Localization of temperature-sensitive transformation mutations and back mutations in the Rous sarcoma virus src gene.

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JOURNAL OF VIROLOGY, May 1986, p. 694-699 Vol.58, No. 2 0022-538X/86/050694-06$02.00/0

Localization of

Temperature-Sensitive Transformation Mutations

and Back Mutations in the Rous Sarcoma Virus src Gene

VALERIE J. FINCHAMANDJOHN A. WYKE*

Imperial CancerResearchFund Laboratories, St. Bartholomew's Hospital, Dominion House, London ECIA 7BE, UnitedKingdom

Received25November 1985/Accepted 3 February 1986

Cloning and sequencing oftwotemperature-sensitive transforming mutations of Roussarcomavirusreveal thattheir lesions are due to distinct but close single amino acid changes near the carboxy terminus of the v-src geneproduct. Back mutations to wild type result from second mutations at either nearby or distant sites.

Temperature-sensitive (ts) transformation mutations of Rous sarcomavirus (RSV) have proved invaluable in

corre-lating the complex phenotype of the transformed cell with the functioning of the viral src oncogene (17). Different v-src mutations have different consequences for the infected cell,

suggesting a pleiotropism in v-src behavior (1, 3, 34), but

ignoranceof the precise mutations has made it impossible to correlate these behavioral differences with defined

alter-ations in the v-src gene product. Site-directed mutagenesis

(5-9, 28, 29, 35) and the use of anti-peptide antibodies (13, 26, 33) are now popular in analyzing the relationship be-tweenv-src structure andfunction,but these procedures are

initially eclectic and can be coarse. A knowledge of the molecular basis of ts mutations has three benefits. (i) It

these mutants were divided, on the basis of their ability to

recombine with one another to yield wild-type virus, into four cooperative transformation groups (37, 38): members within a group recombine with one another at a low but

detectable frequency while they recombine at higher fre-quency with representatives of other groups. We con-structedacoarsemapof thets mutantsbased on their ability to recombine to wild type with v-srcdeletion mutants (11, 12). This mapping, which was consistent with previous

mapping based on recombination frequencies (2), has had twoconsequences. Itshowed that tsmutations are dispersed widely within src, promptingasuccessful searchfor

pheno-typicdifferences between mutantsthat define different func-tional domainsinv-src(30). Moreimportantly for this paper,

LA24 LA31 6862 7129

l

I

I ynv(gp37) ||dr| |

8706 9058

|

drl

lU31

Pstl

I I I

Psil Pstl agli Pstl BgII Pstl

FIG. 1. Salient features of the 3,096-base-pairEcoRIBfragment of thePraguestrain of RSV. Thisfragment, fromnucleotide6144(in env) to9238(inU3), encompasses thewholev-src gene anditsflanking directrepeats (dr) (25). Molecularclones of this fragmentfrommutants, backmutants, and wild-typePrA wereobtained in pAT153 after first cloning Hirtsupernatant DNAfrom infected chick cells (10, 14), or integrated proviral fragments from transformed chickorratcells, in bacteriophageA vectorsbystandardtechniques (18, 19).Majorrestriction enzyme sites used in subcloning as a preliminary to sequencing are indicated. Subcloning in bacteriophage M13 (21) was followed by sequencing bythechaintermination method (4, 24), (see Fig. 2foranexample). Sequences of regions of interestwere confirmed by the Maxamand Gilbert method (20) aftersubcloning in pAT153. The barabove theline showsthepredicted location ofthe mutations inLA24 andLA31(11). Thesolidarrowbelowtheline shows the minimumregion sequenced in itsentirety for allthevirusesstudiedhere. complements these other procedures, defining sites in the

gene where mutations conditionally affect function. (ii) It may identify otherregions ofthe gene as targets for addi-tional analyses;thepropensity oftsmutants toreverttowild typeisofparticularinterestsince thelocationofsecond-site backmutationsmay indicateparts ofthegene productthat interact withoneanother. (iii)Itprovidesusefulinformation

onmutantsthat havebeenusedextensivelyforphysiological studies.

Ourwork has concentratedon asuiteofts mutantsin the

v-src geneof the Prague strain of RSV, subgroup A (PrA). Soon afterisolationfollowing5-azacytidine mutagenesis (36)

*Corresponding author.

theroughlocalization of the causative mutations heldoutthe hope that they could be identified and distinguished from otherinconsequential mutations that the mutagenesis might have caused.

We considered ts LA24 and ts LA31 ideal tests ofthe feasibility of identifying ts mutations. They belong to the

samecooperative transformationgroup(38) andmapwithin about100 basepairstowards the3' endofv-src(11) (Fig. 1).

Both induceamarkedlytsphenotypeand have been used for

many physiological studies. They arenot leaky, so we can

isolate stable spontaneous back mutants from them, as

described previously (39). The v-src genes from LA24, LA31, three spontaneous back mutants, and thePrA

progen-itorweremolecularlycloned(10, 14, 18, 19) (Fig. 1). Chick 694

6269

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TABLE 1. Nucleotide andpredicted aminoacidsequencecomparisons ofPrAanditsmutants

Change from PrC in: Presenceofchangein:

Nucleotide

Base Aminoacid' PrA LA31A 31A.1.4 31A.3.4 LA24A 24A.7b2

6517 T C L-* L(gp37,83) + + NDb ND ND ND

6957 C T Noncoding (indirectrepeat) + + ND ND ND ND

7018 A C Noncoding + + ND ND ND ND

7059 A G' Noncoding - + + + + +

7082 G A Noncoding - + + +

7185 A *G' H R(src,16) + + + + + +

7332 C-T T T(src,69) + + + + + +

7742 A-*G Y Cd(src,205) + + + + + +

7749 C T R R(src,217) + - - - -

-7852 G Ae A T(src,242) + + + + + +

7855 A-C N H(src,243) + + + + + +

7991 A Ge D G(src,288) + + + + + +

8257 G A V M(src,377) - - +

8561 G A G D(src,478) - + + +

8567 G-A R H(src,480) - - - - + +

8602 C T H Y(src,492) - - - + - +

8632 G A D N(src,502) +

8706 G A E E(src,526) + + + + + +

a Data in parentheses are the affected gene and amino acid.

bND, Not determined.

'This change isalso seen in the sequences of SRA and c-src (31, 32).

dSRA andc-srcdifferfrom PrC at nucleotide 7741,substituting arginine for tyrosine(31, 32).

e This change isseeninthe sequences of SRAandc-src (31, 32) as well assomevariants of PrC(25.)

A

_PrA _31A.1.4 _31A._3.4 _24A.7b₂

8558 ;-

AA

C C T

f,I

_

-B570

A C G T G

- /

C-/ A" <

- . . T

A

..~~~C

..c

*. .._.C-c A / T

A C G T A C G T

T -3

C, Nn

C a

* * T I _

.5.~ ~ .^ 6

T __

T

G

-""A

M _s.- o

T

--AA

LA 31A

8260C T C C

G T G T

82 54 T.

A

31A.1.4

G T G C A

G As

T

T w .

T

FIG. 2. Locationofmutations andback mutationsin derivatives of PrA. Allsequenceladders in panel A show anti-parallel strands. The sequences inwhich mutations(0)arelocatedareindicated beside the ladders. (A) In this region (approximately nucleotides 8540to8630), tsLA31A(notshown) is identical insequenceto itsback mutant, 31A.1.4.tsLA24A (not shown) shares with its backmutant,24A.7b2, the

G-to-Atransition atnucleotide8567 butdoes notdisplaythe C-to-Ttransition at nucleotide 8602. (B) In this region (approximately nucleotides 8250to8265), allthe viruses not shownareidenticaltotsLA31A.

8600 -r-G C

8604T

B

VOL.58, 1986 NOTES 695

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696 NOTES

cells transfected with cloned ts mutant proviral DNA pro-duced transformants with ts morphology and protein kinase activity. As anticipated, transformants induced by cloned back mutant DNA lacked temperature dependence (data not shown).

To determine the extent of spontaneous and mutagen-induced variation, we sequenced (4, 20, 24) the entire EcoRI B fragment of LA31 and our current stock of wild-type PrA and compared them with the published sequence of the Prague strain of RSV, subgroup C (PrC [25]). Nucleotide numbering of this EcoRI fragment B followed that of Schwartz et al. (25). Table 1 summarizes the differences between PrA and its derivatives and PrC.

PrA and LA31 show common differences from PrC at 10 sites. Two are noncoding and another three affect the third bases of codons without altering coding. The other five changes alter the coding assignment of v-src at amino acids 16, 205, 242, 243, and 288. PrA differs at two further sites from PrC, LA31, LA24, and their derivatives; one of these differences changes the coding of amino acid 502 of v-src. These latter changes presumably reflect variation that has occurred in our stock of PrA after the isolation of the ts mutants in 1971.

LA31 shows an alteration at nucleotide 8561, changing amino acid 478 of v-src from glycine to aspartic acid. Likewise, in LA24, a change at nucleotide 8567 converts amino acid 480 from arginine tohistidine (Table 1 and Fig. 2). We conclude that these differences account for the temperature sensitivity of the two mutants because they are the only changes within v-src unique to each mutant, they are within the region in which the mutations were located by recombination mapping (Fig. 1), and they are very close to one another, explaining the allocation of LA31 and LA24 to the same cooperative transformation group. Moreover, the mutation in each case is a G-to-A transition, the type of change expected with the use of 5-azacytidine.

These conclusions are strengthened by examination of back mutations to a wild phenotype. The sequences of two of these, LOILA31A.3.4 andLOILA24A.7b2, show that they are identical to their parent ts viruses with one exception; both show the same C-to-T transition at nucleotide 8602, changing amino acid 492 of v-src from histidine to tyrosine (Table 1 and Fig. 2). Thus, an alteration to this amino acid appears to compensate for mutations affecting eitheramino acid 478 or 480. However, this is not the only way bywhich backmutations can occur, forLO/LA31A.1.4 does not differ from parental ts LA31A in this region but shows instead a mutation atnucleotide 8257, changing amino acid 377 from valine to methionine (Table 1 and Fig. 2).

Several interesting points emerge from this work. (i) The sequences of LA24 and LA31 are an impressivevalidation of earlier genetic studies that predicted that LA24 and LA31 bear distinct but closely linked mutations (37, 38). The mutations are 6 nucleotides apart, and this remarkable degree oflinkage can therefore be discerned by examining recombination frequencies. Moreover, the mapping of Fincham et al. (11) of these mutations has been proven accurate, increasing our confidence that the other 12 ts src mutants of PrA that we have studied by genetic means indeed have distinct mutations whose lesions span a large partof the src gene. We suspect that the allocation of these mutants to cooperative transformation groups does not reflect primarily the existence of hot spots for genetic

recombination between groups but gives anindicationof the physical spacing of these mutations in the src sequence.

(ii) Although LA24 and LA31 were obtained after

muta-genesis, itis noteworthy thatextensive silent mutations are not presentintheir genomes, and themutations could have been identified without prior knowledge of their location.

However, mapping studies arevaluable in examining other

mutantsthatdifferfrom thewildtypeatseveralsites (A. W. Stoker, unpublished data). Furthermore, attempts to map certain ts mutants by crosses with deletion mutants have proved equivocal(J. A.Wyke, V. J. Fincham,R. Friis, and

M.Weber,unpublished data).Notably,NY68(15)andsome mutants of the GI and CU series (1, 34) recombine to the wild type with deletionmutants that lack src sequences 5' to nucleotide 8200 (approximately) but cannot do so with mutants lacking sequences 5' to nucleotide 8500

(approxi-mately).This is primafacieevidence that crucial mutations

are located between nucleotides 8200 and 8500 but the wild-typerecombinants either retain somephenotypic pecu-liarities or are ofirregular occurrence. The present study

makes us confident that mapping by recombination is a

powerful andprecise procedure, and wenowconsiderthat these equivocal results may bedue to additional mutations

elsewhere in src thatalsoinfluencethephenotype of infected

cells. Recentfindings (22) confirm this prediction forNY68. (iii) The deduced amino acid sequence ofour wild-type

PrAdiffers from that of PrC (25) at several sites. Some of

these differences are shared withtheSchmidt-Ruppin strain

A(SRA)of RSV andchickenc-src(31, 32)(Table1and

Fig.

3),but overall PrAresembles PrCmore closelythan itdoes

SRA. LA24and LA31 resemble PrA inmostrespects,so our wild-type clonedoes not represent aminorgenotype in the

population.

(iv) The mutations in LA24 and LA31 affect amino acids outside theregions of pp60v-src thatare most

strongly

con-servedamongtyrosine

kinases,

thepresumed active siteand

ATP-binding region (Fig. 3). This is not

surprising,

for although mapping has located several ts mutations in the strongly conserved region, other

mutations,

as in

LA29,

affect pp60v-src kinase activity but map even closer to the

carboxy terminus of the

protein

than those of LA24 and LA31 (11, 30). This accords with the

findings

ofWilkerson andcollaborators (35), who havestudied

portions

ofv-src

by

in vitro mutagenesis and propose that the

carboxy

terminus oftheproteincomprisesa

regulatory

domainthat influences the adjacent, conserved catalytic domain (23). The muta-tions in LA24 and LA31 are near the

junction

of these

postulated domains.

The altered amino acids at

positions

478 and 480 are

themselves

reasonably

conserved in different

tyrosine

kinases (Fig. 3), but the

significance

of their alterations is unclear. Wild-type PrAalso shows a

change

ina conserved

amino acid, atposition 502

(Fig.

3), butthis does not affect its transforming activity. Furthermore, while it is easy to appreciate thatan alteration from

glycine

to

aspartic

acid at position478could result in the

changes

in

protein

stability

or function expected inaconditional mutant, other

changes

in forwardorback mutations are moreconservativeand donot conform to a discernible pattern.

(v) The significance of these mutations should be more

apparent ifinformation on the

tertiary

structure of

pp60v-src

is obtained. Inthe

meantime,

perhaps

the most informative part of this study is the location of spontaneous back mutationstowild type. It may

only

befeasibletoobtain such back mutations when the forward mutation is a

single

base

change, andthis seems a

particular

advantage

ofts mutants obtained in vivo. All three back mutants retain the

original

forward mutation

(Fig. 2),

and the

compensating

second-site

changes substitute amino acids that are

peculiar

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231

V-0 V-0 0 0 00o0 0 0o * 000* 0o

,SKHADGLCHR,LANVCPTSKP,QTQGLAKDAW,EIPRESLRLE,AKLGQGCFGE,VWMGTWNDTT,

291

V

00 0 0000000 0 0 0 0 0 0 00000 0 000

,RVAIKTLKPG,TMSPEAFLQE,AQVMKKLRHE,KLVQLYAVVS,EEPIYIVIEY,MSKGSLLDFL,

351

v

00 0 0 00 0 0000 00 0 0000

,KEMGKYLRL

*PQLVDMAAQI,ASGMAYVOERM,

NYVOH;RDLAA,NILVGENLVC,KVADFGLARLI

SR-A PrC PrA

31A

31A.1.4

31A.3.4

24A 24A. 7b2 SR-A PrC

411

G-G-0000 00 0 0

,IEDNEYTARQ,GAKFPIKWTA,PEAALYGRFT,IKSDVWSFGI,ILLTELTTKGR,VPYPGMVNRE

471

R R E

00C00 000 00 00 00 00 0

,VLDOVERGYR MPCPPECPES,LHDLOMCQCWR,KDPEERPTFK, YLQAQLLPAC,VLEVAE

PrA 31A

31A.1.4

31A. 3.4 24A 24A.7b2

FIG. 3. Predictedsequencesof thecarboxy-terminal300 amino acids ofpp60v-src comparingPrC(25)withSRA(31)and PrAanditsmutant derivatives. Amino acids thatareconserved between v-src,v-yes, andv-fps (0)andmore stronglyconserved residuesthatarealsofound

incyclicAMP-dependent proteinkinase(-)areindicated(dataarethose of Kitamura et al. [16]andShibuyaetal.[27]). Atposition295,the lysineattheATP-bindingsite(A) isindicated;theputativephosphoacceptor tyrosineatposition416(A)is alsoidentified.

SR-A

PrC PrA 31A 31A. 1.4

31A. 3.4

24A

24A.7b2

SR-A

PrC PrA

31A

31A. 1.4

31A. 3.4 24A 24A.7b2

SR-A PrC

PrA 31A

31A. 1.4 31A.3.4

24A 24A. 7b2

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698 NOTES

viruses and are thus not a consequence of recombination with c-src. Back mutations affecting amino acid 492 may affect the same domain or portion ofa domain as the ts forward mutations. However, the back mutation in LOILA31A.1.4. alters an amino acid on the other side of the kinase-active site. This implies a direct or indirect

interac-tion betweentheregionsaroundamino acids377 and 478. A

novel interaction of this kindhas been revealed after

inves-tigating only three spontaneous back mutants. The facility

with which spontaneous back mutants can be obtained from avariety of different ts src mutants suggests that they may

provide useful and unique ways to analyze

structure-function relationships in this gene.

We aregrateful to David Gillespie for advice and discussion, Paul Scotting for commentsonthe manuscript, and Andrea Sterlini for secretarial assistance.

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