Comparisons of the peptide maps of Kunjin virus proteins smaller than the envelope protein.

(1)

Vol.24, No. 2 Printed in U.S.A.

Comparisons

of the

Peptide

Maps

of

Kunjin

Virus Proteins

Smaller than the

Envelope

Protein

PETER J. WRIGHT* ANDE. G. WESTAWAY

Departmentof Microbiology, Monash University MedicalSchool, Prahran,. Victoria, Australia 3181 Received forpublication10May1977

Weanalyzedthemaps of[35S]methionine-labeled tryptic peptidesof theKunjin

virus-specified proteins NV2Y2, NV2, V2, NV1½/2, NV1, andV1. Thepeptidesof

NV1½2 are identical to those ofV2, except for one peptide contained onlyin the

latter. The maps of each of the other proteins are unique, and, consequently,

there isnoevidence ofanyoneprotein beingderived from anotherbyproteolytic

cleavage.Thetryptic peptidemaps of the abovepolypeptideswerealsocompared

withthose ofthelarger Kunjin proteins, NV5, NV4, and V3. The resolution of

the peptides of NV2½2 and NV1 is adequate to exclude any relationships with

NV5 and NV4, but a possible relationship with V3 remains, althoughitseems

unlikelyinthelightof other evidence. Since thepeptidemap of V1 iscomprised

ofonlytwo methionine-containing peptidessimilar inmobilitiesto twopeptides

found in the maps ofNV5, NV4, andV3,aprecursor, if any, of V1 hasnotbeen

positivelyidentified. Thepeptidesof NV2 and V2 (NV1½/2) are notcontained in

digestsofNV5, NV4,orV3,andtherefore, like thelatter, NV2 and V2 (NV11/2)

areindependentproductsof translation from thepositive-strandgenome.

Flaviviruses specify the translation of three

structuralproteins, V3, V2,andV1, andatleast

six nonstructural proteins, NV5, NV4,

NV2Y4,

NV2,

NVlW/!,

andNV1 (7, 12, 13). V3 and NV2

are both

glycoproteins

(2, 9). In the preceding

paper we presented maps of tryptic peptides

derived from proteins prepared from cells

in-fected withKunjin virus, confirmingthat

poly-peptides NV5, NV4, and V3 are unique to

in-fected cells butdemonstrating thatNV3 is

prob-ably host coded (17). No evidence that NV5,

NV4,or V3is acleavage productwas obtained.

The other nonstructural proteins, NV2Y,, NV2,

NVlW>,

andNV1,are comparatively small

poly-peptides of molecular weights less than 22,000

(7,13).NV2 and NV1 aredelayedin appearance

in short pulse-chase experiments, and the size

of

NV1W!

is almost identicalto that of V2, the

core protein of virions (16a). The smallest

pro-tein observed in purified virions,V1, is not

de-tected in infected cells, and ithas been

postu-lated that it results from cleavage of NV2 (8).

With other positive-strand RNA viruses,

smallproteinseither arecleavedfrom a

polypro-teinprecursorduringor soonafter translation,

or are cleaved from the immediate precursors

of structuralproteins duringmaturationof

viri-ons (reviewed in reference 3). Some molecules

of precursorproteins escapeearly cleavage and

accumulate as apparently stable products. Hence, in this report we examine the

relation-ships between the smaller

virus-specified

pro-teins,

NV2Y2,

NV2, V2,

NV1W2,

NV1, and

V1,

as

wellasthepossibility thattheyareproductsof

proteolytic cleavages of NV5, NV4, andV3. To distinguish host from viral

tryptic peptides

in

preparations ofproteins derived from infected

cells,

double-labeled

tryptic peptide

maps were

analyzed aspreviouslydescribed(17).

MATERILS

AND METHODS

Details of all procedures used are given in the precedingpaper(17).

Cells and virus. Kunjin virus-infected Vero cells

werelabeled with[3S]methionine for7h, commencing

23hafter infection. Cells that had been mock infected

were labeled with [3H]methionine under the same

conditions. Kunjin virions and SHA (hemagglutinat-ingparticle of65 to80S) labeled with[3S]methionine

werepurified from infected-cell culture fluids.

Separationofpolypeptides.Radioactive cellular

and viralsamplesweredisruptedwith sodiumdodecyl sulfate-dithiothreitol and heated before

electropho-resis inpolyacrylamide gelswithacontinuous(16) or

discontinuous buffersystem (5). Polypeptides of 35S-labeled infected cellsweresubjectedto coelectropho-resis with those of 3H-labeled uninfected cells; the ratio of 5Scountsperminuteto3Hcountsperminute loaded on gels was 2:1. Proteins ofpurified virions

andof SHAweresubjectedtoelectrophoresiswithout

the additionof[3H]methionine-labeled material. Tryptic peptides. Proteinswere eluted from the

preparative gels and treated with trypsin (18), and

the peptides were separated by electrophoresis and 662

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

PEPTIDES OF KUNJIN PROTEINS. II. 663 thin-layer chromatography on cellulose-coated plates.

Peptides derived from cell-associated viral proteins were eluted from the cellulose, and the ratio of 'S counts perminute to3Hcounts per minute was mea-sured. The criterion for determining whether a peptide wasviral or hostcoded was the same as that described in the preceding paper (17); i.e., peptides were desig-nated as viral if the above ratio was greater than twice the initial ratio in the digest before separation. Throughout the remainder of this paper, when refer-ringto analyzed digests of cell-associated viral pro-teins,we usetheterm"viral peptide" for the peptides satisfying the above ratio requirement. We shall not qualify "peptide" when discussing all of the peptides found in apreparation.

Peptide maps contained DL-alanine and L-valine as markers. To better compare two proteins, a mixture containing the tryptic digests of both polypeptides wasanalyzed. In some of the figures, the viral peptides of the smaller protein in the mixture (the peptides that could beidentified) are marked with arrows or

areshownseparatelyinlinediagrams.

c

V3

_l

6.4

V2

M

d 0 f

-NV5

-NV4

-V3

-NVt2i

i0

-NV 2

0

-NV

II

-NVI

VI ft

RESULTS

Thepeptidemapsof [35S]methionine-labeled, virus-specified proteins thatarefoundinKunjin

virus-infected cells and are smaller than V3

(namely, NV2Yz, NV2,

NV1W2,

and NV1; Fig. 1) are presented in Fig. 2 and 3. Total and net

viral peptide maps are shown for NV2Y2 (Fig.

2A and B), NV2

(Fig.

2C and D),

NV1W2

(Fig. 3C and D), and NV1 (Fig. 3E and F). The molecular weight of each polypeptide and the number of viral

peptides

resolved are listed in Table 1. The number of

[3S]methionine-con-taining viral

peptides

in the two smallest

pro-teins, NV1% and NV1,suggeststhat these

poly-peptides

contain a

higher proportion

(two- to

threefold) ofmethionineresidues than doNV2

and

NV2Y2 (Table

1), and V3, NV4, and NV5

(17).Shapiroetal. (10) have also reported that

NV1isrelatively richer in methionine than other virus-specified proteins of Japanese encephalitis virus.

Thepeptidemapsofthe three structural

pro-teins V3 (51,300

daltons),

V2 (13,500

daltons),

and V1

(8,500

daltons)

are

displayed

in

Fig. 2F,

3A, and2E,respectively. The virion proteins for

peptide mapping

were prepared from purified

Kunjin virions. V3 and V1 are also found in

SHA (7,

12).

In the

SHA;

the

peptidemap ofV1 ofSHA (notshown) is also

identicaltothat ofV1of virions (Fig. 2E).

The lack of similarities in the amino acid

sequencesofNV5,NV4, andV3wasclearfrom

examinationoftheir

peptide

maps

(17).

Weshall

now first compare the

tryptic digests

of the

[image:2.500.255.443.66.276.2]

smaller proteins,

NV2A2,

NV2, V2,

NV1W2, NV1,

FIG. 1. Analysesoff5Slmethionine-labeled

poly-peptidesin 8%polyacrylamidegels with continuous

buffersystem. (a)Kunjin virus. (b) Infectedcells. (c)

NVI, (d) NVJV,, (e)NV2, and (/) NV2Y4 aresamples of thepreparationsusedfor trypticpeptide mapping.

and V1, as indicated

by

the peptide maps of Fig.2 and 3,

and,

second, compare these

poly-peptides

with the

larger proteins,

V3, NV4, and

NV5.

Comparison

of

proteins

smallerthan V3.

Thenetviralmapof

NV2Y2

(Fig. 2B) is different from that of NV2 (Fig. 2D). The differencewas

confirmed

by analyzing

amixture of

NV2VY

and NV2 (Fig. 4A).

NV1W2

possesses at least five peptidesnotfound in

NV2¼2,

asshownby

com-parison of their maps (Fig. 3D and2B,

respec-tively) and from

analysis

of the

protein

mixture (Fig. 4C and D). The

peptides

ofNV1 (Fig. 3F) andV1 (Fig. 2E) were notfound in

NV2A2 (Fig.

2B). Thus, V1, NV1,

NVlW2,

and NV2 are not

products ofproteolytic cleavage of

NV242.

Thepeptides of V1 (Fig. 2E), NV1 (Fig. 3F),

and

NV1Y2

(Fig. 3D) are distinct from those of

NV2 (Fig. 2D). The

peptides

of V1

(Fig. 2E)

andNV1 (Fig. 3F) are notfound in the mapof

NV1W2

(Fig. 3D), and V1

(Fig. 2E)

isnotderived

from NV1 (Fig. 3F). Taken together, these

re-sults show that viral

proteins NV2Y/!,

NV2,

NV1W2,

NV1, and V1 each haveauniqueamino

acid sequence and noneis derived

by

cleavage

of alarger

polypeptide

inthegroup.

Comparison of NV1% with V2. The maps of

NV1W2

(Fig.

30)

and V2

(Fig. 3A)

arealmost

identical. The

only

obvious difference between

the twois the presenceofanadditional

peptide

in V2 (arrow,

Fig. 3A).

The

designation

of the

viral

polypeptide

of

approximately

13,500

dal-VOL. 24,1977

on November 10, 2019 by guest

http://jvi.asm.org/

(3)

664 WRIGHT AND WESTAWAY

A

NV2;2

4w

Qtep

C

NV2

0s

0>

E

1vIrion

Ob

0D

0

_

**

A

FIG. 2. Tryptic peptidemapsof ?S~methionine-labeled proteins. Electrophoresiswasfrom leftto right; chromatographywasfromtoptobottom. Theopencircles mark thepositions ofalanine and valinemarkers.

(A)NV2i4 prepared from infectedcells.(B)Netviralmapof NV2I4. (C)NV2prepared from infectedcells.(D)

Net viralmapofNV2.(E) V1and(F)V3prepared from purifiedvirions.

e

_C

B

NV2,

L

D

NV2

C/A.9.Xz

F

V3,VIj

J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:3.500.65.459.68.623.2]

(4)

B

V3eelI

o t

I

DO

_ V

e

a

w }

o .

S

t

[image:4.500.55.446.36.637.2]

*4 at

FIG. 3. Tryptic peptidemapsof

rSlmethionine-labeled

proteins. (A) V2prepared frompurifiedvirions. Arrow indicatesdifference between V2 and NV1½. (B) V3from infectedcells.Fordesignation ofhost and viralpeptides,seereference17. (C)NVJVpreparedfrom infectedcells. (D)NetviralmapofNV1½2. (E) NVI preparedfrominfectedcells. (F)Net viralmapof NVI.

665

A

V2yirion

"a

& #P ov

e

40

c

NV

I'

_NV12

0

Co

C) 0

0

0 5F9

0

E

NV1

0 F

NV1

0

CD

C

0

0 C

on November 10, 2019 by guest

http://jvi.asm.org/

(5)

[image:5.500.61.253.75.236.2]

TABLE 1. Numberof

P5S~methionine-labeled

tryptic peptides found in cell-associated viralpolypeptides

Viral poly- Initial Totalno.of No of net

peptide wt ratio td viral pep-pttpeptdes' tides"

NV2V, 21,000 2.4 25 7

NV2 19,000 2.9 11 4

NV1A4 13,500 1.1 17 11

NV1 10,300 4.2 15 6

aFromreference13.

bRatio of'S counts per minuteto

3H

countsper minute intrypticdigestimmediatelybeforeseparation ofpeptides.

cNumber ofpeptides for which the IS/3H ratio

wasdetermined.

dPeptides for which the 35S/3H ratiowas greater than twice the initial ratio.

tonsfound in infectedcellswaschangedto

NVW1Y

from V2 because this polypeptide migrates

slightly ahead ofV2 fromvirions when subjected

toelectrophoresisinsodiumdodecyl sulfate-con-taining polyacrylamide gels (13, 15, 16a). The maps ofFig. 3 also imply that V2 is a slightly largerproteinthanNV1'/2 and showclearlythat V2contains the aminoacidsequence ofNV1Y2.

A comparisonof Fig. 3A andC enables vali-dation of the ratio methodusedfor

discriminat-ing between host-coded and virus-coded

pep-tidesinmapsofpolypeptidesfrominfectedcells.

Inthiscase,onlytwoofthelikelygenuine viral

peptides in

NVW1X

areexcluded(oneimmediately above thetop markerand one inthetop

right-hand group ofthree), possibly because of the

presence of overlapping 3H-labeled host

pep-tides. This confirmsthe

findings

with V3:that

the exclusion technique based on the

35S/3H

ratio is conservative and does not mistakenly

include hostpeptidesin the netviralmap (17).

Comparisons with V3. Inspection of the

peptidemapsofV3(Fig.2F and3B) and

NV2Y,

(Fig. 2A and B) suggests that the peptides of

NV2½2 could be common with those of V3. Therefore, a mixture ofdigests of V3 from

in-fected cells and NV2/2was analyzed (Fig. 4E),

and,

although

the seven NV21/2 peptides were

tentatively identified (Fig. 4F), they were not

completely resolved from those of V3. The

ex-periment wasrepeatedseveraltimes, but inno case was resolution improved. However, it is

clear by analysis ofthe mixtures that the

pep-tides of NV2 (four offour; Fig. 4B) andNV11/2

(eightofeleven;Fig. 5A) are distinct from those ofV3(Fig. 2Fand3B).

The two major peptides ofV1 (Fig. 2E) and

somepeptidesof V3 (Fig. 2Fand3B) migrated

to similar positions in the maps. The pair of

peptidesin the topright of the NV1 map (Fig.

3F) is in a position correspondingto a heavily

labeled area of the V3 map (Fig.3B). The

pep-tide at the extreme left ofFig. 3Fcorresponds

to one found in V3 of infected cells (Fig. 3B).

The positions of the three otherNV1peptides

werecompared withpeptidesin similarpositions

in the V3 map (Fig.3B) byinspectingpatterns

of mixed NV4 and V3 from infected cells (17)

and mixed NV4 and NV1 (see Fig. 5F), thus

using NV4peptidesasmarkers. These

compar-isons demonstratedslightdifferences in

migra-tion between the three NV1peptidesand

possi-blecorrespondingV3peptides.Becauseatleast

threepeptides (ofthe six identified in Fig. 3F)

ofNV1 were resolved from those ofV3,we can

tentativelyconclude that NV1 isnotderivedby cleavage of V3. However, since

cleavage

of a

precursorproteinmay generateatleast two new

peptides in the product, and changes in the

glycosylationlevel(V3is aglycoprotein)of pep-tides may occur, altering their mobility during separation, ourresults havenotcompletely

ex-cludedaprecursor-product

relationship

between

V3of infectedcells and NV1.

Thus, NV2 and NV1Y4 are not derived by cleavage ofV3; NV1 andpossibly

NV2Y;4

seem

unlikely to be derived from V3, but the two

majorpeptides of V1 could be derived by

cleav-age of V3. This

possibility

is discussed more

fully below.

Comparison

with NV4. The peptide maps

ofan NV4 tryptic digest (Fig. 5B) mixed with digests of

NV2Y;,

NV2, NV1Y2, and NV1 are

displayed in Fig. 5C through

F,

respectively. Peptidesthat werefound inthe netviralmaps

of the smallest proteins and were resolved in

the mixtures are indicated by arrows. No

evi-dence was obtained that theseproteinsare

de-rived by cleavage of NV4.

Comparison

of the

map of V1 (Fig. 2E) with that of NV4(Fig. 5B)

does not exclude the

possibility

that V1 is

de-rivedfromNV4.

Comparison

with NV5. An

analysis

of a

mixture (Fig. 6B) ofNV5 (same preparationas

in Fig. 6A) and

NV2Y/,

(Fig. 2A andB) reveals

no

relationship

between these

polypeptides.

The

samplesof NV2(Fig.6C)andof NV1 andNVW1Y (Fig. 6D) used forcomparisons with NV5 were

prepared in 13% discontinuous gels and

con-tained apartlyalteredbackgroundofhost

poly-peptides compared with preparations from 8%

continuous gels. NV1 and NV1 migrate as a

single band in the discontinuous system (17).

Viral peptidesasidentified inFig. 2D, 3D, and

3F forNV2,

NVJA4,

andNV1, respectively, are

marked in Fig. 6C and D. In mixed analyses

with NV5, the peptides of NV2 were resolved

from those of NV5 (Fig. 6E) as were those of

J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

(6)

-*0

ak

6

io

i

_tr

_0

0 E V3+NV2I

9.

CF-1 a

.Ol

Is

Sb

r0

.0

0

00c

e

f

0 .2

7

0

o

0

0 O

C

FIG. 4. Tryptic peptidemapsofrS~methionine-labeledproteins. (A) Mixture ofNV2Y, (20,000 cpm)and

NV2(20,000cpm). Viralpeptides ofNV2(four of four, from Fig. 2D) identifiedin the mixturearemarked withanarrow. (B)MixtureofV3(20,000 cpm) from infectedcells andNV2(11,000 cpm). Viral peptides of NV2(four of four)aremarked. (C)MixtureofNV2Y4 (5,600 cpm) and NV1V,(3,200 cpm). (D) Viral peptides of

NVIP4 (5 of 11, from Fig. 3D) identifiedin themixturein (C). (E) Mixture ofV3(26,000 cpm) from infected

cells(samepreparation asinFig. 3B) andNV2-" (24,000 cpm). (F) Viralpeptides ofNVZ4 (seven ofseven,

from Fig. 2B) identifiedin themixture in(E).

667

B V3+NV2

S

A NV2+NV2!

2

C

NV212+NV1I

0

D

0

F

e6

a

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.500.65.459.59.601.2]

(7)

AS

V3

1~

B

V3tNV12 NV4

.~

~

~~0

_~~ a

* ~ t 0

C

D~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~D

NVN 24 NV4+NV2

._

E ~ F

NY4+NYI; NV4 +NYI

2~~~~ ~ ~ ~ ~

a

~1. 9

FIG. 5. Trypticpeptide maps of r3S~methionine-labeled proteins. (A) Mixture of V3 (20,000 cpm) from

infectedcells and NVZ½ (9/00) cpm). Viralpeptides of NV1½ (8 of 11,

fr-om

Fig. 3D) are identified with

arrows.(B)NV4fr-ominfectedcells. Fordesignation ofhost and viralpeptides,seereference17. (C)Mixture

ofNV4(8,300cpm)and NV2½(5,600cpm). Viralpeptides NV2½2 (seven ofseven,fromFig. 2B) aremarked.

(D) MixtureofNV4(12,000 cpm) andNV2 (11/KY)cpm). Viralpeptides ofNV2(four of four, from Fig. 2D) aremarked. (E)Mixture ofNV4(8,300cpm) andNV1½%(3,200 cpm). Viralpeptides ofNVJ½1 (9of11,

fr-om

Fig. 3D)aremarked. (F)MixtureofNV4(12,000cpm)# and NV1 (12,000cpm). Viralpeptides ofNV1 (fiveof

six,from Fig. 3F)aremarked.

668

on November 10, 2019 by guest

http://jvi.asm.org/

[image:7.500.71.442.42.597.2]

(8)

V,, .#

i.I.; (

i

9'.

.-D

co

I

D

NVI+NV11

Oe

0

...

a

9

_w-a

I

L

I

.-0do

l -P.

o

w* 040

0

F

NYS+NV1

+NVIj

40

0-.-t_.

p4-

0-I

4-0

[image:8.500.70.429.29.627.2]

v

FIG. 6. Trypticpeptidemapsof[fS~methionine-labeledproteins. (A)NV5from infected cells.For

desig-nationofhost andviralpeptides, seereference17. (B)Mixture of NV5 (17,000cpm)and NV2½ (15,000cpm).

Viralpeptides of NV2% (sixofseven,from Fig. 2B) are indicated. (C) NV2from infected cells (different preparation fromthat inFig.2C);A, viral peptides.(D) Mixture ofNVJ andNVIJ½;0,peptidesof NVJ; E.

peptides ofNV1½h. (E)MixtureofNV5(17,000cpm) and NV2(14,000 cpm);samepreparationasin (C); NV2

peptidesaremarked. (F)MixtureofNV5(17,000 cpm) and NVJ plusNV½1h (14,000cpm);samepreparation asin(D);peptidesmarkedasin(D).

669

S

A

NV5

B NV5+NV22₂

.

c

NV2

-4-g

S

E NYV5 NV2

C

*ra

d

V

010

on November 10, 2019 by guest

http://jvi.asm.org/

(9)

NV1 (Fig. 6F). Because at least threepeptides

of NV1W? were identified in a mixed run with

NV5 (Fig. 6F), we tentatively conclude that

there isnorelationshipbetween these two

pro-teins. The mapsofV1 (Fig. 2E) and NV5 (Fig.

6A) have peptides in similar positions at the

right of the top marker. Thus, NV5 isapossible

precursorof V1.

DISCUSSION

ThepeptidemapsofNV2i4, NV2,NV1W,,NV1,

and V1 demonstrate thatnoneofthese

polypep-tides was derived from another by proteolytic

cleavage. However,NV1 and NV2weredelayed

in appearance during pulse-chase experiments

(16a), and evidence has been obtained that

NV1i2 and NV2/2 are products of

post-transla-tional modification of newly synthesized

pro-teins in infectedcells synchronized by removal

ofahypertonicblock(14a). V1is found invirions

and SHA butnot in infected cells(7, 13).

There-fore,toobtain more informationonthe

forma-tion ofNV2Y2, NV2, NV1i/2, NV1, and V1, the

peptidemaps of these proteinswere compared

with thoseofthelarger virus-specified proteins,

NV5, NV4, and V3, eachofwhich hasaunique

peptidemapandsoisindependently coded (17).

The results of the mapping experiments

pre-sented in thispaper aresummarized inTable2.

The overall conclusion is that the amino acid

sequencesof theKunjin virus-specified proteins,

NV5, NV4, V3, NV2, and NV1i2, are unique.

These proteins, with a total molecularweight

of250,000 (13), account for 60% ofthe coding

potential of the Kunjin virus genome of 4.2 X

106 daltons (1). The peptides of NV21/2, NV1,

andV1, possibly representing afurther 10% of

the total coding capacity,were not adequately

resolvedfromthoseof V3 (for NV2Y2, NV1, and

V1) or NV5 and NV4 (for V1) to rigorously

excludeNV2/2, NV1, and V1asproducts of

post-translationalcleavage.

Since thepeptides of NV2i4, NV1, and V1 do

not overlap and arenot resolved from those of

V3, itmaybe thatNV2YI, NV1, and V1 are all

derived by cleavage of V3. However,in infected

cells treated with proteolysis inhibitors,the

for-mation of NV2½2 andNV1isnotinhibited(16a).

During a 1- or 2-min pulse of [35S]methionine

with cells reinitiatedintranslationafter reversal

of a hypertonic block, label was incorporated

intoV3, NV2½2, andNV1,withsimilaramounts

of label being incorporated into NV2'/2 and NV1

(14a). If NV2Y2 and NV1 are derived from one precursor,thepolypeptide arisingnearerthe

N-terminal end of thatprecursor should be more

heavily labeled. Further evidence on the

sug-gested cleavages from V3 comes from

experi-ments with puromycin. The presence of

puro-mycin (160

tg/ml)

inhibits the production of

NV212, NV1, and infectious virus (V1 is found onlyin virionsorSHA) despite ongoing

synthe-sis of V3 (10, 16a). After removal of the

puro-mycin, infectious virus is again produced but

does not incorporate V3 labeled with amino

acids during the inhibition period (16a). These

findingsmaybeinterpreted inatleasttwoways.

They are compatible with the suggestion that,

undernormalconditions,someV3moleculesare

cleaved to NV2Y2, NV1, and V1, but that the

cleavage is inhibited in the presence of

puro-mycin, perhaps becausearequired unstable

pro-tein is not made. Because of the pulse-label

incorporationinreinitiationexperiments and the

lack of effect ofproteolysis inhibitors,wefavor

the notion that NV2Y2 and NV1 synthesis is

unrelated to that of V3 and that puromycin

affects V3 incorporation into virions in some

othermanner.Toconvincingly demonstrate the

presenceorabsence ofanyrelationship between

V3 andNV21/2, NV1, orV1, it will benecessary

tofurtheranalyze the tryptic peptides of these

proteins. Peptides must be labeled with amino

acids other than methionine and separated by

differenttechniques.

Shapiroetal. (8) proposedthat NV2 is cleaved

to produce V1, based on the presence of NV2

in particles (T forms) released from LLC-MK2

cellsmaintained inmedium containing Tris and

infected withdengue-2orJapanese encephalitis

TABLE2. Uniqueness of Kunjinvirus-specifiedproteins smaller than V3

Possible Excludedasproducts

precursor NV2½/2 NV2 NV1½/2(V2) NV1 V1

NV5a Yes,6Bb Yes,6E Yes, 6F Yes, 6F Uncertain, 6A, 2E

NV4 Yes,5C Yes,5D Yes,5E Yes, 5F Uncertain, 5B, 2E

V3 Uncertain,'4E Yes,4B Yes, 5A Uncertain,d3B, 3E, 3F Uncertain, 3B, 2E NV5,NV4,and V3 are unrelated to each other (17).

Source(figure)ofdata.

Peptide mappingwasinconclusive.However,puromycin (160 ,ug/ml) inhibits the synthesis of NV5butnot that ofNV1 /2 (10, 16a).

dPeptide mappingwasinconclusive.Puromycin (160 ,ug/ml) inhibits the synthesis ofNV2'/2andNV1 but not that of V3

(10,16a).

J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

(10)

PEPTIDES OF KUNJIN PROTEINS. II. 671

virus,andonthepresence of NV2 in intracellular

virus (Iforms)obtained from chick cells infected

with Japaneseencephalitisvirus. In our

experi-ments, we compared the peptide maps of [3S]

methionine-labeled tryptic peptides of V1

de-rived fromKunjinvirusand fromSHA with the map of NV2 derived from infected cells. The two majorlabeled peptides of V1 are not

con-tained inNV2, indicating that V1 isnotderived from NV2 by proteolytic cleavage unless the

amino acidsequenceofV1 isnotNorC terminal in NV2 but is internal and includes only one

tryptic cleavage site. To rigorously exclude a

relationship between NV2 and V1, it may be

necessary to examine tryptic peptides of NV2

fromTforms,Iforms, and SHA becauseof the

considerable heterogeneity inmigration through

gels ofNV2 (2,9,16a). We have been unableto

obtain sufficient NV2 from SHA for peptide mapping, since the amount of this protein in

the

particle

issmallandvariable(7, 12, 14).

In further search of a precursor of V1, we

compared the peptide mapsofNV5, NV4, and V3withthemapof V1.Quite obviously, V3 and V1 contain

peptides

migrating

to similar

posi-tions. However,

defining

a precursor of V1 is

not easy because V1 has

only

twomajor [3S]

methionine-containing peptides. The

larger

pro-teins, NV5, NV4, and V3, have

fairly complex

peptidemaps,increasing the

probability

of find-ingby chancespotsin thesameareaofthemap

as one of the two V1

peptides,

but the spots

may not

necessarily

represent

peptides

of the

same structureas those ofV1. IfV1 is derived

from one of NV5, NV4, or V3

by proteolytic

cleavage, then the other

products

of the

cleavage

havenotbeenidentifiedininfected cells; NV5, NV4, and V3 are stable in

pulse-chase

experi-ments

(16a).

IfV1 is cleaved fromV3, then V1

isderived from V3 molecules other than those foundin

purified

virus, since thereis noloss of the "V1

peptides"

from these molecules. If V1 is derived from NV5 or NV4, then the larger protein is almost

certainly

multifunctional. It has been

suggested

that NV5 and NV4 maybe polymerase

proteins

(14a). The existence of such

a

large

stable

protein

only

as aprecursor ofV1

would beout of

keeping

with thegenetic econ-omy of the virus. In summary, although the tryptic peptide maps are consistent with a hy-pothesis that NV5 or NV4 or V3 could be a

precursor ofV1, no other supporting evidence

isavailable.

Clearly,

NV1W,

and V2 are related

proteins,

the amino acid sequence of

NV1YW being

con-tainedinV2.Theextrapeptidein V2is

consist-entwith itslargersizebasedonits

migration

in

acrylamide

gels

(15). The

only

feasibleprecursor

of

NV1Y, (and

V2) from the

mapping

experi-ments is NV5; however, because treatment of

infected cells with puromycin (160,g/ml)

in-hibits the synthesis ofNV5 but notthat of

NVW1Y,

(10,16a), we conclude that

NV1½

isnot derived

bycleavageof NV5.

Thus, theonlytwopost-translational cleavage

productssuggested by the tryptic peptide maps are

NV1W2

(from V2) and V1 (fromNV5, NV4, orV3).Thepossibility that

NV2Y;

andNV1are

derived by cleavage of V3 also arises from the

peptide maps; however, this possibility arises

mainly

because oflack of peptide resolution in

heavilylabeled areasof themaps, andthere is

considerable other evidence implyingthat such acleavage does not occur (see above). In

con-trast totheseexperiments with

flavivirus-speci-fiedproteins, trypticpeptide mapshave readily demonstrated precursor-product relationships between proteins specified by other positive-strand viruses, namely, the picornaviruses and alphaviruses. For example, post-translational cleavage of viral proteins has been confirmed

by

peptide mapping

for

poliovirus

(4), Semliki

Forestvirus(11), and Sindbis virus

(6).

Peptide

mapsof

Kunjin

virus-specified proteins

demon-strate alack of

precursor-product relationships

among them

(with

the

possible exceptions

noted

in Table 2); they are consistent with the

pro-posal,

based on

experiments

with inhibitors of

proteolytic

cleavage

and on

experiments

using pactamycinor

hypertonic

saltto control

trans-lationevents, that

during

the

replication

of fla-viviruses thetranslation of the

majority

of viral polypeptidesisinitiated

internally

onviral RNA

asthe

single

template (14a).

ACKNOWLEDGMENT

This work wassupported by a grant from the National

Healthand Medical Research Council of Australia.

LITERATURE CITED

1. Boulton,R.W.,and E.G.Westaway.1972.

Compari-sonsoftogaviruses:Sindbis virus(group A)andKunjin

virus(groupB). Virology49:283-289.

2. Boulton,R.W.,and E.G.Westaway.1976.Replication

of the flavivirus Kunjin: proteins, glycoproteins and maturation associated with cell membranes.Virology

69:416-430.

3. Hershko,A.,and M.Fry.1975.Post-translational cleav-age ofpolypeptidechains:role inassembly.Annu.Rev. Biochem. 44:775-797.

4. Jacobson, M. F., J. Asso, and D. Baltimore. 1970. Furtherevidence on the formation ofpoliovirus pro-teins.J. Mol. Biol.49:657-669.

5. Laemmli, U. K. 1970. Cleavage of structural proteins duringtheassemblyof the head ofbacteriophageT4. Nature(London)227:680-685.

6. Schlesinger, M.J., and S. Schlesinger. 1973. Large-molecular-weightprecursors of Sindbis virusproteins.

J. Virol.11:1013-1016.

7. Shapiro, D.,W. E.Brandt, R. D.Cardiff,and P. K. Russell. 1971. TheproteinsofJapanese encephalitis

virus.Virology44:108-124.

8. Shapiro, D.,W. E.Brandt,and P. K. Russell. 1972. VOL. 24,1977

on November 10, 2019 by guest

http://jvi.asm.org/

(11)

Change involvingaviralmembraneglycoprotein during morphogenesis of group B arboviruses. Virology 50:906-911.

9. Shapiro, D.,K. A.Kos,and P. K.Russell.1973.

Japa-neseencephalitis virusglycoproteins. Virology56:88-94.

10. Shapiro, D.,K. A. Kos,andP.K.Russell. 1973. Protein synthesisinJapanese encephalitis virus-infected cells Virology 56:95-109.

11. Simons, K., S. Keranen, and L. Kaariainen. 1973. Identification ofa precursor for one of the Semliki forestvirus membraneproteins.FEBSLett.29:87-91. 12. Stollar,V. 1969. Studiesonthe nature ofdengue viruses. IV. The structural proteins oftype 2 dengue virus. Virology39:426-438.

13.Westaway, E. G. 1973. Proteins specified by group B

togavirusesinmammaliancellsduringproductive infec-tions. Virology51:454-465.

14. Westaway,E.G.1975.The proteins ofMurrayValley encephalitisvirus.J. Gen. Virol. 27:283-292.

14a.Westaway, E.G.1977.Strategyof the flavivirusgenome:

evidenceformultiple internal initiationoftranslation of proteins specified by Kunjin virus. Virology 80:320-335.

15. Westaway,E.G.,J.L.McKimm,and L.G. McLeod. 1977. Heterogeneity among flavivirus proteins

sepa-ratedinslabgels.Arch. Virol.53:305-312.

16.Westaway,E.G.,and B.M.Reedman.1969.Proteins of thegroupBarbovirusKunjin.J.Virol. 4:688-693. 16a.Westaway. E. G., and M.Shew. 1977. Proteins and

glycoproteins specified bytheflavivrusKunjin.

Virol-ogy80:309-319.

17. Wright,P. J.,D. S.Bowden, andE. G.Westaway. 1977.Uniquepeptidemapsof thethree largestproteins

specified bytheflavivirusKunjin.J.Virol. 24:651-661. 18. Wright, P.J.,andG. diMayorca. 1975.Virion

poly-peptide composition of the human papovavirus BK: comparisonwithsimianvirus40andpolyomavirus.J. Virol. 15:828-835.

J. VIROL.