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Host

Range Mutants of Polyoma Virus*

Thomas L. Benjamin

PUBLIC HEALTHRPSEARCH INSTITUTE OF THE CITY OF NEW YORK, INC., NEW YORK, N.Y.10016

Communicated by James D. Watson, June 29, 1970

Abstract. Aline ofpolyoma-transformed mouse cells has been isolated which isfullysusceptible to lytic infection by polyoma virus. Thisline has been used

toselect virusmutantswhich have lost most or all of their ability to grow in the untransformedparentallinewhile retaining the ability to grow in the transformed derivative. These virus mutants are also defective in theirability to transform cells of rat or hamsterorigin. Since the DNAextracted from the mutants has the samehost rangeasthewholevirus, the mutants appear to be blocked at some intracellular stepwhich isrequiredboth forthe completionofvirusdevelopment inmousecells andfor transformation in rat orhamstercells.

It now seems clear that neoplastic transformation of mammalian cells by the small DNA-containing tumor viruses, polyoma and simian virus 40 (SV40), is accompanied by persistence of viral genes, and that some of these genes continue to be expressed in transformed cells in the absence of in-fectious virus. 2

However,

the nature of those gene functions which continue

tobe expressed bythe cell-associated viral genome, and the role such functions might have indirectingthe transformedstate, areunknown. TheDNAcontent

of these viruses is small (3 X 106daltons), corresponding toperhaps 5-8 genes; radiobiological evidence indicates that only a fraction (50-60%) of the small geneticcontent isrequired to cause the neoplastic response comparedto that

re-quiredforvirusreproduction.3 Thus,whatever these viruses do to cause

trans-formationtheydoby puttingintoplay onlyasmall number of discretefunctions.

To furtherunderstand this process, it is crucial firsttoidentifywhat these func-tions are, andsecondtodetermine how the relevant viral functions interactwith the hostcelltoalteritsgrowth.

Thesearch for conditionally lethalmutants of these virusesoffers apromising approach for studying the biochemical changes involved in transformation.

Temperature-sensitive mutants of polyoma virus have already been isolated

and studied.46 The existence of another class of polyoma mutant showing a conditional lethality of the host range type has been predicted7-namely,

mu-tantsblocked in their growthin normalcells, butstill ableto grow incells pre-viously transformed by polyoma. Thistype of host range selection was chosen because it seemed possible that the survival of the mutants in the permissive (transformed) cell would be based on complementation of the conditionally defective viral function(s) by the homologous function(s) of the cell-associated viral genome; thus, if any viralgenes expressed in the transformed cell are re-quired for the initiation or maintenance of the transformed state (and also for

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virus reproduction), then mutants defective in these crucial function(s) would

be found. The present communication describes the isolation and preliminary characterization of such mutants.

Materials and Methods. Cell lines: The 3T3 line8 of Swiss mouse embryo

fibroblast served as a normal line from which the polyoma-transformed line 3T3-Py-3

wasisolated (see Results andDiscussion).

Mutagenesis: (a) Hydroxylamine: Hydroxylamine wasusedon apurified stock ofsmallplaque virusby mixing0.4 ml of the virus in Tris-buffered saline and 0.1 ml of

2 MNH20H+ 2 MNaCl, pH 7.0,incubatingat450Cfor90min,anddialyzing againsta large volumeof bufferat4VC. Thesurvival of plaque-forming units after thistreatment wasabout 5%. Aliquots of thehydroxylamine-treated virus werethen passed once at low multiplicity through 3T3-Py-3 to allow segregation of possible mutational

hetero-zygotes. Theselysateswerethen usedforplaqueisolations.

(b) Nitrosoguanidine: N-methyl-N'-nitro-N-nitrosoguanidine was added 17 hr after infection ofababymousekidneyculturewith smallplaque polyoma virusat afinal con-centration of 314 1ug/ml. Receptor-destroying enzyme and anti-viral antiserum were present from the third to seventeenth hr to remove residual virus from the inoculum. Thetotalyieldof virus50hrafterinfectionwasabout3%of the untreated control. The crudelysate from the NG-treated culturewasuseddirectlyforplaqueisolations.

Plaquescreening and mutantisolations: Appropriate dilutions of the

mutagen-ized lysates were plated on sub-confluent monolayers of 3T3-Py-3, and single plaques

were picked on the fifth to eighth day. Each plaque isolate was then tested for its ability to produce plaques on confluent monolayers of 3T3, or in some instances on mouse embryo secondary cultures. Those isolates were kept which failed to produce distinct plaquesafter 10-14 days of incubation; three further sequential single-plaque

isolations on 3T3-Py-3 were carriedout with each such isolate. Wild type unselected plaques on3T3-Py-3fromanonmutagenized virus stockwerekeptascontrols. Stocks

of all virus isolates were grown on 3T3-Py-3. All isolations, screening, and growth of stocksweredoneat37.0-38.5'Cunless statedotherwiseinthetext.

Results and Discussion. Isolation and characterization of polyoma-trans-formed 3T3 lines: A cell line with thefollowing properties was required as a permissive host for the isolation of virus mutants:

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transformed by polyoma andvirus-free, (2) highly susceptibleto productive reinfection by polyoma, and (3) capable ofbeing used in a plaque assay. Whereas polyoma transformants of nonpermissive hosts (rat or hamster) are known to remain nonpermissive, rare transformants which arise after infection of the lytic host

(mouse)

might retain susceptibility to lytic infection. Previous reports9'10 showing polyoma-transformed mouse cells to have increased resistance to reinfection might be explainedonthebasis ofastrongselection for viral resistance which accompanied their isolation rather than anintrinsic"immunity."

Suitable transformants were therefore soughtin the followingway. 3T3 cells exposed topolyoma at a multiplicity of 10-20plaque-formingunits (PFU)/cell werefirst washedtoremoveunadsorbed virus and thenplatedin agarsuspension cultures." High-titer rabbit anti-viral antiserum and receptor-destroying enzyme were added to the soft agar layer to protect the rare emerging trans-formants from being reinfected and killed by the large amounts of virus being released by themajority of cells. After 9 days in the agar, a number of small clones were picked and re-cloned in liquid media containing antiserum and re-ceptor-destroying enzyme. Clones with transformed morphology were grown, tested for thepresence of infectiousvirus, and re-cloned if necessary until

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virus-free. Nine such clones with various degrees of transformed morphology were eventually isolated. When these were tested for susceptibility to wild type polyoma in plaque assays, three clones gave no clear plaques and had apparently lost susceptibility to reinfection, while another three could not be maintained in a viable state under agar long enough for plaques to develop. The other three did allow the development of plaques initially. However, two of the last three clones could not be maintained reproducibly under agar on repeated passage; the third one, 3T3-Py-3, continued to remain viable under agar long enoughtopermit plaque isolations. The presence of polyomaTantigen(s) could be demonstrated in3T3-Py-3 by complement fixation and the presence of virus-specific RNA byDNA-RNA hybridization.

Isolation of viral mutants: Roughly 900 plaques from the NG-treated and 360 from the HA-treated virus stocks were picked (see Materials and Methods). When tested on 3T3or mouse embryo secondary cultures, a total of60 isolates showed either no plaques or turbid plaques appearing several days later than those ofwild type. These werethen grown in 3T3-Py-3 totiters of 5 X 106 to 2 X 107 PFU'ml and tested for plaques on 3T3 and 3T3-Py-3. Fifty-six be-havedlike "partial" mutants,givingclearplaqueswithin5or6dayson 3T3-Py-3, and turbid plaques on 3T3 after 9 or 10 days. The remainingfour failed to

give discernible plaqueson3T3,even atinputsseveral orders ofmagnitudehigher than required to give plaques on 3T3-Py-3. Further studies were confined to

thesefour virus isolates: NG-18,NG-23, HA-33,andNG-59.

Growth abilities of the mutants in 3T3 and 3T3-Py-3: In contrast to wild type, which has roughly the same efficiency of plating on 3T3 and 3T3-Py-3, the fourmutantsgive discernibleplaquesonlyonthe transformed line (Table 1). TABLE 1. Plaquetitersofmutants onST3 and3T3-Py-3.

3T3-Py-3 3T3 3T3

Virus 37.50C,7days 37.51C,7days 31.50C,26days

NG-18 1.6 X 107 <10' -3 X 105

NG-23 6 X 106 <101 -2 X

106

HA-33 7 X 106 <101 -3 X

104

NG-59 5 X 106 <101 -2 X 104

Wildtype 1 X 108 7 X 107 3 X 1(8

Monolayers of 3T3 infected with the mutants at multiplicities approaching 1

PFU/cellorhigher (ascalculatedfrom titerson 3T3-Py-3) showsomecelldeath,

as evidenced by lack of neutral red staining. The absence of distinguishable plaquesindicates,however,that themutants arehighlyrestrictedintheirgrowth in 3T3 cells. When the assay on 3T3 is done at 31.50 instead of

37.50C,

very turbid plaques appear after long periods of incubation.

Though

difficult to determine accurately, the

plaque

counts inthese cultures neverapproachthose

on 3T3-Py-3. Both the cell killingat 37.50C and thelimitedgrowthat 31.50C suggest that themutants canadsorbtoand carryoutlimited functions in normal

cells.

A better estimate of the degree ofgrowth restriction in 3T3 cells isobtained by measuring the average burst size after a

single cycle

of growth (Table

2).

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TABLE 2. Average burstsizesof mutants on3TS and STS-Py-3.* Virus 3T3 3T3-Py-3 NG-18 0.63 4.2 X 102 NG-23 1.4 4.5 X 102 HA-33 0.19 6.0 X 102 NG-59 2.1 2.0 X 102 Wildtype 3.0 X 102 5.1 X 102

*Cellswereinfected at amultiplicityofapproximately0.1 PFU/celland harvested48 hrlater.

Cells and mediaweresonicated, and thevirus wasassayedon3T3-Py-3. Thenumbers givenare

ratiosof output to input virus.

of the mutants, while in 3T3-Py-3, the mutants give values approximately the same as wild type and several hundred times higher than in 3T3. If 3T3 cells infected by the mutants are incubated for a time equivalent to two wild type growth cycles, essentially the same results are obtained.

To testdirectly whether the reduced burst sizes in 3T3 could be due to failure in adsorption, penetration, or uncoating, infections were done with the DNA's extracted from two of the mutants (Table 3). The results show the same host TABLE 3. DNAinfectivityofmutants.*

Virusyields

DNAInput(PFU) 3T3 3T3-Py-3

NG-18 (9 X 102) 3 X102 3 X 104 + DNaset 0 0 HA-33(2.2 X 103) 4 X 103 1.6 X 106 + DNase 0 0 Wild type(1.1 X 103) 1.6 X 106 2 X 106 +DNase 0 0

*Cultureswerefirstwashed with2ml Hanksbuffercontaining200;&g/mlDEAE-Dextran. The

DNA,in 0.1 ml Hanksbuffercontaining750;g/mlDEAE-Dextran, wasaddedfor20min at room temperature. Thecultureswerethen washed with Hanksand fed. DNAinput titersweremeasured

on3T3-Py-3inthesameway. Virusyields44hrafterinfection withthe DNAweremeasuredon

3T3-Py-3.

tDNase treatmentwascarried out with 0.05;sg/mlenzymefor 10 min at37.50C.

range pattern ofinfectivity asinexperiments withwholevirus. That the DNA fromthe mutants does give rise in 3T3 cellstosome infectious virus can be ex-plained on the basis of incomplete loss of function (leakiness), or by the en-capsidation ofinputDNA with a

bypass

ofessential earlyfunction(s), or both. It is clear in any case that the restricted growth of the mutants in 3T3 cells is notduesimplytoafailure in any steppriortouncoating.

Sincethe mutants wereisolatedondividing cells of3T3-Py-3 and screened for absence of growth on

stationary

cells of

3T3,

the

possibility

existed of having selected mutants requiringan

actively

dividingcell in whichto grow butbeing indifferentto the transformed state of its host. This wastested by comparing virus growth inexponential andstationary 3T3 cells (Table 4). Acomparison ofresultsbetween themutantsandwild type shows that3T3 cells in exponential growth donotbecomepermissivefor the mutants.

Since the mutants can propagatestably in 3T3-Py-3, recombination withthe cell-associated viral genes (marker rescue) occurs onlyinfrequentlyif at all, and

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TABLE 4. Growthof the mutants in dividing andnondividingSTScells.

-- Totalvirus per culture*

Input virus (PFU) 3T3,day 1 3T3, day 4

NG-18 (1.2 X 105) 1.5 X 105 6.4 X 103

NG-23 (6 X 104) 1.2 X 105 8.4 X 104

HA-33 (5 X 104) 7.0 X 103 7.5 X 102

NG-59 (5 X 104) 1.6 X 105 1. 5 X 104

Wild type(2 X 105) 2. 1 X 108 2.4 X 107

*Culturesof3T3 in60-mmdishescontaining 3.5 X 106 cells in exponential growth on day 1 and 1.9 X 106nondividingcells on Day 4 were infectedand harvested 48 hr later. Cells and media were sonicated and the viruswasassayedon3T3-Py-3.

Properties ofthe virusparticles: Themutant virus particles have properties associated withthecapsid which are not detectablydifferent from those of wild type particles. Both mutant and wild type viruses have PFU:HAU ratios in the range of 2-4 X 104. The mutants possess neutralization antigen(s) similar to wild type, as shown by their inactivation by antiserum prepared against wild typeparticles.

Transformation: All four of the mutants fail to transform rat embryo fibro-blasts (Table 5). The frequency of transformation is lower by a factor of at TABLE 5. Transformation ofRECI-3.*

Transforming Efficiency of

PFU in Transformants units wild type

Virus inoculum perculture perPFU (%)

NG-18 5 X 106 0 <2.0 X 10-' 0.07

NG-23 9 X 105 0 <1.1 X 10-6 0.4

HA-33 3 X 106 0 <3.3 X 10-' 0.12

NG-59 7 X 105 0 <1.4 X 10-6 0.52

Wildtype 3 X 105 80 2.7 X 10-4 100

*REC1-3is anestablished lineofratembryofibroblast. Theagarsuspension methodwas used

tomeasuretransformation."

least 200

compared

to wild type. Similar resultswere also obtainedusing the BHKline ofbabyhamsterkidney cells.11

Basis of the host range

properties

of the mutants: A

plausible explanation

in general terms for the host range

properties

of the mutants can be based on

complementation-the

transformed cell

expressing (either

continuously

orupon infection) some

function(s)

homologoustothat of the virus and

required by

the the latterbothtogrow in and totransform normal cells. The

positive

correla-tion in all four cases studied between the selected property of transformed cell dependence in virus growth and the predicted (unselected) property of being unable to cause transformation suggests that the cell-associated function(s) which allows themutantstogrow is also

implicated

somehow in thetransformed state. This

function(s)

could be associated with any of the

"integrated"

viral genes themselves, or with some cellular gene(s) which is expressed specifically in

polyoma-transformed cells,

or

perhaps

generally

in transformed cells of diverse origin. Further clarification should come from

experiments

now in progress on the abilities of other cell types

(including spontaneously

or SV40-transformed lines and

phenotypic

revertants of

polyoma-transformed

lines)

to support,thegrowthofthemutants.

(6)

Iwould like to thank Dr. J. Piatigorskyfor stimulating discussions, and D. Tidabackand S.Ching for experimental assistance. Portions of this work were carried out in theDivision of Biology at the California Institute of Technology, at the Institute for Biomedical Research, and at the Public Health Research Instituteof the City of NewYork,Inc.

Abbreviation: PFU,plaque-formingunits; HAU, hemagglutination units.

* The work waspartlysupportedbyU.S.Public Health Servicegrant CA11473-01of the

National Cancer Institute.

1Benjamin, T. L., J.Mol.Biol., 16, 359 (1966).

2Westphal, H., andR.Dulbecco, Proc.Nat. Acad. Sci. USA, 59,1158 (1968).

3Benjamin,T.L., Proc. Nat. Acad. Sci. USA, 54, 121 (1965). 4Fried, M., Proc. Nat. Acad.Sci., USA, 53, 486(1965).

6 Eckhart, W., Virology,38, 120 (1968).

6DiMayorca,G., J.Callender,G. Marin, and R.Giordano, Virology, 38, 126(1968). 7Benjamin,T.L., in SubviralCarcinogenesis,1st Int. Symp. Tumor Viruses, Nagoya, Japan, (1968) p. 62.

8Todaro,G.,andH. Green, J. Cell Biol., 17, 299 (1963).

9Vogt, M., and R. Dulbecco, Proc. Nat. Acad. Sci. USA, 46, 365 (1960).

10

Hellstrbm,

I.,K.E.Hellstrom,andH.0. Sjogren, Virology, 16, 282 (1962).

References

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