.g., Lautenberger et al., 1980), it is unlikely on statistical grounds that a small segment of host cell DNA would contain a BglI site. The several defectives which have been characterized in sufficient detail all have a number of BglI sites equal to the number of viral replication origins (Lee and Nathans, 1979; Gutai and Nathans, 1978a, b; Wakamiya et al., 1979; McCutchan et al., 1979; this study). By the same sort of logic, the enzyme BamHI is a probe for the viral termination region. It has a single site at position 0.143 of the wild-type genome and it is retained in the defectives which do not contain host cell substitutions (Carroll and O’Neill, 1978). The assay consists of comparing a BglI ECORI
FIG. 2. BglI/BamHI screens on defective-containing SV40 stocks from Al72 and TC-‘7 ceils. DNA samples were run undigested (-) and digested with BglI (G), BamHI (B), and sometimes BcZI (C); restriction enzyme fragments of pBR322 DNA were included as length standards (stds). (a) From a DNA transfection of Al72 cells, showing defectives with single BamHl sites and duplicated BgZl sites. (b) PP3SV40, P3 lo-” in Al72 cells, showing simple deletion defectives with single BglI, BamHI, and BclI sites. (c) NPPSV40 P6 in A172. (d) From a lo-? dilution of the sample in c, in A172. (e) PP3SV40, 100 PFU/flask, then P2 lo-” in A172, series 2 (D’Neill and Carroll, 1978). (f:I PP3SV40, P16 10” in TC-7. (g) NPPSV40, P2 10” in TC-7. (h) Al72 P6 virus [as in (c)l, P4 10” in TC-7. The positions of supercoiled (I), nicked circular (II) and linear (III) forms of nondefective SV40 DNA are indicated. Electrophoresis was in 1.0% agarose gels.
digest with a BamHI digest of a mixed population of defective genomes. If each subunit in a reiteration mutant contains one origin and one terminus, then BglI and BamHI would yield identical bands, reflecting the subunit size. In many cases, BclI, which has a single site at map position 0.188, was also used as a termination region probe.
A172 and Monkey Cell Defectives
FIG. 1. Schematic diagram of the SV40 genome. The origin of replication and the so-called termination region are indicated, as are cleavage sites for several key restriction enzymes used in this study.
One generalization we can make based on the BglI and BamHI digests has to do with the early stages in the generation of defectives and seems to be true of stocks passed in both TC-7 and Al72 cells. The first altered genomes to arise seem to be the result of simple deletions of nonessential DNA sequences; they are smaller than the wild-type and have a single site each for BglI and BamHI (Fig. 2b). Another form we see early in a passage series car-
464
CARROLL
ries a small duplication which includes the replication origin (Mertz et al., 1975). Such defectives have two, asymmetric BglI sites, yield large and small fragments on BgZI digestion, and retain a single BamHI site (Fig. 2a). The distinctive reiteration mutants seem to arise in later passages. We have not followed carefully the progress from one form to another in any one series of infections, but we have seen the simple deletions and origin duplications only at early stages in the generation of new defective types. Our previous findings that defectives accumulated in Al72 cells retained multiple origins and termini were based on studies of defectives that may have been related to each other (Carroll and O’Neill, 19’78).We wanted to see whether this characteristic was generalizable to independently generated and selected defectives. When populations of A172-derived defectives were examined by BglI and BamHI digestion, we found, in most cases, that the digests matched essentially band for band, indicating equal reiterations of origin and termination regions (Fig. 2, c-e). This was true of defectives from several independent passage series (O’Neill and Carroll, 1978) (e.g., Fig. 2e). In contrast, populations of defectives accumulated during high multiplicity passage on monkey (TC-7) cells showed evidence of multiple BgZI sites (and therefore origins), but characteristically had one or no BamHI sites (Fig. 2, f-h). There was little or no correspondence between BglI and BamHI (or Bc.?I)bands, and often uncleaved, form I defectives could be seen in the BamHI digests. The electrophoretic analyses were confirmed by electron microscopic analysis (Table 1). When linear and circular molecules were counted in complete BamHI digests, a high percentage of undigested circles was found in TC-7 defectives, but many fewer in the A172 defectives. It should be pointed out that where there are multiple BamHI sites in some defectives, a single molecule will yield several linear pieces and the undigestible circles in the population will be somewhat underestimated. Also, many of the linear molecules
ET AL.
in the TC-7 populations correspond to the wild-type helper DNA.
Fates of Characterized Defectives in Al 72 Cells As a sort of converse approach to characterization of defectives generated and accumulated in Al72 cells, we obtained stocks of several well-characterized, monkey cell-derived defectives from other laboratories and tested the ability of these defectives to persist in Al72 cells. The defectives used are illustrated in Fig. 3. Two of these (ev-1101 and d5) contain monkey cell sequences in addition to reiterated origins, while the other two (a; and a3) have both reiterated viral origins and BamHI regions. We knew from experiments with mixed defectives that Al72 cells would select from among TC-7 defectives a different subpopulation from that preferred on continued passage in monkey cells. As a general observation, the same was true of these characterized defective stocks. However, when BgZI and BamHI digests of the Al’IBselected derivatives were examined, it was not generally true that equal reiterations of origin and terminus were found. Rather, multiple origins but one or no termini characterized the Al72 population, as it had the original monkey cell population. ev-1101. This cloned defective was quite pure in the stock we received from Nathans and persisted at a level roughly equal to standard virus when passed in TC-7 cells at high multiplicity. After several passages, ev-1101 was joined by a second defective (Fig. 4). When passed in Al72 cells, ev-1101 was lost from the virus stock. This occurred at low and at high multiplicity, although more slowly at high input. After three or four diluted passages in Al72 cells a faint smear of heterogenous defectives became evident, just as in passage of wt SV40 (O’Neill and Carroll, 1978). Restriction enzyme digests verified, when it could be seen, that the remaining discrete defective in all stocks was ev-1101. d. This defective represented only a small proportion of all viral genomes in
SV40 DEFECTIVES TABLE
IN Al72 CELLS
465
1
ELECTRON MICROSCOPIC ANALYSES OF DEFECTIVE DNAs
Virus
Host
Passage
Percentage circles after BamHI digestion
NPPSV40 PP3SV40 PP3SV40 A172P6SV40 NPPSV40 PP3SV40 PP3SV40
TC-7 TC-7 TC-7 TC-7 A172 Al72 A172
P4, 10” PlO, 10” P15.10” P4,lO” P9, 1o-3 Pl, 10m3,series 2 P3, 10m3,series 2
42.2 29.5 40 28.8 8.4 4.5 2.2
Sample 32 ‘76 37 33 24 41 74
Note. Each defective-containing DNA sample was digested to completion with BarnHI, then spread for electron microscopy. A total of 400 (or morej molecules were counted in each specimen and classified as linear or circular. The input virus in sample 33 was the stock from six passage of NPPSV40 in Al72 cells (all but the initial passage at low multiplicity). Samples 41 and 74 are from one of the very low initial input series described earlier (O’Neill and Carroll, 1978). NPPSV40 was the large-plaque stock prior to plaque purification; PP3SV40 was a triply plaque-purified isolate of NPPSV40 (O’Neill and Carroll, 1978).
the stock from Fareed and was present as both the fourfold (d4) and fivefold (d5) reiteration (Fig. 5). When we passed this stock undiluted on TC-7 cells, d4 and d5 were overtaken by two abundant defectives, both apparently fivefold reiterations, and several minor bands. d was lost during low multiplicity passage in Al72 ev-1101
(eA%),
00TC-7 -- ‘0
d
a'
a
cells, but was retained, particularly as d4, upon high-multiplicity passage in these cells. A variety of restriction enzymes digests showed the major A172-retained defective to be identical to authentic 4. cc’. The characterized defective a; was only one among many defectives in the stock received from Fareed, but was the major one having reiterated EcoRI sites
t.1"
i
c;
1
.I*,.* I .,,.I3 '
Al72 ‘: “: “0 -w-v0 “0 0
, 34 I)4 ,
.n 43
FIG. 3. Maps of the repeating units of characterized SV40 defectives isolated from monkey cells. Open boxes denote SV40 sequences, while thin lines are nonviral DNA. The map of ev-1101 is adapted from Lee et al. (1975), those for d and a’ from Davoli et al. (1977), while the revised map of a is based on our own detailed mapping (see Fig. 9). The brackets and subscripts indicate the reiteration number of these subunits in the major circular defective species. The maps are aligned to their &II sites, which are indicated by arrows; arrowheads show the locations of BornHI sites in a’ and a; and map coordinates at the junctions of normally noncontiguous segments are given.
FIG. 4. Supercoiled DNAs from passage of an ev1101 stock on TC-7 and Al72 cells. The positions of wild-type viral DNA and of the fivefold reiteration mutant ev-1101 are indicated. The faster-moving band in some of the Al72 samples is very likely the fourfold version of the same repeating unit. Electrophoresis was from top to bottom in a 1.0% agarose gel.
466
CARROLL
I’2IQ’
P2IQ-$
A172 PS IQ’
P5 IQ’
P1IQ-’ ’
FIG. 5. Analysis of DNAs from passage of a d5 stock in TC-7 and Al72 cells. Undigested DNAs (-) are compared with BgS (G) and BornHI (B) digests. The position of genuine d is indicated. One slot contains pBR322 restriction fragments used as length standards. Electrophoresis was in a 1.0% agarose gel.
(Fig. 6). On high-multiplicity passage in TC-7 cells, a; was retained at a low level, while other defectives came to dominate the virus stock. In Al72 cells at low multiplicity, monkey cell-derived defectives were gradually lost from the ai stock. Those still present at the second and fourth diluted passage had one or no sites TC-7 ~A172
I
FIG. 6. Analysis of DNAs from passage of an ai stock in TC-7 and Al72 cells. Shown are undigested DNAs (-) and digests with BgZI (G), BamHI (B), and EcoRI (E). The position of genuine a’ was assigned on the basis of its size relative to pBR322 length standards and its production by digestion with BamHI and EcoRI (Davoli et al., 1977). Electrophoresis was in 1.0% agarose gels.
ET AL.
TC-7 I P510~
P2 100
A’72 y-J P5100
FIG. 7. Analysis of DNAs from passage of an a3 stock in TC-7 and Al72 cells. Shown are undigested DNAs (-) and digests with BgZI (G), BamHI (B), and EcoRI (E). The fragments a, b, c, and d were identified by restriction enzyme mapping following recovery from preparative gels. Electrophoresis was in 1.0% agarose gels.
for BumHI and EcoRI, and did not appear to include a’. With undiluted passage in Al72 cells, defectives were amplified until they represented >80% of viral DNA after five passages. Again, BumHI and EcoRI cut the defectives once or not at all, and a’ was lost. Some of the BglI bands in these A172 (high m.o.i.) defectives corresponded to bands present in the original or the TC7 stocks, but evidently different subsets were preferentially amplified. a. a3 was the major, but not the only, defective in the stock obtained from Fareed (Fig. 7). This stock, derived from an earlier passage of the DAR variant of SV40 in the same series that yielded up d5, also contained a low level of that defective, as well as the defectives b4 and c5 (Ganem et al., 1976; Davoli et al., 1977).4 4 Our identification of b and c in the a3 stock is based on: (1) knowledge that they were in the stock initially (Ganem et al., 1976); (2) their sizes and restriction maps (Davoli et al., 1977). Some details of our restriction maps differ from the previous report, but are probably within variations between laboratories. The most striking discrepancy is our finding of only about 29% viral sequences in b and 25% in c (see below). Davoli et al. (1977) concluded that most of the sequences in b and c were viral; but it must be observed that this was based on very low cpm of total viral DNA hybridized.
SV40 DEFECTIVES
At high multiplicity in TC-‘7 cells, the reiteration mutants with subunits a, b, c, and d are retained, but the most abundant defectives seem to contain only a single &ZI site (and a single BamHI site). Upon passage at low or high multiplicity in Al72 cells, the defective b3 came to dominate. This was present at a low level in the original defective stock, but was selectively amplified in the human cells. b. Because of its evident advantage in Al72 cells, we characterized the subunit b in some detail. It has a single EcoRI site, but no BamHI site, in each repeat, and we thought b might have been derived from a by deletion. However, detailed restriction enzyme mapping of b (Figs. 8, 9) showed no homology to a, except in the immediate vicinity of the viral replication origin. In addition, the remainder of the restriction map did not correspond to any other region of the nondefective viral genome. Nevertheless, because extensive rearrangements of viral sequences have been found in some defectives (Gutai and Nathans, 1978a, b) we performed blot hy-
FIG. 8. Restriction enzyme digests of the sort used to map a, b, and e. (A) Digests of b and c with: (1) Bgl[ + EcoRI; (2) BglI + EcoRI + HindIII; (3) BgZI + t’indII1; (4) BglI + EcoRI + PstI; (5) BglI + EcoRI + HincII. Comparisons between similar digests of b and c indicate the nature of the deletion by which the two differ. (B) Digests of a, wt SV40, and b with Bgl[ + BstNI. Electrophoresis was in 2.0% polyacrylamide, 0.5% agarose gels.
IN Al72 CELLS
467
bridizations to determine whether viral sequence other than those around the origin were present in b (Fig. 10). These showed that only a contiguous region of about 250 bp of b, from the B&N1 site at 0.691 to a little short of the HueIII-BstNISau961 cluster, was derived from SV40. We assumed that the apparently nonviral DNA in b may have been derived from the monkey cell genome. We did one simple blot hybridization experiment to determine whether b, like many other defectives (Gutai and Nathans, 197813;Wakamiya et al., 1979; McCutchan et al., 1979; Rosenberg et al., 1977), contained sequences related to the monkey (Ysatellite DNA. This analysis was also applied to d and ev-1101. None of these showed detectable hybridization to the cysatellite probe. Rosenberg et al. (1977) have previously reported that d5 and ev-1101 do not hybridize to this satellite. b3 was preferred to three other discrete defectives in Al72 cells. Two of these we know to be a3 and d4/d5. Interestingly, when we characterized the remaining BglI fragment (c in Fig. 7), we found it to be related to b by a simple deletion of about 200 bp, including a Hind111 and a H&c11 site (Figs. 8,9). Thus, it appears that deletion of these nonviral sequences makes c less fitted than b for propagation in Al72 cells. Repo. There is in the literature a description of one SV40 defective which contradicts the rule that reiteration mutants contain either reiterated viral termination regions or reiterated host cell substitutions in addition to reiterated viral origins (Fig. 11). That is the defective (Repo) constructed in vitro by Shenk and Berg (1976) to contain only the viral origin. When passed undiluted in TC-7 cells, we found that Repo never made up more than a very small proportion of all viral DNA. Shenk has had the same experience with Repo (Shenk and Berg, 1976; T. Shenk, personal communication), indicating that it is not a healthy defective. Repo rapidly fell below the level of detectability on passage in Al72 cells, at low or high multiplicity. We also found, by examining restriction enzyme digests (Fig. ll), that Repo was not a pure species. At least two reiterated
468
CARROLL ET AL.
760
I I
390
I
B611 + PstI + HhaI
495 I 470
-1
I
I
BglI + HaeIII
e 685
BglI + Pet1 + Hind111
610 I
BglI + BstNI + HincII
Jm 610
I
+ Hind111
285
345 I
BatNI + Him11
FIG. 9. Detailed restriction maps of the defective subunits a, c, and b (see Figs. ‘7 and 8). In each case, parentheses enclose one full subunit, and portions of adjacent units are shown by dashed lines. In the map of a, coordinates of the corresponding sites in the wild-type SV40 genome are given. Below the map of b, the results of blot hybridizations to SV40 DNA are summarized. For each digest, the relevant fragments are indicated as bars; shaded bars showed hybridization to SV40 [@PjDNA, while open bars did not. Sizes of these fragments are given in base pairs. Gaps represent fragments too small to be reliably detected in these experiments. There was also very faint hybridization to the rightmost Bgfl + BstNI + HincII fragment, suggesting the presence of a very small amount of SV40 DNA between the B&N1 and H&z1 sites.
subunits are present, apparently in separate defective molecules, as judged by examination of partial BQZI digests. DISCUSSION
Cells of the human glioblastoma line, 8172, are unusual in two respects: (1) unlike most human cell lines, they support lytic growth of SV40 (O’Neill, 1976; O’Neill and Carroll, 1978); and (2) they accumulate defective viral genomes to very high levels, even on low-multiplicity passage of the virus (O’Neill and Carroll, 1978). Recently, in an extensive survey, we found that both these characteristics are shared to some extent by other lines of human cells (O’Neill and Carroll, in preparation). Another striking feature of the A172derived defectives we characterized initially was retention of multiple copies of
both the viral replication origin and termination regions (Carroll and O’Neill, 1978). We have now demonstrated that this property is shared by the majority of defectives accumulated during passage of standard SV40 in Al72 cells, i.e., they have equal reiterations of viral B&I (origin) and BamHI (terminus) sites. We should point out that this is not generally true of the other human cells which accumulate defectives on low-multiplicity passage (O’Neill and Carroll, in preparation). The degree of substitution with nonviral sequences may reflect rates of recombination between cellular and viral DNAs in the various cell types. The finding of retained viral termini led us to propose that this region plays an important role in cis in the propagation of the defectives (Carroll and O’Neill, 1978). Examination of other defective genomes
SV40 DEFECTIVES IN Al72 CELLS
indicated that a wide range of monkey cell DNA sequences could substitute in performing the same function, at least in monkey cells. We also knew that Al72 cells select from stocks passed in monkey cells defective genomes different from the ones preferred in the monkey cell cultures (O’Neill and Carroll, 1978); and the A172selected genomes have reiterated BumHI sites (D. Carroll, unpublished results). It appeared that the sequences required to perform the non-origin function were different in monkey and human cells. Thus, we were interested in studying the fate of individual, well-characterized, monkey cell-derived defectives on passage in AI72 cells. We made the initial assumption that sequences in addition to those around the replication origin are necessary for efficient propagation. If monkey cell sequences cannot perform this function adequately in human cells, defectives lacking the viral termination region but containing host cell substitutions (ev-1101, Lee et al., 1975; dg, Davoli et al., 1977) would be lost on passage in Al72 cells. If the monkey sequences functioned well, the defec-
FIG. 11. Restriction enzyme digests of viral DNA from a stock containing the defective, Repo. (1) BQA; (2) Hind111 + BQA; (3) HindIII. Strong bands are from nondefective SV40 DNA; faint doublets are from the defective. The wild-type Hind111 fragments are labeled. Electrophoresis was in a 2.0% polyacrylamide, 0.5% agarose composite gel.
tives would be retained. We expected the defectives carrying the viral terminus (a3, a:; Davoli et al., 1977) to be retained in Al72 cells. Of the two host-substituted defectives, ev-1101 was rapidly lost on low or high multiplicity passage in Al72 cells. h/d6 was lost slowly at low multiplicity, but seemed to have excellent competitive capability (the & isomer particularly) when passed undiluted in Al72 cells. To our surA prise, both the terminus-containing defecFIG. 10. Blot hybridizations of ‘?-labeled SV40 tives, a3 and a:, were rapidly replaced by probe to restriction fragments of the defective b. (A) other defective species on both diluted and and (B) represent two separate experiments. (1) undiluted passage in A172 cells. HeteroSV4O/HaeIII; (2) b/B& + HiudIII; (3) b/&II + PstI geneous defectives were selected out of the + Hhd; (4) b/&a + PstI + HiudIII; (5) SV40/ HindIII; (6) SV4O/HindIII; (7) b/R@ + Hoe111 a; stock; and the particular genome b,, which is unrelated to a3, was amplified + HindIII; (8) b/R&I + B&N1 + H&11; (9) SV40/ from the a3 stock. bs was amplified indeBQZI + BstNI; (10) b/MN1 + HincII. Electrophoresis pendently in high- and low-multiplicity was in a 1.5% agarose gel.
470
CARROLL ET AL.
passage series, indicating a strong selective advantage, not just a fortuitous event. None of the defectives selected from the above stocks in Al72 cells showed reiterated BarnHI sites (termini). We conclude that some nonviral DNA sequences can substitute in function for the viral termination region-in Al72 cells. Those in ev-1101 cannot; those in d5 can to some extent; those in b3 work particularly well. And we see more definitively than before (O’Neill and Carroll, 1978) that different families of non-origin sequences are selected in the monkey and human cells. Since the wild-type helper virus is identical in both cell types, this suggests an interaction with host enzymes or structures. In fact, given the passage history of the DAR virus stock (Sack et al., 1973), the nonviral sequences in b3 could easily be derived from the human genome. In fact, the host cell specificity of defective genomes is evident even among green monkey cell lines. The defectives in the stocks containing dr,, a;, and a3 were initially selected on primary kidney cells (Fareed et al., 1974; Ganem et al., 1976). When we passed these stocks on the established kidney line, TC-7, those defectives were replaced by others, particularly ones with less highly reiterated &$I sites (see Figs. 6, 7). The argument that a function is required in addition to that (those) provided by the viral replication origin, appears intact. With the exception of the sickly Repo (Shenk and Berg, 1976), all characterized defectives carry other reiterated viral or cellular sequences. What their function may be remains a matter of speculation. Lee and Nathans (1979) demonstrated that defectives have an advantage in replication, not in packaging, so the possibility of an assembly function seems remote. It should be noted that the defectives used in that study all contained reiterated host cell sequences, so their accumulation cannot be attributed solely to origin functions. Earlier studies of Brockman et cd. (1975) showed that the actual site at which bidirectional DNA replication terminates is at the meeting of the two forks moving
at equal rates, independent of the sequence at that point. Still, the termination region could be: (1) an entry or recognition site for a molecule or complex required for initiation of replication; (2) similarly for replication termination or resolution of daughter molecules; or (3) a termination site for early transcription which, if left unchecked, might interfere with the progress of replication. Comparisons of nucleotide sequences of host cell substitutions in a number of different defectives from the same host cell type may shed some light on the precise region required. ACKNOWLEDGMENTS We are grateful to Drs. Daniel Nathans, George Fareed, and Thomas Shenk for providing stocks of defectives and to Dr. Maxine Singer for a gift of a satellite-containing plasmid DNA. This work was supported by NIH Grants CA23123 to D.C. and CA15141 to F.J.O., and by Veterans Administration research funds. REFERENCES BROCKMAN, W. W., GUTAI, M. W., and NATHANS, D.
(1975). Evolutionary variants of Simian Virus 40: Characterization of cloned complementing variants. Virology 66.36-52. CARROLL,D., and O’NEILL, F. J. (1978). Genome maps of Simian Virus 40 defectives propagated in human glioblastoma cells. Virology 87,120-X29. CHEN, C. W., and THOMAS,C. A., JR. (1930). Recovery of DNA segments from agarose gels. And Bioch. 101.339-341. DAVIS, R. W., 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. 413423. Academic Press, New York. DAVOLI, D., GANEM, D., NUSSBAUM,A. L., FAREED, G. C., HOWLEY, P. M., KHOURY, G., and MARTIN, M. A. (1977). Genome structures of reiteration mutants of Simian Virus 40. Virology 77,110-X24. FAREED, G. C., and DAVOLI. D. (1977). Molecular biology of papovaviruses. Annu. Rev. Biodwm. 46, 471-522. FAREED, G. C., BYRNE, J. C., and MARTIN, M. A. (1974). Triplication of a unique genetic segment in a Simian Virus IO-like virus of human origin and evolution of new viral genomes. J. Mol. Biol. 87, 275-233. FEDOROFF, N. V., and BROWN, D. D. (1978). The nu-
SV40 DEFECTIVES IN A172 CELLS cleotide sequence of oocyte 5s DNA in Xenopus Zaevis. I. The AT-rich spacer. CeU 13, ‘701-716. FIERS, W., CONTRERAS,R., HAEGEMAN, G., ROGIERS, R., VAN DE VOORDE, A., VAN HEUVERSWYN,H., VAN HERREWEGHE,J., VOLCKAERT,G., and YSEBAERT,M. (1978). Complete nucleotide sequence of SV40 DNA. Nature (London) 273,113-Z%. GANEM, D., NUSSBAUM,A. L., DAVOLI, D., and FAREED,G. C. (1976). Isolation, propagation and characterization of replication requirements of reiteration mutants of Simian Virus 40. J. MoZ. Biol. 101,57-83. GUTAI, M. W., and NATHANS, D. (1978a). Evolutionary variants of Simian Virus 40: Nucleotide sequence of a conserved SV40 DNA segment containing the origin of viral DNA replication as an inverted repetition. J. Mol. Biol. 126,259-274. GUTAI, M. W., and NATHANS, (1978b). Evolutionary variants of Simian Virus 40: cellular DNA sequences and sequences at recombination joints of substituted variants. J. Mol. Biol. 126,275~288. KELLY, T. J., and NATHANS, D. (1977). The genome of Simian Virus 40. Advan. Virus Res. 21,85-173. LAUTENBERGER,J. A., WHITE, C. T., HAIGWOOD,N. L., EDGELL, M. H., and HUTCHISON,C. A. III (1980). The recognition site of type II restriction enzyme BglI is interrupted. Gene 9,213~231. LEE, T. N. H., and NATHANS, D. (1979). Evolutionary variants of Simian Virus 40: Replication and encapsidation of variant DNA. Virology 92,291~298. LEE, T. N. H., BROCKMAN,W. W., and NATHANS, D. (1975). Evolutionary variants of Simian Virus 40: Cloned substituted variants containing multiple initiation sites for DNA replication. Wok 66, 53-69. MCCUTCHAN, T., SINGER, M., and ROSENBERG,M. (1979). Structure of Simian Virus 40 recombinants that contain both host and viral DNA sequences. II. The structure of variant 1103and its comparison to variant CVP8/1/P2 (EcoRI res). J. BioZ. Chem. 254, 3592-3597. MERYZ, J. E., CARBON, J., HERZBERG, M., DAVIS, R. W., and BERG, P. (1975). Isolation and characterization of individual clones of Simian Virus 40 mutants containing deletions, duplications and insertions in their DNA. Cold Sprint Harbor Symp. Quant. Biol. 39, 69-84. O’NEILL, F. J. (1976). Propagation of Simian Virus 40 in a human cell line. Virology 72, 287-289.
471
O’NEILL, F. J., and CARROLL,D. (1978). Appearance of defective Simian Virus 40 following infection of cultured human glioblastoma cells. Virology 87, 109-119. REDDY, V. B., THIMMAPPAYA, B., DHAR, R., SUBRAMANIAN, K. N., ZAIN, B. S., PAN, J., GHOSH,P. K., CELMA, M. L., and WEISSMAN, S. M. (1978). The genome of Simian Virus 40. Science 206.494-502. RIGBY, P. W. J., DIECKMANN, M., RHOADES,C., and BERG, P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113.237251. ROSENBERG,M.. SEGAL, S., KUFF, E. L., and SINGER, M. F. (1977). The nucleotide sequence of repetitive monkey DNA found in defective Simian Virus 40. CeU 11, 845-857. SACK,G. H., JR., NARAYAN, O., DANNA, K. J., WEINER, L. P., and NATHANS, D. (1973). The nucleic acid of an SV40-like virus isolated from a patient with progressive multifocal leukoencephalopathy. Virology 51,345-350. SHENK,T. E., and BERG,P. (1976). Isolation and propagation of a segment of the Simian Virus 40 genome containing the origin of DNA replication. Proc. Nat. Acad Sci. USA 72,1513-1517. SOUTHERN,E. M. (1975). Detection of specific species among DNA fragments separated by gel electrophoresis. J. MoL Biol. 98, 503-517. TJIAN, R. (1978). The binding site on SV40 DNA for a T antigen-related protein. CeU 13, 165-179. VAN HEUVERSWYN,H., and FIERS, W. (1979). NUcleotide sequence of the Hind-C fragment of Simian Virus 40 DNA. Comparison of the 5’-untranslated region of wild-type virus and of some deletion mutants. Eur. J. B&hem. 166,51-60. VOGELSTEIN,B., and GILLESPIE, D. (1979). Preparative and analytical purification of DNA from agarose. Proc. Nat. Acad Sci. USA 76,615-619. WAHL, G. M., STERN, M., and STARK, G. (1979). Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc. Nat. Acad Sci. USA 76, 3683-3687. WAKAMIYA, T., MCCUTCHAN,T., ROSENBERG,M., and SINGER, M. (1979). Structure of Simian Virus 40 recombinants that contain both host and viral DNA sequences. I. The structure of variant CVP8/ l/P2 (EcoRI res). J. Biol. Chem. 254, 3584-3591.
112.461-4’71 (1981)
SV40 Defectives Selected during Low Multiplicity A 172 Human Glioblastoma Cells
Passage
on
DANA CARROLL,**’ JOANNA L. HANSEN,* EDWARD B. MARYON,* AND FRANK J. O’NEILL**+ Departments of +CeUu.iar, Viral and Molecular Bidogy, and tPat*, University crf Utah Medial Center and *Research Service, Veterans Administration Hospital, Salt L&e Ci&, Utah 8blS2 Accepted Fekry
13, 1981
The gliobastoma line, A172, is unusual among human cell lines in being permissive for SV40 infection, and differs from many host cell types in allowing the accumulation of viral defectives on low multiplicity passage. We have characterized these Al72-derived defectives and find that most are reiteration mutants, containing multiple copies of both the viral replication origin (including the J3glI site at map position 0.669) and sequences from the opposite side of the standard genome (around the BamHI site at map position 0.143). This distinguishes them from defectives accumulated during high-multiplicity passage in monkey cells, which generally have only reiterated origins. When well characterized, monkey cell-derived defectives (ev-1101, d6, az, 4) were passed in Al72 cells, the characterized defectives were lost, and sometimes replaced by other defectives present in the same stock. Surprisingly, this was true even when the characterized defective carried sequences from the BamHI region of standard SV40. These findings are taken to support the notion that sequences in addition to the viral replication origin are positively selected during defective propagation, but that different requirements for such sequences are set by monkey cells and Al72 cells.
INTRODUCTION
Studies of simian virus 40 (SV40) defectives have served to help define and emphasize some functions of the standard virus (Kelly and Nathans, 1977; Fareed and Davoli, 1977). Since they are carried in the presence of wild-type helper, defectives need retain, and often are found to amplify, only those portions of the genome which cannot function in tram, but are required for propagation. In addition, the virus utilizes host cell machinery extensively for the replication of its DNA, so insights can be gained into cellular functions. Quite sensibly, all highly evolved SV40 defectives retain multiple copies of the viral replication origin, but have lost most other wild-type sequences. These reiterated origins confer a competitive replication advantage on the defectives, with i To whom reprint
requests should be addressed. 461
respect to standard virus, and account in large measure for their interference activity (Lee and Nathans, 1979). In earlier experiments, we found that Al72 human glioblastoma cells accumulate SV40 defectives, even on low multiplicity passage (O’Neill, 1976; O’Neill and Carroll, 1978). Analysis of several of these A172-derived defective genomes (Carroll and O’Neill, 1978) showed that they had retained and reiterated, in addition to the viral replication origin, viral sequences from the region opposite the origin on the wild-type genome which we call the termination region.2 Examination of characterized defectives from other sources revealed that, with a single exception, they ’ By this designation, we do not imply a function for this region, but merely acknowledge the fact that bidirectional replication of standard virus, and possibly early transcription, normally terminate in this vicinity. 0042-6822/81/100461-11$02.00/O Copyright All rights
0 1981 by Academic Press. Inc. of reproduction in any form reserved.
462
CARROLL
carried reiterated copies of the viral termination region or of host cell-derived DNA sequences (Carroll and O’Neill, 1978). This led us to suggest that these sequences, like the replication origin, perform in cis an essential function for virus propagation, or at least contribute to the competitive advantage. These sequences are proposed to be selected during defective evolution and not simply carried along passively with the origins. The function served by the termination region is probably also essential to standard virus, and can be served by a wide variety of monkey chromosomal DNA sequences. In this study, we have examined several populations of SV40 defectives arising in Al72 cells to see how general the retention of the termination region is. We found that a majority of A172-derived defectives, but only a minority of monkey cellderived defectives, have equal reiterations of viral origin and termination regions. We have also tested the ability of wellcharacterized monkey cell-derived defectives to propagate in Al72 cells. The human cells selectively amplified different defectives from those preferred monkey cells, but this selection was not simply correlated with the presence of the viral termination region. MATERIALS
AND METHODS
Cells and viruses. General aspects of culturing Al72 (human glioblastoma) and TC-7 (African green monkey kidney) cells and their SV40 infections have been given previously (O’Neill and Carroll, 1978): Stocks containing the characterized defectives a3, dg, a; (Davoli et al., 1977) were obtained from Dr. George Fareed; the ev1101 strain (Lee et al., 1975) was provided a We have observed that when Al72 cells are allowed to grow to high densities on consecutive passages, they undergo a sort of transformation, following which they saturate at higher densities than the original line, no longer support efficient growth of SV40, and no longer amplify defectives on low multiplicity passage. We have not studied this phenomenon extensively, but find it can be avoided by passaging the Al72 cells more frequently and growing them in 5%, instead of 10%. serum.
ET AL.
by Dr. Daniel Nathans; and Repo (Shenk and Berg, 1976) by Dr. Thomas Shenk. Independent passage series were started from these stocks: (1) undiluted on TC-7; (2) undiluted on A172; (3) diluted lo3 at each passage on A172. Intracellular viral DNA was purified from Hirt supernates by banding in ethidium bromide-CsCl gradients (O’Neill and Carroll, 1978). Analysis of viral DNAs. Restriction enzymes were purchased from New England Biolabs (Beverly, Mass.) or Bethesda Research Laboratories (Rockville, Md.). Analytical digestions of 0.1-1.0 pg of viral DNA samples were done under conditions specified by the suppliers. Electrophoretic analyses were performed in 1.0% agarose, 1.5% agarose, or composite 2.0% polyacrylamide, 0.5% agarose gels, as previously described (O’Neill and Carroll, 1978; Carroll and O’Neill, 1978). For detailed restriction mapping, the intact defective b3 and the BglI fragments a, b, c, and d were recovered from 1.0% agarose gels by one of two techniques. Most commonly, the gel segments were dissolved in saturated NaI and the DNA bound to, then eluted from, glass powder (Vogelstein and Gillespie, 1979) or glass fiber filters (Chen and Thomas, 1980). Some samples were eluted electrophoretically and purified by passage over a small DEAE-cellulose column (Fedoroff and Brown, 1978). For Southern blot hybridizations, nicktranslated 32P probes were prepared as described by Rigby et al. (1977). DNA fragments were blotted from agarose gels onto Schleicher and Schuell BA83 nitrocellulose strips according to Southern (1975). Hybridizations were performed in 50% formamide, 5 X SSC, 10% dextran sulfate, as described by Wahl et al. (1979). The plasmid pCa1004 (R. Thayer, M. Singer, and T. McCutchan, in preparation) containing a 344-bp insert of African green monkey satellite DNA was provided by Dr. Maxine F. Singer. DNAs were spread for electron microscopy using the formamide procedure of Davis et al. (1971) and were examined in a JEOL-100s electron microscope. SV40 map positions are based on the published DNA sequence of strain 776
SV40 DEFECTIVES
(Reddy et al., 1978; Fiezs et al., 1978; Van Heuverswyn and Fiers, 1979) and are given as fractional distance from the EcoRI site, measured in the usual direction (Kelly and Nathans, 1977).
463
IN Al72 CELLS y------A112
,
-JC
J--,
RESULTS
The BgWBamHI Assay Digestion with the restriction
enzyme
BgtI provides a simple assay for the SV40 replication origin. There is a single BglI site in the nondefective genome (Fiers et al., 1978; Reddy et al., 1978), included in the T antigen binding region at map position 0.659 (Tjian, 1978; Fig. 1). Since the BgZI recognition site is (effectively) a hexanucleotide sequence (e.g., Lautenberger et al., 1980), it is unlikely on statistical grounds that a small segment of host cell DNA would contain a BglI site. The several defectives which have been characterized in sufficient detail all have a number of BglI sites equal to the number of viral replication origins (Lee and Nathans, 1979; Gutai and Nathans, 1978a, b; Wakamiya et al., 1979; McCutchan et al., 1979; this study). By the same sort of logic, the enzyme BamHI is a probe for the viral termination region. It has a single site at position 0.143 of the wild-type genome and it is retained in the defectives which do not contain host cell substitutions (Carroll and O’Neill, 1978). The assay consists of comparing a BglI ECORI
FIG. 2. BglI/BamHI screens on defective-containing SV40 stocks from Al72 and TC-‘7 ceils. DNA samples were run undigested (-) and digested with BglI (G), BamHI (B), and sometimes BcZI (C); restriction enzyme fragments of pBR322 DNA were included as length standards (stds). (a) From a DNA transfection of Al72 cells, showing defectives with single BamHl sites and duplicated BgZl sites. (b) PP3SV40, P3 lo-” in Al72 cells, showing simple deletion defectives with single BglI, BamHI, and BclI sites. (c) NPPSV40 P6 in A172. (d) From a lo-? dilution of the sample in c, in A172. (e) PP3SV40, 100 PFU/flask, then P2 lo-” in A172, series 2 (D’Neill and Carroll, 1978). (f:I PP3SV40, P16 10” in TC-7. (g) NPPSV40, P2 10” in TC-7. (h) Al72 P6 virus [as in (c)l, P4 10” in TC-7. The positions of supercoiled (I), nicked circular (II) and linear (III) forms of nondefective SV40 DNA are indicated. Electrophoresis was in 1.0% agarose gels.
digest with a BamHI digest of a mixed population of defective genomes. If each subunit in a reiteration mutant contains one origin and one terminus, then BglI and BamHI would yield identical bands, reflecting the subunit size. In many cases, BclI, which has a single site at map position 0.188, was also used as a termination region probe.
A172 and Monkey Cell Defectives
FIG. 1. Schematic diagram of the SV40 genome. The origin of replication and the so-called termination region are indicated, as are cleavage sites for several key restriction enzymes used in this study.
One generalization we can make based on the BglI and BamHI digests has to do with the early stages in the generation of defectives and seems to be true of stocks passed in both TC-7 and Al72 cells. The first altered genomes to arise seem to be the result of simple deletions of nonessential DNA sequences; they are smaller than the wild-type and have a single site each for BglI and BamHI (Fig. 2b). Another form we see early in a passage series car-
464
CARROLL
ries a small duplication which includes the replication origin (Mertz et al., 1975). Such defectives have two, asymmetric BglI sites, yield large and small fragments on BgZI digestion, and retain a single BamHI site (Fig. 2a). The distinctive reiteration mutants seem to arise in later passages. We have not followed carefully the progress from one form to another in any one series of infections, but we have seen the simple deletions and origin duplications only at early stages in the generation of new defective types. Our previous findings that defectives accumulated in Al72 cells retained multiple origins and termini were based on studies of defectives that may have been related to each other (Carroll and O’Neill, 19’78).We wanted to see whether this characteristic was generalizable to independently generated and selected defectives. When populations of A172-derived defectives were examined by BglI and BamHI digestion, we found, in most cases, that the digests matched essentially band for band, indicating equal reiterations of origin and termination regions (Fig. 2, c-e). This was true of defectives from several independent passage series (O’Neill and Carroll, 1978) (e.g., Fig. 2e). In contrast, populations of defectives accumulated during high multiplicity passage on monkey (TC-7) cells showed evidence of multiple BgZI sites (and therefore origins), but characteristically had one or no BamHI sites (Fig. 2, f-h). There was little or no correspondence between BglI and BamHI (or Bc.?I)bands, and often uncleaved, form I defectives could be seen in the BamHI digests. The electrophoretic analyses were confirmed by electron microscopic analysis (Table 1). When linear and circular molecules were counted in complete BamHI digests, a high percentage of undigested circles was found in TC-7 defectives, but many fewer in the A172 defectives. It should be pointed out that where there are multiple BamHI sites in some defectives, a single molecule will yield several linear pieces and the undigestible circles in the population will be somewhat underestimated. Also, many of the linear molecules
ET AL.
in the TC-7 populations correspond to the wild-type helper DNA.
Fates of Characterized Defectives in Al 72 Cells As a sort of converse approach to characterization of defectives generated and accumulated in Al72 cells, we obtained stocks of several well-characterized, monkey cell-derived defectives from other laboratories and tested the ability of these defectives to persist in Al72 cells. The defectives used are illustrated in Fig. 3. Two of these (ev-1101 and d5) contain monkey cell sequences in addition to reiterated origins, while the other two (a; and a3) have both reiterated viral origins and BamHI regions. We knew from experiments with mixed defectives that Al72 cells would select from among TC-7 defectives a different subpopulation from that preferred on continued passage in monkey cells. As a general observation, the same was true of these characterized defective stocks. However, when BgZI and BamHI digests of the Al’IBselected derivatives were examined, it was not generally true that equal reiterations of origin and terminus were found. Rather, multiple origins but one or no termini characterized the Al72 population, as it had the original monkey cell population. ev-1101. This cloned defective was quite pure in the stock we received from Nathans and persisted at a level roughly equal to standard virus when passed in TC-7 cells at high multiplicity. After several passages, ev-1101 was joined by a second defective (Fig. 4). When passed in Al72 cells, ev-1101 was lost from the virus stock. This occurred at low and at high multiplicity, although more slowly at high input. After three or four diluted passages in Al72 cells a faint smear of heterogenous defectives became evident, just as in passage of wt SV40 (O’Neill and Carroll, 1978). Restriction enzyme digests verified, when it could be seen, that the remaining discrete defective in all stocks was ev-1101. d. This defective represented only a small proportion of all viral genomes in
SV40 DEFECTIVES TABLE
IN Al72 CELLS
465
1
ELECTRON MICROSCOPIC ANALYSES OF DEFECTIVE DNAs
Virus
Host
Passage
Percentage circles after BamHI digestion
NPPSV40 PP3SV40 PP3SV40 A172P6SV40 NPPSV40 PP3SV40 PP3SV40
TC-7 TC-7 TC-7 TC-7 A172 Al72 A172
P4, 10” PlO, 10” P15.10” P4,lO” P9, 1o-3 Pl, 10m3,series 2 P3, 10m3,series 2
42.2 29.5 40 28.8 8.4 4.5 2.2
Sample 32 ‘76 37 33 24 41 74
Note. Each defective-containing DNA sample was digested to completion with BarnHI, then spread for electron microscopy. A total of 400 (or morej molecules were counted in each specimen and classified as linear or circular. The input virus in sample 33 was the stock from six passage of NPPSV40 in Al72 cells (all but the initial passage at low multiplicity). Samples 41 and 74 are from one of the very low initial input series described earlier (O’Neill and Carroll, 1978). NPPSV40 was the large-plaque stock prior to plaque purification; PP3SV40 was a triply plaque-purified isolate of NPPSV40 (O’Neill and Carroll, 1978).
the stock from Fareed and was present as both the fourfold (d4) and fivefold (d5) reiteration (Fig. 5). When we passed this stock undiluted on TC-7 cells, d4 and d5 were overtaken by two abundant defectives, both apparently fivefold reiterations, and several minor bands. d was lost during low multiplicity passage in Al72 ev-1101
(eA%),
00TC-7 -- ‘0
d
a'
a
cells, but was retained, particularly as d4, upon high-multiplicity passage in these cells. A variety of restriction enzymes digests showed the major A172-retained defective to be identical to authentic 4. cc’. The characterized defective a; was only one among many defectives in the stock received from Fareed, but was the major one having reiterated EcoRI sites
t.1"
i
c;
1
.I*,.* I .,,.I3 '
Al72 ‘: “: “0 -w-v0 “0 0
, 34 I)4 ,
.n 43
FIG. 3. Maps of the repeating units of characterized SV40 defectives isolated from monkey cells. Open boxes denote SV40 sequences, while thin lines are nonviral DNA. The map of ev-1101 is adapted from Lee et al. (1975), those for d and a’ from Davoli et al. (1977), while the revised map of a is based on our own detailed mapping (see Fig. 9). The brackets and subscripts indicate the reiteration number of these subunits in the major circular defective species. The maps are aligned to their &II sites, which are indicated by arrows; arrowheads show the locations of BornHI sites in a’ and a; and map coordinates at the junctions of normally noncontiguous segments are given.
FIG. 4. Supercoiled DNAs from passage of an ev1101 stock on TC-7 and Al72 cells. The positions of wild-type viral DNA and of the fivefold reiteration mutant ev-1101 are indicated. The faster-moving band in some of the Al72 samples is very likely the fourfold version of the same repeating unit. Electrophoresis was from top to bottom in a 1.0% agarose gel.
466
CARROLL
I’2IQ’
P2IQ-$
A172 PS IQ’
P5 IQ’
P1IQ-’ ’
FIG. 5. Analysis of DNAs from passage of a d5 stock in TC-7 and Al72 cells. Undigested DNAs (-) are compared with BgS (G) and BornHI (B) digests. The position of genuine d is indicated. One slot contains pBR322 restriction fragments used as length standards. Electrophoresis was in a 1.0% agarose gel.
(Fig. 6). On high-multiplicity passage in TC-7 cells, a; was retained at a low level, while other defectives came to dominate the virus stock. In Al72 cells at low multiplicity, monkey cell-derived defectives were gradually lost from the ai stock. Those still present at the second and fourth diluted passage had one or no sites TC-7 ~A172
I
FIG. 6. Analysis of DNAs from passage of an ai stock in TC-7 and Al72 cells. Shown are undigested DNAs (-) and digests with BgZI (G), BamHI (B), and EcoRI (E). The position of genuine a’ was assigned on the basis of its size relative to pBR322 length standards and its production by digestion with BamHI and EcoRI (Davoli et al., 1977). Electrophoresis was in 1.0% agarose gels.
ET AL.
TC-7 I P510~
P2 100
A’72 y-J P5100
FIG. 7. Analysis of DNAs from passage of an a3 stock in TC-7 and Al72 cells. Shown are undigested DNAs (-) and digests with BgZI (G), BamHI (B), and EcoRI (E). The fragments a, b, c, and d were identified by restriction enzyme mapping following recovery from preparative gels. Electrophoresis was in 1.0% agarose gels.
for BumHI and EcoRI, and did not appear to include a’. With undiluted passage in Al72 cells, defectives were amplified until they represented >80% of viral DNA after five passages. Again, BumHI and EcoRI cut the defectives once or not at all, and a’ was lost. Some of the BglI bands in these A172 (high m.o.i.) defectives corresponded to bands present in the original or the TC7 stocks, but evidently different subsets were preferentially amplified. a. a3 was the major, but not the only, defective in the stock obtained from Fareed (Fig. 7). This stock, derived from an earlier passage of the DAR variant of SV40 in the same series that yielded up d5, also contained a low level of that defective, as well as the defectives b4 and c5 (Ganem et al., 1976; Davoli et al., 1977).4 4 Our identification of b and c in the a3 stock is based on: (1) knowledge that they were in the stock initially (Ganem et al., 1976); (2) their sizes and restriction maps (Davoli et al., 1977). Some details of our restriction maps differ from the previous report, but are probably within variations between laboratories. The most striking discrepancy is our finding of only about 29% viral sequences in b and 25% in c (see below). Davoli et al. (1977) concluded that most of the sequences in b and c were viral; but it must be observed that this was based on very low cpm of total viral DNA hybridized.
SV40 DEFECTIVES
At high multiplicity in TC-‘7 cells, the reiteration mutants with subunits a, b, c, and d are retained, but the most abundant defectives seem to contain only a single &ZI site (and a single BamHI site). Upon passage at low or high multiplicity in Al72 cells, the defective b3 came to dominate. This was present at a low level in the original defective stock, but was selectively amplified in the human cells. b. Because of its evident advantage in Al72 cells, we characterized the subunit b in some detail. It has a single EcoRI site, but no BamHI site, in each repeat, and we thought b might have been derived from a by deletion. However, detailed restriction enzyme mapping of b (Figs. 8, 9) showed no homology to a, except in the immediate vicinity of the viral replication origin. In addition, the remainder of the restriction map did not correspond to any other region of the nondefective viral genome. Nevertheless, because extensive rearrangements of viral sequences have been found in some defectives (Gutai and Nathans, 1978a, b) we performed blot hy-
FIG. 8. Restriction enzyme digests of the sort used to map a, b, and e. (A) Digests of b and c with: (1) Bgl[ + EcoRI; (2) BglI + EcoRI + HindIII; (3) BgZI + t’indII1; (4) BglI + EcoRI + PstI; (5) BglI + EcoRI + HincII. Comparisons between similar digests of b and c indicate the nature of the deletion by which the two differ. (B) Digests of a, wt SV40, and b with Bgl[ + BstNI. Electrophoresis was in 2.0% polyacrylamide, 0.5% agarose gels.
IN Al72 CELLS
467
bridizations to determine whether viral sequence other than those around the origin were present in b (Fig. 10). These showed that only a contiguous region of about 250 bp of b, from the B&N1 site at 0.691 to a little short of the HueIII-BstNISau961 cluster, was derived from SV40. We assumed that the apparently nonviral DNA in b may have been derived from the monkey cell genome. We did one simple blot hybridization experiment to determine whether b, like many other defectives (Gutai and Nathans, 197813;Wakamiya et al., 1979; McCutchan et al., 1979; Rosenberg et al., 1977), contained sequences related to the monkey (Ysatellite DNA. This analysis was also applied to d and ev-1101. None of these showed detectable hybridization to the cysatellite probe. Rosenberg et al. (1977) have previously reported that d5 and ev-1101 do not hybridize to this satellite. b3 was preferred to three other discrete defectives in Al72 cells. Two of these we know to be a3 and d4/d5. Interestingly, when we characterized the remaining BglI fragment (c in Fig. 7), we found it to be related to b by a simple deletion of about 200 bp, including a Hind111 and a H&c11 site (Figs. 8,9). Thus, it appears that deletion of these nonviral sequences makes c less fitted than b for propagation in Al72 cells. Repo. There is in the literature a description of one SV40 defective which contradicts the rule that reiteration mutants contain either reiterated viral termination regions or reiterated host cell substitutions in addition to reiterated viral origins (Fig. 11). That is the defective (Repo) constructed in vitro by Shenk and Berg (1976) to contain only the viral origin. When passed undiluted in TC-7 cells, we found that Repo never made up more than a very small proportion of all viral DNA. Shenk has had the same experience with Repo (Shenk and Berg, 1976; T. Shenk, personal communication), indicating that it is not a healthy defective. Repo rapidly fell below the level of detectability on passage in Al72 cells, at low or high multiplicity. We also found, by examining restriction enzyme digests (Fig. ll), that Repo was not a pure species. At least two reiterated
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760
I I
390
I
B611 + PstI + HhaI
495 I 470
-1
I
I
BglI + HaeIII
e 685
BglI + Pet1 + Hind111
610 I
BglI + BstNI + HincII
Jm 610
I
+ Hind111
285
345 I
BatNI + Him11
FIG. 9. Detailed restriction maps of the defective subunits a, c, and b (see Figs. ‘7 and 8). In each case, parentheses enclose one full subunit, and portions of adjacent units are shown by dashed lines. In the map of a, coordinates of the corresponding sites in the wild-type SV40 genome are given. Below the map of b, the results of blot hybridizations to SV40 DNA are summarized. For each digest, the relevant fragments are indicated as bars; shaded bars showed hybridization to SV40 [@PjDNA, while open bars did not. Sizes of these fragments are given in base pairs. Gaps represent fragments too small to be reliably detected in these experiments. There was also very faint hybridization to the rightmost Bgfl + BstNI + HincII fragment, suggesting the presence of a very small amount of SV40 DNA between the B&N1 and H&z1 sites.
subunits are present, apparently in separate defective molecules, as judged by examination of partial BQZI digests. DISCUSSION
Cells of the human glioblastoma line, 8172, are unusual in two respects: (1) unlike most human cell lines, they support lytic growth of SV40 (O’Neill, 1976; O’Neill and Carroll, 1978); and (2) they accumulate defective viral genomes to very high levels, even on low-multiplicity passage of the virus (O’Neill and Carroll, 1978). Recently, in an extensive survey, we found that both these characteristics are shared to some extent by other lines of human cells (O’Neill and Carroll, in preparation). Another striking feature of the A172derived defectives we characterized initially was retention of multiple copies of
both the viral replication origin and termination regions (Carroll and O’Neill, 1978). We have now demonstrated that this property is shared by the majority of defectives accumulated during passage of standard SV40 in Al72 cells, i.e., they have equal reiterations of viral B&I (origin) and BamHI (terminus) sites. We should point out that this is not generally true of the other human cells which accumulate defectives on low-multiplicity passage (O’Neill and Carroll, in preparation). The degree of substitution with nonviral sequences may reflect rates of recombination between cellular and viral DNAs in the various cell types. The finding of retained viral termini led us to propose that this region plays an important role in cis in the propagation of the defectives (Carroll and O’Neill, 1978). Examination of other defective genomes
SV40 DEFECTIVES IN Al72 CELLS
indicated that a wide range of monkey cell DNA sequences could substitute in performing the same function, at least in monkey cells. We also knew that Al72 cells select from stocks passed in monkey cells defective genomes different from the ones preferred in the monkey cell cultures (O’Neill and Carroll, 1978); and the A172selected genomes have reiterated BumHI sites (D. Carroll, unpublished results). It appeared that the sequences required to perform the non-origin function were different in monkey and human cells. Thus, we were interested in studying the fate of individual, well-characterized, monkey cell-derived defectives on passage in AI72 cells. We made the initial assumption that sequences in addition to those around the replication origin are necessary for efficient propagation. If monkey cell sequences cannot perform this function adequately in human cells, defectives lacking the viral termination region but containing host cell substitutions (ev-1101, Lee et al., 1975; dg, Davoli et al., 1977) would be lost on passage in Al72 cells. If the monkey sequences functioned well, the defec-
FIG. 11. Restriction enzyme digests of viral DNA from a stock containing the defective, Repo. (1) BQA; (2) Hind111 + BQA; (3) HindIII. Strong bands are from nondefective SV40 DNA; faint doublets are from the defective. The wild-type Hind111 fragments are labeled. Electrophoresis was in a 2.0% polyacrylamide, 0.5% agarose composite gel.
tives would be retained. We expected the defectives carrying the viral terminus (a3, a:; Davoli et al., 1977) to be retained in Al72 cells. Of the two host-substituted defectives, ev-1101 was rapidly lost on low or high multiplicity passage in Al72 cells. h/d6 was lost slowly at low multiplicity, but seemed to have excellent competitive capability (the & isomer particularly) when passed undiluted in Al72 cells. To our surA prise, both the terminus-containing defecFIG. 10. Blot hybridizations of ‘?-labeled SV40 tives, a3 and a:, were rapidly replaced by probe to restriction fragments of the defective b. (A) other defective species on both diluted and and (B) represent two separate experiments. (1) undiluted passage in A172 cells. HeteroSV4O/HaeIII; (2) b/B& + HiudIII; (3) b/&II + PstI geneous defectives were selected out of the + Hhd; (4) b/&a + PstI + HiudIII; (5) SV40/ HindIII; (6) SV4O/HindIII; (7) b/R@ + Hoe111 a; stock; and the particular genome b,, which is unrelated to a3, was amplified + HindIII; (8) b/R&I + B&N1 + H&11; (9) SV40/ from the a3 stock. bs was amplified indeBQZI + BstNI; (10) b/MN1 + HincII. Electrophoresis pendently in high- and low-multiplicity was in a 1.5% agarose gel.
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passage series, indicating a strong selective advantage, not just a fortuitous event. None of the defectives selected from the above stocks in Al72 cells showed reiterated BarnHI sites (termini). We conclude that some nonviral DNA sequences can substitute in function for the viral termination region-in Al72 cells. Those in ev-1101 cannot; those in d5 can to some extent; those in b3 work particularly well. And we see more definitively than before (O’Neill and Carroll, 1978) that different families of non-origin sequences are selected in the monkey and human cells. Since the wild-type helper virus is identical in both cell types, this suggests an interaction with host enzymes or structures. In fact, given the passage history of the DAR virus stock (Sack et al., 1973), the nonviral sequences in b3 could easily be derived from the human genome. In fact, the host cell specificity of defective genomes is evident even among green monkey cell lines. The defectives in the stocks containing dr,, a;, and a3 were initially selected on primary kidney cells (Fareed et al., 1974; Ganem et al., 1976). When we passed these stocks on the established kidney line, TC-7, those defectives were replaced by others, particularly ones with less highly reiterated &$I sites (see Figs. 6, 7). The argument that a function is required in addition to that (those) provided by the viral replication origin, appears intact. With the exception of the sickly Repo (Shenk and Berg, 1976), all characterized defectives carry other reiterated viral or cellular sequences. What their function may be remains a matter of speculation. Lee and Nathans (1979) demonstrated that defectives have an advantage in replication, not in packaging, so the possibility of an assembly function seems remote. It should be noted that the defectives used in that study all contained reiterated host cell sequences, so their accumulation cannot be attributed solely to origin functions. Earlier studies of Brockman et cd. (1975) showed that the actual site at which bidirectional DNA replication terminates is at the meeting of the two forks moving
at equal rates, independent of the sequence at that point. Still, the termination region could be: (1) an entry or recognition site for a molecule or complex required for initiation of replication; (2) similarly for replication termination or resolution of daughter molecules; or (3) a termination site for early transcription which, if left unchecked, might interfere with the progress of replication. Comparisons of nucleotide sequences of host cell substitutions in a number of different defectives from the same host cell type may shed some light on the precise region required. ACKNOWLEDGMENTS We are grateful to Drs. Daniel Nathans, George Fareed, and Thomas Shenk for providing stocks of defectives and to Dr. Maxine Singer for a gift of a satellite-containing plasmid DNA. This work was supported by NIH Grants CA23123 to D.C. and CA15141 to F.J.O., and by Veterans Administration research funds. REFERENCES BROCKMAN, W. W., GUTAI, M. W., and NATHANS, D.
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SV40 DEFECTIVES IN A172 CELLS cleotide sequence of oocyte 5s DNA in Xenopus Zaevis. I. The AT-rich spacer. CeU 13, ‘701-716. FIERS, W., CONTRERAS,R., HAEGEMAN, G., ROGIERS, R., VAN DE VOORDE, A., VAN HEUVERSWYN,H., VAN HERREWEGHE,J., VOLCKAERT,G., and YSEBAERT,M. (1978). Complete nucleotide sequence of SV40 DNA. Nature (London) 273,113-Z%. GANEM, D., NUSSBAUM,A. L., DAVOLI, D., and FAREED,G. C. (1976). Isolation, propagation and characterization of replication requirements of reiteration mutants of Simian Virus 40. J. MoZ. Biol. 101,57-83. GUTAI, M. W., and NATHANS, D. (1978a). Evolutionary variants of Simian Virus 40: Nucleotide sequence of a conserved SV40 DNA segment containing the origin of viral DNA replication as an inverted repetition. J. Mol. Biol. 126,259-274. GUTAI, M. W., and NATHANS, (1978b). Evolutionary variants of Simian Virus 40: cellular DNA sequences and sequences at recombination joints of substituted variants. J. Mol. Biol. 126,275~288. KELLY, T. J., and NATHANS, D. (1977). The genome of Simian Virus 40. Advan. Virus Res. 21,85-173. LAUTENBERGER,J. A., WHITE, C. T., HAIGWOOD,N. L., EDGELL, M. H., and HUTCHISON,C. A. III (1980). The recognition site of type II restriction enzyme BglI is interrupted. Gene 9,213~231. LEE, T. N. H., and NATHANS, D. (1979). Evolutionary variants of Simian Virus 40: Replication and encapsidation of variant DNA. Virology 92,291~298. LEE, T. N. H., BROCKMAN,W. W., and NATHANS, D. (1975). Evolutionary variants of Simian Virus 40: Cloned substituted variants containing multiple initiation sites for DNA replication. Wok 66, 53-69. MCCUTCHAN, T., SINGER, M., and ROSENBERG,M. (1979). Structure of Simian Virus 40 recombinants that contain both host and viral DNA sequences. II. The structure of variant 1103and its comparison to variant CVP8/1/P2 (EcoRI res). J. BioZ. Chem. 254, 3592-3597. MERYZ, J. E., CARBON, J., HERZBERG, M., DAVIS, R. W., and BERG, P. (1975). Isolation and characterization of individual clones of Simian Virus 40 mutants containing deletions, duplications and insertions in their DNA. Cold Sprint Harbor Symp. Quant. Biol. 39, 69-84. O’NEILL, F. J. (1976). Propagation of Simian Virus 40 in a human cell line. Virology 72, 287-289.
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O’NEILL, F. J., and CARROLL,D. (1978). Appearance of defective Simian Virus 40 following infection of cultured human glioblastoma cells. Virology 87, 109-119. REDDY, V. B., THIMMAPPAYA, B., DHAR, R., SUBRAMANIAN, K. N., ZAIN, B. S., PAN, J., GHOSH,P. K., CELMA, M. L., and WEISSMAN, S. M. (1978). The genome of Simian Virus 40. Science 206.494-502. RIGBY, P. W. J., DIECKMANN, M., RHOADES,C., and BERG, P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113.237251. ROSENBERG,M.. SEGAL, S., KUFF, E. L., and SINGER, M. F. (1977). The nucleotide sequence of repetitive monkey DNA found in defective Simian Virus 40. CeU 11, 845-857. SACK,G. H., JR., NARAYAN, O., DANNA, K. J., WEINER, L. P., and NATHANS, D. (1973). The nucleic acid of an SV40-like virus isolated from a patient with progressive multifocal leukoencephalopathy. Virology 51,345-350. SHENK,T. E., and BERG,P. (1976). Isolation and propagation of a segment of the Simian Virus 40 genome containing the origin of DNA replication. Proc. Nat. Acad Sci. USA 72,1513-1517. SOUTHERN,E. M. (1975). Detection of specific species among DNA fragments separated by gel electrophoresis. J. MoL Biol. 98, 503-517. TJIAN, R. (1978). The binding site on SV40 DNA for a T antigen-related protein. CeU 13, 165-179. VAN HEUVERSWYN,H., and FIERS, W. (1979). NUcleotide sequence of the Hind-C fragment of Simian Virus 40 DNA. Comparison of the 5’-untranslated region of wild-type virus and of some deletion mutants. Eur. J. B&hem. 166,51-60. VOGELSTEIN,B., and GILLESPIE, D. (1979). Preparative and analytical purification of DNA from agarose. Proc. Nat. Acad Sci. USA 76,615-619. WAHL, G. M., STERN, M., and STARK, G. (1979). Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc. Nat. Acad Sci. USA 76, 3683-3687. WAKAMIYA, T., MCCUTCHAN,T., ROSENBERG,M., and SINGER, M. (1979). Structure of Simian Virus 40 recombinants that contain both host and viral DNA sequences. I. The structure of variant CVP8/ l/P2 (EcoRI res). J. Biol. Chem. 254, 3584-3591.