Nucleotide sequence changes in polyoma ts-a mutants: correlation with protein structure.

0022-538X/81/030871-05$02.00/0 Vol.37, No.

Nucleotide

Sequence Changes

in

Polyoma

ts-a

Mutants:

Correlation

with

Protein

Structure

PRESCOTT L. DEININGER, PATRICIA LAPORTE,ANDTHEODORE FRIEDMANN*

Department ofPediatrics, University of California, San Diego, La Jolla, California92093

The mutations in three polyoma ts-a mutants have been determined. Two

mutants,ts-25and ts-52,have different single-base changesatthesameposition

(2883) in the early region correspondingtoaconservedglycineresiduevery near

the C-terminus of the polyoma large T antigen. Mutant ts-48 hasasingle-base

changeatposition 2341,aswellas asecondchangeatposition 1228, in the region

oflarge Tantigen shared with medium T antigen.

A thorough understanding of the

structure-functionrelationshipof abiologically active

pro-tein requires detailed infonnation of the

enzy-matic and otherbiologicalfunctions, knowledge

of itsprimarystructurethrough amino acid

se-quencing,adescription of secondary and tertiary

structuralfeatures through X-ray

crystallogra-phy and otherphysicalmethods, the

identifica-tion of funcidentifica-tionaldomainsand residuesthrough

characterization ofmutants, and evolutionary

studies of functionally and genetically related

proteins to identify conserved regions of the proteins. Suchanalyses require readysourcesof

purified proteins

and

well-developed genetic

sys-temsin additionto assaysforprotein function.

Within the past several years, the

availability

of

purifiedsimian virus 40(SV40) largeTantigen

(13,26) and thedescriptionof itsproteinkinase

and ATPaseactivities(11, 27), together withthe

likely availability of other papovavirus tumor

antigens,have

begun

tomake

possible

both an

examination of thestructure-function

relation-ships of thepapovavirustumor(T)

antigens

and

an

understanding

of the mechanisms of cell

transformation mediatedby these proteins. Of

majorimportance hasbeen the

development

of

methodsforthe rapid determination of the

nu-cleotidesequence oflarge DNAmolecules (17, 21), by which the nucleotide sequences of the entire genomes ofSV40 (5, 19),

polyoma

virus

(2, 8, 24), and BK virus (22) have been

deter-mined. These studies providedvaluable

infor-mation aboutthenature andstructure ofallof

the virus-coded proteins,

including

the T

anti-gens,longbeforepurified proteinsbecame

avail-able. Using nucleotide sequence information

alone,wehave identified

functionally

important regionsofthese molecules fromanidentification

of conserved regions in the viral genomes (7).

Otherworkers have examined the effects of

mu-tations at avarietyofpositionsin thegenomes

onthe function of viral geneproducts.

We wish to present here the nucleotide

se-quenceofpolyomagroupAmutantsthataffect

the structure and expression of the polyoma

large Tantigens and a correlation of the altered

sequences with characteristics of the protein

structure.

MATERIALS AND METHODS

Wild-type strain 3 polyoma is the strain whose complete nucleotide sequence has been described and analyzed in detail in thislaboratory (2, 8). The tem-perature-sensitive (ts) mutants ts-25, ts-48, and ts-52 were obtained from Walter Eckhart, TheSalk Insti-tute, La Jolla, Calif., and have been characterized extensively (3). All three mutants have the polyoma ts-aphenotype; i.e., they failto transform cellsat a nonpermissive temperature, andtheyare also

condi-tionallydefective in the induction oflargeTantigen

and in the inductionof viral DNAsynthesis.Allviral DNAswere prepared after infectionof 3T6cells by

previouslydescribed methods(9).

Preparationof clonedfragments.DNApurified

from themutants wascleaved with MboI and cloned

byligationinto theBamHI site ofplasmidpBR322;

selection ofampicillin-resistant colonies and detection ofappropriate polyomainsertsweredonebythe

hy-bridization method of Grunstein and Hogness (12),

using nick-translated (16) wild-type polyomaHaeIII fragment7as aprobe. Similarly,thePstIfragments

werecloned into the PstI site of pBR322, and inserts were detected as above by selecting tetracycline-re-sistantcolonies andbycolony hybridization with wild-typepolyoma HpaII fragment6.

Full-genome cloneswerepreparedbycleaving mu-tantDNAwith BamHI andligatinginto the BamHI siteofpBR322. Theresulting colonieswerescreened as described above. Plasmid DNA was prepared in SF8 Escherichia coli(4) and purifiedin cesium chlo-ride-ethidium bromidegradients (1, 20). HaeIII and

HpaII fragments were prepared, labeled, and

se-quencedasdescribedpreviously(9). All cloned

mate-rialswerepreparedand used in accordancewith Na-tionalInstitutes of HealthandUniversityof Califor-nia, SanDiego,Biosafety Committee guidelines.

Nucleotide sequencing. Details of thesequence

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872 DEININGER, LAPORTE, AND FRIEDMANN determination of wild-type polyoma have been re-portedpreviously (2, 8, 9). The nucleotide sequences of themutant DNAsweredeterminedbythe method of Maxamand Gilbert(17)andincluded theG-specific

methylene blue reaction for G(6)andthepotassium

permanganatereaction for T(C.Houck, personal com-munication). Details offragment preparation,labeling,

andpurification, gel electrophoresisand

autoradiog-raphy,informationstorage, and sequencecomparisons have beenpublishedpreviously from this laboratory (7-9).

RESULTS

Marker rescue experiments from other

labo-ratories havepreviously mapped the mutations

ofseveralgroup mutantsof

polyoma

(18). ts-a waslocalizedto a

fragment

between theHindIII siteatnucleotideposition1671 andanHhaI site atposition 2331, whereas mutants ts-25, ts-48,

and ts-52 have all been localized to the HhaI

fragment spanning the

region

from positions

2331 to 2913 bythenumbering convention used

by Friedmannet al. (9). Thesequencechanges

inthemutantsexaminedinthisstudyare

sum-marizedinTable1.Eachof themutantsshowed

onlyasingle mutation within the markerrescue fragment,indicating that the mutations

given

in

Table 1 are responsible for the ts

phenotype.

Mutant ts-48is, in

addition,

knowntobemissing theHpaIIcleavage siteatthejunction ofHpaII fragments 7 and 8. Wehave sequenced across

this site from theMboI siteatposition1207and havefoundatransition from the

wild-type

Cto a T atposition 1228(W. Eckhart, S. Delbruck,

P. Deininger, T. Friedmann, and T. Hunter, Virology, in press).

Figure1shows the amino acid distributionof the polyoma largeT antigen and the positions

of themutations determinedinthisstudy. The

positionsatwhichallthreepapovavirus

nucleo-TABLE 1. Nucleotide and amino acid sequence changes in polyoma ts-a mutants

Mutant Nucleo-

Nucleo-

Amino acidchangeand tide no. chne position

change

ts-25 2883 G- T Gly-.Cys(largeT antigen residue 778)

ts-48 2341 G- T Ser- Illeu(largeT

antigen residue

597)

1228 C-T Thre- Ileu (large T

antigen residue 597)

ts-52 2883 G A Gly-+Ser(large T

antigen residue 778)

tide sequences are identicalinthelargeT

anti-genareindicated at the top of Fig. 1.

DISCUSSION

The ts-amutantscharacterized in thisstudy

were generatedby nitrous acid mutagenesis of

the polyoma genome, making it possible, and

evenlikely, that some of these mutants contain

multiple alterations in large T antigen and other

viralproteins, as in the case of ts-48, in which

wehave found one alterationaffectingonly large

Tantigen, oneaffectingboth thelargeTantigen

and the medium T antigen (see below), and

anotheraltering the VP2,3 terminator (Eckhart

etal., in press). The use of the marker rescue

data and analysis ofwild-type revertants makes

it possible to determine the region containing

the alterations responsible for the phenotypic

changes brought about by an altered large T

antigen (18). Inthe cases of allthree mutants,

there is only a single-base-pair change within

the mapped region, indicating that, for these

mutants, it is asingle amino acid change that

affects the tsphenotype and that other possible

alterations outside the marker rescue region

have little or noeffect onT-antigenfunction.

The mutation in ts-48 that confers the ts-a

ptenotype

is a transversion from aG to a T at

position 2341, resulting in a substitution of an

isoleucine residue for the normally occurring

serine residue inlargeTantigen (Table 1).This

mutation is inoneof themosthighlyconserved

regions of the papovavirus large T antigens, although the wild-type polyoma serineresidue

itself has not been conserved in the two other

papovaviruses (Fig. 1). The serine residue is

locatedamid anumber ofhydrophobicresidues,

mostly leucine and alanine,in a general region

that also contains a number ofthe positively

charged residues lysine and arginine (Fig. 1). Themoreshort-rangeenvironment, however,in

the immediatevicinity of thets-48mutationis

devoid ofchargedresiduesover a stretchof12

amino acids. Thus, the change ofaserineto an

isoleucinemayenrichthisalready hydrophobic region.

The ts-52mutation responsible for the altered

phenotype is a G-to-A transition at position

2883, very near the portion of the sequence

encoding the C-terminus of the T antigen at

position 2919. This region in polyoma virus

shows relatively poor conservation with the

other papovaviruses, but the alteration itself

doesrepresent thechangeof aconserved glycine

(Fig. 1)toserine. Mutant ts-25 contains a G -.

Ttransversion at the same position as the ts-52

mutation, resultingin achange from glycine to

cysteinein themutant protein. These alterations

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POLYOMA ts-a MUTANTS 873

tS - 48

tSi

tS - 48 tS- 52

I

111imin i I miiii0 NI mI IsI I

11llEhil I--1 1 fI * I II* I E"l 1 1111111 I1I

HIS II 11n I

LYS 1111 I III

ARG GLU ASP GLN ASN GLY SER THRE PRO ALA VAL ILEU LEU MET TYR 1I

11111 1i I11 1

II 1111 1 11 11111 1111

liil l IIlIl l

I 1 1111 | 11 |I1

lI l il l l l l

1111 1 11 I1

PHE III

TRP I 11

CYS

100

11

I I

II

I mII iiiii

I I1 lI 11 II III 11111 I II liii I III

I 111 I IIII

III I 1111 III11 I I I

l111i 1111 11 11

11 II

1III

I 11 11I I

Hl1 I II 11

III1

mII

1iII I 11 11I II I 11 III I11 III' II I I I

IrI

11111 11 III II

11 I

II II 11

I lMIi 11

II I I

III I 111111

II

I IIII1 1111 I I

lI III I I

I I I lI I

II I

I Hll1 III I III II Il

I I II III I I

11 I 11111111i 11 11111 111111 1 11111

II I II I I 11 11 II I I

11 III 11I

11 I l11111 I I I I I 11 III I I III

111 I I

I1I

II III I1l I

200 300 400 500 600 700 800

AMINO ACIDNUMRFR

FIG. 1. Amino acid distributionofthepolyoma largeTantigen. Thearrowsindicate theposition ofthe

mutations ints-25, ts-48,andts-52. Thepositionsthatareconserved inallthreepapovavirus largeTantigens

areindicatedatthe top. Thelargegaprepresents theuniqueportion ofthepolyoma large Tantigen. The

asterisksindicate thepositions ofthesequencechangesinfourSV40tsAmutants(23),andthedashedarrow

indicates thets-apolyomamutantcharacterizedbyThomas etal.(25).Thets-25sequenced byThomas etal.

is mutatedatthesamesiteasthets-25sequenceinourstudy.

occurin therelatively highlynegatively charged

C-terminus of thelarge T antigen and place a

serineresidue withinanegatively charged

clus-ter. The C-terminus of the wild-type large T

antigen contains eight negatively charged

resi-dues andonlyonepositivelycharged residue.

We have detected in ts-48asecondearly

re-gion point mutation outside the markerrescue

region, a C-to-T transition at position 1228

whichcausesachange from threonineto

isoleu-cine in large T antigen atamino acid position

219(Fig. 1), andachangefromprolinetoserine

inmedium Tantigen.This mutation lieswithin

adomainofthepolyoma largeTantigenthatis

unusual in several respects. Most notable are

theverymarkedclusteringofglycineandserine

residues, the large numberofprolineand

thre-onine residues, and the deficiency of charged

residues. This region contains aportionof the

coding regionfor the polyomamedium T

anti-gen,and since there is noequivalent regionin

theBK virusorSV40genomes,itcanbe

consid-ered aninsert, relative to those genomes. The

markerrescue results indicate that neither the

changeinmediumTantigennorthechangein

thelarge T antigen caused by the mutation at

position 1228 isresponsible for the ts-a

pheno-type.However,it ispossible that the medium T

antigen, notrequired for lytic infection of

per-missive cells,or evenlarge T antigen functions

arealtered ina mannernotdetected bythe lytic

infectionassayused for markerrescue.

Theregion of the polyoma medium T antigen

affected by the ts-48 mutationatposition 1228

is within the unusual glutamic acid-, aspartic

acid-, and proline-rich region of the molecule

that characterizes the C-terminal half of medium

T antigen. The mutation also lies within the

deletedregion of d11013 and d123 andnearthe

deletions ofd11014 anddll015, a series of

mu-tantsthat generally reduce the transformation

efficiency or the transformation phenotype in

rat cells (10, 14). We do not know whether

aspectsof the defectincelltransfornationof

ts-48mayberelatedtotheaminoacidsubstitutions

in medium Tantigen.

Analysis of the conserved regionsofthelarge

T antigens of the three papovaviruses reveals

further common andpossibly important

struc-tural features. In the 79 amino acid residue

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874 DEININGER, LAPORTE, AND FRIEDMANN

region of thelargeTantigenN-terminaltothe

proximal large Tsplice, none of the 13 amino acid differences between BK virus and SV40

occur atpositions thatareconserved inpolyoma

virus and SV40. The latter conserved residues

thereforeseem toberelatively important. Sim-ilarly,throughouttherestof the

large

Tantigen molecules, most of the amino acid differences

between SV40 and BK virus alsooccurat

posi-tionsnotconservedin

polyoma

virus and

SV40,

indicatingthat the conserved sitesare

important

forproteinfunction.Of the22aminoacid

differ-ences between BK virus and SV40 atresidues

conserved in polyoma virus and

SV40,

14 are functionally conservative, i.e., Lys -. Arg,

Asp-*Glu, hydrophobic-*

hydrophobic.

These

findings suggest that most of the amino acid

residues of thelargeTantigens

play

important and similar structure-function roles and are thereforeconserved.Therearetwosmall

regions

that may beexceptions,onejust N-terminalto

the ts-48 mutation at nucleotide 1228, where

several ofthe

polyoma-SV40

conserved residues

arealteredin BKvirus,and the otheratthe C-terminal tailin thevicinityof the ts-52mutation.

Thomas and hercolleagueshavedetermined

thenucleotide sequencealterations

responsible

for thets

phenotype

inthe

prototypical

group A

mutant ts-aand ints-25

(25).

Theirts-25shows

a change identical to that described

here,

whereasts-acontainsa

change

at

position

2193, correspondingtoamino acid547inFig. 1.

The mutations in four tsA mutants ofSV40

havebeen identified

by

Seifetal. (23),and the

positions of those mutations are indicated by asterisks in

Fig.

1. All four fall in part ofthe

sequence near that ofpolyoma mutants ts-48

and ts-a, as identified

by

Thomas et al. (25),

suggesting particular importance for that por-tionofthemolecule.It isof interesttonotethat, whereas the mutationsin ts-48 andts-25 cause

changes to more hydrophobic amino acid resi-dues, the mutation ints-52results in a change fromglycinetoserine,tworesidues withlittleor nodifference in theirhydrophobicity(15).

ACKNOWLEDGMENTS

WethankWilliamFolk forallowing us to see his results beforepublication and Walter Eckhart, Tony Hunter, and Russell Doolittle for their helpful discussions. We also thank H.U.Bernardforthets-52whole-genome clone, Abby Esty for technicalassistance,andDorothyMillerforadministrative help.

Thiswork was supported by Public Health Service grant CA24288 from theNational Cancer Institute, and P.L.D. was

supported byPublic HealthService training grant GM07199 from the National InstitutesofHealth.

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