Reovirus type 3 genome segment S4: nucleotide sequence of the gene encoding a major virion surface protein.

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0022-538X/84/120984-04$02.00/0

Copyright

©

1984, American Society for

Microbiology

Reovirus Type 3

Genome Segment S4: Nucleotide Sequence of the

Gene

Encoding

a

Major Virion Surface Protein

MICHAEL GIANTINI,* LAURIE S. SELIGER, YASUHIRO FURUICHI, ANDAARON J. SHATKIN

Roche Institute

of

Molecular

Biology,

Roche

Research Center, Nutley, New Jersey 07110

Received 18July 1984/Accepted 2 September 1984

A

full-length cDNA

copy

of reovirus double-stranded RNA

genome segment

S4

which codes

for

a

major

virion structural

polypeptide, r3, has been completely

sequenced.

The 1,196-nucleotide cDNA

containsasingle

longopenreading

frame

in

the plus strand extending

1,095

nucleotides

from

the

5'-proximal A-T-G

toasingle

stop

codon. This

corresponds

to

translation of

92%

of the

S4

gene.

The inferred

a3

polypeptide

is

hydrophilic

and consists of

365 amino

acids, totalling 41,164 daltons.

Human reovirus

type

3 is the

prototype

of animal and

plant viruses that contain segmented, double-stranded RNA

genomes

(12). One of the

ten

reovirus

genomesegments,

S4,

codes for viral

polypeptide c3

(21, 22), which is the

most

abundant

protein in virions and

a

major constituent of the

outer

capsid shell (31). An important structural feature of

reovirions is the

presence

of

a

second, inner capsid. It

consists of several

polypeptides,

including RNA polymerase

and

capping

enzymes,

that enclose the double-stranded

RNAs

to

form

a

stable, protease-resistant

core

(7, 30). In

contrast to

the inner

capsid, reovirus

outer

shell

polypep-tides, notably cr3,

are

highly susceptible

to

chymotrypsin

digestion (11, 29). Although the polymerase and capping

enzymesarepresent

in

intact virions in

an

active

configura-mRNA

translation

at

late times after infection

(18). Another

3

among

the reovirus

polypeptides is its double-stranded

RNA

binding activity, suggestive of

a

role in virus

morpho-genesis (9). The S4

gene

also influences initiation of reovirus

persistent infections in tissue culture cells (1). To gain

insight into the multifunctional role of cr3 in the reovirus

replicative cycle and

to

investigate the basis for its

chymo-trypsin sensitivity,

we

have deduced the amino

acid

se-quence

of

polypeptide cr3 by determining the complete

nucleotide

sequence

of

genomesegment

S4.

A

cDNA

copy

of the S4 double-stranded RNA

was

cloned

by insertion into the PstI site of pBR322 and transfection of

Escherichia coli RR1

cells essentially

as

described

previ-ously (10). Plasmid DNA

was

amplified, purified, digested

0 100 200 300

400

500 600 700 800 900 1000 1100 1196 NUCLEOTIDE

a I I I a a a a a I I I a a a I I a a I a I I I i

0-

o---0

+ STRAND

-STRAND

II I I

~~~~~~~~~~~~~~~~~~~RESTRICTION

I

lll l l l l

~~~~~~~~~~~~SITE

PTt Ta MH F MI Hf X H MbTa Hf P

FIG. 1. Strategy for sequencingreovirusgenomesegment S4 cDNA. Closed circlesonthe left andrightof restrictionfragments represent the 32P-labeled 5' and 3'ends,respectively,thatwereused forsequencingtheplusstrand(oppositeorientation for the open circles and minus strand). Restriction sites usedwere: P, PstI; Tt, TthIIItype I;Ta, TaqI; M, MspI; H, HincII; F, FokI; Ml,

M1lu;

Hf,Hinfl; X, XhoI;and Mb, MboI. In additiontoendlabelingwithpolynucleotide kinase andby repair synthesis (19),PstIendswerealso3' labeledbyincubation with [ot-32P]ddATP and terminal transferase (32).

tion

(33),

only after proteolytic removal of c3 does the

core-associated RNA

polymerase synthesize full-length viral

mRNAs.

Apparently the

outer

shell

polypeptides impose

structural constraints that

prevent

elongation of

nascent

transcripts by the virion-associated

RNA

polymerase (3).

In

addition

to

its

role

as a

structural

polypeptide, cr3

inhibits host protein synthesis in

infected

mouseL

cells (27)

and

may

be

involved in

switching from cellular

to

viral

*Corresponding author.

with PstI, and

analyzed by

agarose

gel

electrophoresis

to obtain the

full-length

S4 insert for DNA

sequencing.

Restric-tion

fragments

were 32P-labeled at the 5' and

3'

ends

by

kinase and

repair reactions, respectively (19). Both strands

were

sequenced in their

entirety by using the

strategy detailed in

Fig.

1.

The

plus (mRNA sense)

strand and the inferred amino acid sequence of

polypeptide

cr3 are shown in

Fig.

2. The

DNA sequence agrees

completely

with the 5'-terminal 54 residues determined

previously

for reovirus s4 mRNA

(28)

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NOTES 985

10 20 30 47 62 77 92

GCTATTTTTG CCTCTTCCCA GACGTTGTCG CA ATG GAG GTG TGC TTG CCC AAC GGT CAT CAG GTC GTG GAC TTG ATT MC MC GCT TTT GMGGT CGT GTA TCA MET Glu ValCys Leu Pro Asn Gly His Gln ValVa1 Asp Lou Ile AsnAsn Ala PheGlu Gly Arg Vas Sor

107 122 137 152 167 182 197

ATC TAC AGC GCG CM GAG GGA TGG GAC AM ACA ATC TCA GCA CAG CCA GAT ATG ATG GTA TGT GGT GGC GCC GTC GTT TGC ATG CAT TGT CTA GGT GTT IleTyr Ser AlaGln Glu Gly Trp Asp Lys Thr IleSer Ala Gln Pro Asp MET MET ValCys Gly Gly Ala Va1Vas Cys MET His Cys Leu Gly Va1

212 227 242 257 272 287 302

GTT GGA TCT CTA CMCGC MGCTG MGCAT TTG CCTCAC CAT AGA TGT MT CMCAG ATC CGT CAT CAG GAT TAC GTC GATGTA CAG TTC GCAGAC CGT Vsi Gly Ser Leu Gln Arg Lys Lou Lys His Lou Pro His His Arg Cys Asn Gln Gln IleArg His Gln Asp Tyr Vsi AspVal Gln PheAisAsp Arg

317 332 347 362 377 392

GTT ACT GCT CAC TGG MGCGGGGT ATG CTG TCC TTC GTT GCG CAG ATG CAC GAG ATGATG MT GAC GTG TCG CCA GAT GAC CTG GAT CGT GTG CGT ACT

Vsl Thr Ala His Trp Lys Arg Gly MET Lou Ser Phe ValAlaGln MET His Glu METMET Asn Asp Vsi Ser Pro Asp Asp Lou Asp Arg Vsi Arg Thr

407 422 437 452 467 482 497

GAG GGA GGT TCA CTA GTG GAG CTG MC CGGCTT CAG GTT GAC CCA MT TCA ATG TTT AGA TCA ATA CAC TCA AGT TGG ACA GAT CCT TTG CAGGTGGTG

Glu Gly Gly Ser Lou Vas Glu Lou Asn Arg Lou Gln Vsa Asp Pro Asn Sor MET PheArg Ser IleHis Ser Ser Trp Thr Asp Pro Leu Gln Vsl Vsi

512 527 542 557 572 587

GAC GAC CTT GAC ACT MG CTG GAT CAG TAC TGGACA GCC TTA AAC CTG ATGATC GAC TCA TCC GAC TTG ATA CCC MC TTT ATG ATG AGA GAC CCATCA

Asp Asp Leu Asp Thr Lys Leu Asp Gln Tyr Trp Thr slaLeu Asn Leu MET IleAsp Ser Ser Asp Lou IlePro Asn Ph. NET MET Arg Asp Pro Ser

602 617 632 647 662 677 692

CAC GCG TTC MAT GOT GTGAM CTG GAG GGA GAT GCT CGT CAAACC CMTTC TCC AGGACT TTT GAT TCGAGA TCG AGTTTGGMTGG GGT GTG ATG GTT

His Ala Phe Asn Gly Val Lys Leu Glu Gly AspAlaArg Gln Thr Gin Phe Ser Arg Thr PheAsp Ser Arg Ser Sor Leu Glu Trp Gly Vsl MET Val

707 722 737 752 767 782 797

TAT GAT TAC TCT GAG CTG GAG CAT GAT CCA TCG MG GGC CGT GCT TAC AGA MG GOA TTG GTG ACG CCA GCT CGA GAT TTC GGT CAC TTT GGATTA TCC

Tyr Asp Tyr Ser Glu Leu Glu His Asp Pro Ser Lys Gly Arg Ala Tyr Arg Lys Glu Leu Vas Thr Pro Ais Arg Asp Phe Gly His Phe Gly Leu Ser

812 827 842 857 872 887

CAT TAT TCT AGGGCG ACT ACC CCA ATC CTT GGA MGATG CCG GCC GTA TTC TCA GGA ATGTTG ACT GGG MCTGT AM ATGTATCCA TTC ATT MA GGA His Tyr Ser Arg Ais Thr Thr Pro IleLou Gly Lys MET Pro AlaVsl Phe Ser Gly MET Lou Thr Gly Asn Cys Lys NET Tyr Pro PheIle Lys Gly

902 917 932 947 962 977 992

ACG GCT MG CTG MG ACA GTG CGC MG CTA GTG GAG GCA GTC MT CAT GCT TGG GGT GTC GAG MG ATT AGA TAT GCT CTT GGG CCA GGT GGC ATG ACG

Thr Msa Lys Leu Lys Thr Vas Arg Lys Leu Vsl Glu Ais ValAsn His AlaTrp Gly Vas Glu Lys IleArg Tyr AlsLou Gly Pro Gly Gly NET Thr

1007 1022 1037 1052 1067 1082

GGA TGG TAC MT AGG ACT ATG CM CAG GCC CCC ATT GTG CTA ACT CCT GCT GCT CTC ACAATGTTC CCA GAT ACC ATC MGTTT GGGGAT TTG MT TAT Gly Trp Tyr Asn Arg Thr NET Gin Gln Ala Pro Ile Val Lou Thr Pro Ais Ais Lou Thr NET Phe Pro Asp Thr Ile Lys Phe Gly Asp Lou Asn Tyr

1097 1112 1127 1140 1150 1160 1170 1180 1190 1196

CCA GTG ATG ATT GGC GAT CCG ATG ATT CTT GGC TMACACCCCCAT CTTCACAGCG CCGGGCTTGA CCMCCTGGT GTGACGTGGG ACAGGCTTCATTCATC Pro Val NET 11eGly Asp Pro MET Ile Leu Gly

FIG. 2. Nucleotide sequence of genome segment S4

coding

strandandinferred amino acid sequence of

polypeptide

u3.

and also

contains

the

3'-terminal

pentanucleotide

common to

reovirus

plus strands

(6)-properties

expected of

the

full-length

clone. The S4 gene

contains

no

obvious

TATA

initiation

box

or

A-A-U-A-A-A

cleavage signal,

a

finding

that

is

consistent with

end-to-end

transcription

without

polyadenylation by

a

reovirus-specific

RNA

polymerase.

Two

points concerning

the

nucleotide sequence data

are

noteworthy.

First,

the

TaqI

recognition

sequence

TCGA

at

positions

553

to

556 apparently

is

modified

since

mapping

and

radiolabeling experiments

showed

no

TaqI cleavage

at

this

site.

A

reasonable

explanation

for this enzyme

resist-ance

is

N6-methylation

of

an

adenine in the

TaqI

site.

Probably this

occurs at

nucleotide 553 in the

complementary

strand because the

TaqI

recognition

sequence

overlaps

a

GATC sequence

at

positions

551

to

554 which is

recognized

for adenine N6

methylation by

E.

coli dam

methylase (8).

Second,

the

sequencing

data

document the presence of four

EcoRII restriction sites

(CCAGG)

located

at

nucleotides

383,

652,

981 and 1165.

Except for

position 981,

the

internal

+

STRAND

G G/A C T/C

- STRAND

3 3 G G/A CT/C

,G

G

-'0

-T

A----.C~~~~~~~~~~~

.5.

5'5

FIG. 3.

Radioautograph indicating

cytosine methylation

at an

EcoRlI recognition

sequence in reovirus S4 cDNA. A

TaqI

site

(position 280)

in the

plus

strand andHincll site

(position

440)

in the minus strand were

5'-end

labeled.

Fragments

were cleaved

(19),

electrophoresed

in 7%

polyacrylamide sequencing gels,

and

auto-radiographed

without

intensifying

screen.Both sequencesread5'to 3' fromthebottom ofthe film.

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cytosines in each of

these EcoRII

sites

are not

observed

in

radioautographs

of the

relevant

sequencing

gels

but

are

replaced by

a

blank space, e.g., at position 384

in

the

plus

strand and

386 in the minus strand

(Fig. 3). In

contrast,

the

complementary G residues

present

in the

opposite strand

are

readily evident. Presumably methylation of

the

C residues

at

positions 384,

653,

and

1,166

(386, 655 and

1,168 in the

minus

strand)

prevents

chemical cleavage and results in

one-nucl-eotide

gaps

in the

sequencing gels (23).

Although

there

is

total agreement between the DNA and

mRNA

5'-terminal

sequences,

comparison of the

DNA

sequence

with S4 double-stranded

RNA sequences (2)

re-vealed

differences in both the

3'-

and

5'-terminal regions.

The

nucleotide differences in

the RNA

do

not

affect

the

longest translational reading frame,

but the

absence of

a

codon, AAC

at

positions

78 to

80,

results

in loss

of

one

of

a

pair

of asparagine residues

at

positions

78 to

83 (Fig.

2).

The

differences between the S4 cDNA

and

genomic

RNA

se-quences may

be

due to

variability

among

virus isolates.

However,

differences which

we

have

found with both

the

sequences

of Antczak

et

al.

(2) and

McCrae

(20) are

in

the

last

few

RNA

residues

sequenced and may

reflect

difficulties

in

interpreting bands in

the

upper

part

of autoradiographs.

Ribosome protection studies of reovirus

s4

viral

mRNA

have already indicated

that the

initiator

codon

for

a3 is the

5'-proximal A-U-G

(14).

This

triplet lies in

a

consensus

sequence,

i.e., Pur-X-X-A-U-G-G, considered favorable for

initiation of protein synthesis

(13). The DNA sequence

reported here reveals that

this initiator codon is followed

by

an

open

reading frame extending 1,095 nucleotides

to a

stop

codon

at

position 1,128 (Fig. 2). Examination of

all

other

phases in both the minus

and

plus strands revealed

no

additional

open

reading frames capable of

yielding

a

polypep-tide

greater

than

40 amino acids.

The

single

open

reading

frame found in the S4

genes contrasts

with the reovirus

Si

gene

which codes for the

crl

protein

but

also contains

a

second

out-of-phase, overlapping reading frame

(R.

Bassel-Duby,

A. Jayasuriya,

and B.

Fields, unpublished data).

Recent

studies demonstrate

that

both

frames in the

sl

mRNA are

translated

(5;

H. Ernst

and

A.

J. Shatkin,

unpub-lished

data).

The

S4

gene

is similar

to

the reovirus

S3 RNA segment

(25) in that

92%

of each is

apparently translated. However,

the

longest open reading frame reported for

segment

S2

corresponds

to

only

75% of the total

gene (4). Assuming

that

translation of s4 mRNA starts

from

nucleotide

33,

polypep-tide a3

consists of 365 amino acids including

40 acidic

and 37

basic

residues,

14 histidines,

and 21

methionines-an

unu-sually high number. The total molecular

weight of

a3 is

41,164, close

to the

value

of

41,500 estimated from

elec-trophoretic migration

in

sodium

dodecyl

sulfate-polyacrylam-ide

gels

(26).

The sequence

-(Val,Val,Leu)-COOH reported

for

a3

purified from

virions

(24)

is not present in

the

inferred

amino acid

sequence

(Fig.

2).

Chymotrypsin

treatment of reovirions results in the

rapid

hydrolysis of

u3, and two

small

polypeptides of

14,000

and

11,500 daltons

are

released after

only

brief

exposure to

the

protease (11).

Fragments of this size

conform

to

the location

of some of

the

chymotryptic

sites in

u3, but

the inferred

polypeptide

sequence

includes

a

total of

14 phenylalanine,

11 tyrosine,

and 7

tryptophan residues in

addition

to

many

other

potential cleavage

sites.

Surprisingly, these potential

chymotryptic sites

are

distributed

throughout

a3,

and many

of

the aromatic residues

are

located in

hydrophilic regions

estimated by

the

procedure

of Kyte

and Doolittle

(16)

(Fig.

4).

Consequently,

the

sequence does not allow

prediction of

primary chymotryptic cleavage site(s)

in the

virion

outer

shell a3

polypeptides.

Other features of a3 include six

cysteine residues:

five

near

the

N

terminus

and one near the

carboxyl end.

There

is

a

single potential

glycosylation site, Asn-Arg-Thr

at

residues

325 to

327,

which

on

the

basis of incorporation of

radiola-beled amino

sugars

is used

ineffectively (17)

or not at

all (15).

A

hydrophilic

region between amino acids

63 and

72 con-tains

seven

positively charged residues

counting histidine

and

may be

to

the RNA

binding capacity of a3.

Finally,

comparison of

the

hydropathicity profiles of a3

and

aNS, another RNA-binding

protein (9), shows

two

regions of

strong

similarity in

terms

of

hydrophobic-hydro-philic domains:

a

region from amino acid residues

48 to 88

and

another

from

residue 318

to

the

carboxyl terminus.

In

addition, both of these reovirus polypeptides contain

a

high

proportion

(6%)

of

methionines.

It may

be

possible

to test

whether

these

structural similarities

are

related to RNA

binding

capacity by analyzing site-specific

mutants

gener-ated

by

in

vitro

manipulation of

cDNA

clones.

FIG. 4. Hydropathicity profile of polypeptidecr3 generated bycomputerbytheprocedure of KyteandDoolittle (16). Thelocations of aromatic amino acids correspondingtopotentialchymotrypsin cleavage sitesareindicated

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NOTES 987 Wethank A. R. Bellamy and M.Richardson for technical advice

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ERRATUM

Reovirus Type 3 Genome Segment S4: Nucleotide Sequence of the Gene

Encoding

a

Major

Virion

Surface Protein

MICHAEL

GIANTINI, LAURIE S. SELIGER, YASUHIRO FURUICHI, AND AARON J. SHATKIN

Roche InstituteofMolecularBiology,RocheReseacrch Center,Nutley, New Jersey 07110