A small highly basic protein is encoded in overlapping frame within the P gene of vesicular stomatitis virus.

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0022-538X/93/063103-08$02.00/0

A

Small Highly

Basic Protein

Is

Encoded

in Overlapping

Frame

within the

P

Gene of Vesicular Stomatitis

Virus

CHRISTINAF. SPIROPOULOU ANDSTUARTT. NICHOLt*

Cell andMolecular BiologyProgram and Department of Biochemistry,

University

of

Nevada, Reno,

Nevada 89557 Received 21 January 1993/Accepted2 March 1993

Vesicular stomatitis virus (VSV) has served for severaldecades astheprototype rhabdovirusandamodel

RNAvirus. Extensive studiesupheld the original view of VSV genetics with simply fivegenes(N, P, M, G, and L), eachencodingasingle unique protein. Wenowreportthefirstunambiguous demonstration of the existence ofanadditional unique protein encoded inanoverlapping frame within the virus Pgene. Experiments using

antipeptideseraspecific forthepredicted secondopenreading frame have demonstratedthesynthesis oftwo

N-terminallynestedforms ofthe protein in virus-infected cells. Themajor form is 55amino acids in length, whereas the minor form has 10 additional N-terminal amino acids. Ribosome initiation of

synth,esis

of these proteins appears tooccuratAUG codons, 68 and 41 bases, respectively, downstream of the P protein AUG initiation codon.The proteinsarefound in the cytoplasm of the infected cell butareundetectable in purified virions, consistent with their beingnonstructural proteins. Both the major and minor forms of the proteinare highlybasicandarginine rich, reminiscentof theCandC'proteins encoded in overlapping frame closetothe 5' terminus ofthe P mRNAof several paramyxoviruses. The potentialtoencode small, highly basic proteins

within thePmRNA5' terminusis highlyconserved amongthe vesiculoviruses. In recentyears, negative-strand RNA viruses have been

shown to utilize anumber of diverse mechanismsto

maxi-mize the codingpotential of their relatively smallgenomes.

Thesemechanismscanbebasically divided intotwo

catego-ries: mRNA processing or modification, and alternative

translation strategies (4, 7, 13, 17, 24, 37). The use of

overlapping open reading frames (ORFs) to generate more than one unique protein from a single mRNA has been

described for several of these viruses. The synthesis of

influenza B virus NA and NBglycoproteinsandSendai virus P and Cproteins fromoverlapping frameswere amongthe

first well-characterized examples (17, 35). Since then,

nu-merousotherexampleshave beenfound,themoststrikingof

which is theexistence ofmultiplefunctional ORFs within the P mRNAof several paramyxoviruses (12, 25).

Vesicular stomatitis virus (VSV), the prototype

rhabdo-virus, has been intensively studied over the past several decades and has served as a model negative-strand RNA virus. To date, these extensive studies have upheld the

originalviewof VSVgeneticswithsimplyfivegenes (N,P, M, G, and L), each encodinga single unique protein (40).

However, recent molecular evolutionary analysis led to speculationthatanadditional ORF(ORF2)mayexist within the P gene of VSV NewJersey (NJ) serotype viruses (6). Briefly,thisgenecodes for thepolymerase-associated phos-phoprotein P, a virus structural protein essential for viral transcription and replication (2). Three distinct functional domains have been defined(9). Theamino-terminal domain I ishighlyacidic andcontains theconstitutively

phosphory-lated sites. Domains II and III are basic and apparently

function inbindingtothepolymerase proteinLand N-RNA template, respectively (14). The ability to functionally

re-* Correspondingauthor.Electronic mail address: stnl@ciddvdl.

em.cdc.gov

tPresent address: Special PathogensBranch, G-14, Divisionof

Viral and Rickettsial Diseases, Centers for Disease Control,

At-lanta,GA 30333.

placedomainI with ,B-tubulin in virus in vitro transcription

assays suggested that only the overall acidic character and associated phosphorylationofthis domainwererequired to maintain function(10). This led tothe expectation that the

evolution of this domain may not be tightly constrained. However, phylogenetic analysis of nucleotide sequences of the Pgenesof18 VSV NJisolatesrevealed that theencoded

domain Iwasmorehighly conservedthaneither domainIIor III (6).

Detailed analysis of the rates of substitution of first-, second-, and third-base codon positions within the P gene revealed a statistically significant (P < 0.001) reduction in third-base variability between bases 50 and 250 of the domain I-encodedregion. Itwasspeculatedthat the colocal-ization of a potentially functional second ORF (65 amino acids in length) to this region could explain the high se-quenceconservation observed(thethirdbasepositionof the Preadingframecorrespondstothe second basepositionin the overlapping reading frame) (6). Inthis study, wereport direct evidence of the existence oftwoN-terminallynested

proteins derived from theP-gene second ORF of VSVNJ,

the cytoplasmic localization of the protein(s) within virus-infectedcells, lack ofdetection of theprotein(s) in purified virions,andapparentconservation in several different vesic-uloviruses.

MATERIALS AND METHODS

Growth of viruses. Baby hamster kidney (BHK-21) cells

weregrown asmonolayercultures inEagleminimalessential

medium containing 5% calfbovine serum. The VSV strain used in thisstudywastheOgdenstrain of the NJserotype. Theoriginsof the isolatespresentedintheevolutionarydata have been describedpreviously (5, 6).

Generation of antisera. ThepeptidesCRMPMNLRKDER INISKTSS(P1),a20-merrepresentingamino acids8 to 27 of thepredicted secondoverlapping readingframe(ORF2)and

SKIKEINQLRHIIRKKNRQ (P2), a 19-mer representing

3103

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AACAGATATCATGGACAGTGTTGATAGGCTCAAGACTTACTTAGCCACTTATGATAATTTGGATTCTGCCTTGCAGGATGCCAATGAATC 90

M D S V D R L K T Y L A T Y D N L D S A L Q D A N E S

Pprotein -> M I I W I L P C R M P M N L

ORF2protein ->

TGAGGAAAGACGAGAGGATAAATATCTCCAAGACCTCTTCATCGAAGATCAAGGAGATAAACCAACTCCGTCATATTATCAGGAAGAAGA 180

E E R R E D K Y L Q D L F I E D Q G D K P T P S Y Y Q E E E

R K D E R I N I S K T S S S K I K E I N Q L R H I I R K K N

ATCGTCAGATTCAGATACTGATTATAATGCTGAACATCTTACGATGCTGTCACCGGATGAAAGAATAGACAAGTGGGAAGAAGATTTGCC 270

S S D S D T D Y N A E H L T M L S P D E R I D K W E E D L P ...

R Q I Q I L I I M L N I L R C C H R M K E *

FIG. 1. Nucleotide sequence and deduced amino acid sequence of predicted ORF2. The first 270 nucleotides of the P mRNA are presented. ORF2 beginsatposition51and is terminatedbythe TAGcodonatposition 250. The deduced amino acidsequenceof ORF2 is indicated below thenucleotide sequence.The firsttwopotential N-terminal methioninesareshown inbold.

amino acids 28to46ofORF2,weresynthesizedandcoupled

to keyhole limpet hemocyanin (KLH) with maleimidoben-zoyl-N-hydroxysuccinimideester.Antipeptideantiserawere

raised inrabbits,and thefinalbleedsweretakenat9weeks postimmunization. These sera were affinity purified (31) beforeuse in theexperiments described.

Protein labeling and immunoprecipitation. VSV NJ-in-fected cellswerelabeledwith 100,uCiof[35S]methionine per mlat5.0 hpostinfection(p.i.)for 45 minin methionine-free minimal essential medium supplementedwith 2% dialyzed

serum. The infected cellswere harvested in radioimmuno-precipitation assay (RIPA) buffer (150 mM NaCl, 1.0% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl sul-fate [SDS], 1 mMEDTA, 10 mMTris [pH 8.0]) containing 1% SDS, 1% 2-mercaptoethanol, 1% aprotinin, 2 mM phenymethylsulfonylfluoride, leupeptin (1 ,ug/ml), and pep-statin(1

p,g/ml).

Celllysateswerediluted1/10in RIPA buffer and added to the antibody-protein A-Sepharose (Sigma) complexfor 3 hat4°C,washed four times with RIPAbuffer, andboiled inprotein samplebuffer for 4 min (34). Proteins

were analyzedon Tricine-SDS-polyacrylamide gels, as op-posedtoglycine-SDS-gels,because oftheir superiorability

toseparate low-molecular-weight proteins. Electrophoresis utilized4% T-3%Cstackingand16.5%T-6%Cseparating gels(whereTrepresentstotalmonomerconcentrationandC represents cross-linking monomer concentration) as de-scribed previously (34). Fluorography and autoradiogram exposure of dried gels were carried out by standard tech-niques. When required, exactquantitationofradioactivity in specificbandswasobtained byusing the Molecular Dynam-ics 425 series PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, Calif.).

For

[35S]methionine

labelingofvirions, cells were infected and labeled overnight. The culture medium was then har-vested, and cell debris was removed by centrifugation at 3,000xgfor 10 min. Viruswaspelletedfrom the mediumby centrifugation at 95,000 x g for 1.5 h at 4°C, then resus-pended in TEN (50mMTris-HCl [pH 7.6], 100 mMNaCl, 0.5 mMEDTA),andpurifiedon a5to40%sucrosegradient by bandingat150,000 xgfor 35 min. Thepurified virus band

wasdiluted with TEN and pelleted at 95,000xg for 1.5 h at 4°C.Afraction of the viruspellet was resuspended in RIPA buffer for immunoprecipitations. Another fraction of the viruspelletwasresuspended in a small volume of protein gel samplebuffer andboiled for 3 min for use as a virion marker.

In vitro translationandcDNA clones. The VSV NJ P-gene fragment potentially encoding ORF2 was reverse tran-scribed, polymerase chain reaction (PCR) amplified (Perkin ElmerCetus), and cloned by using oligonucleotide primers P255(-)

(5'-GGTGGAAAGGATCCTFTCAGGCAAATCT

TCTTCCC-3', with aBamHIsite) and P15(+)(5'-CCATTC CCGAATTCCAGTGTTGATAGGCTCAAGAC-3', with an

EcoRI site). To construct a fragment with mutation of the first AUGto a GUG,primer

P255(-)

wasused in combina-tion with primer

P45(+)

(5'-CCATTCCCGAATTCGTG

ATAAT'TGGATTCTGCC-3', withanEcoRI

site) (see

Re-sults).ThePCRfragmentswere

purified by using

aMermaid kit (Bio 101), proteinase K digested,

phenol-chloroform

extracted, and ethanol

precipitated

asdescribed

previously

(11). Theywere then digestedwith the appropriate restric-tionenzymesandclonedinto

plasmid

pBs+,

using

standard protocols(26). Coupledinvitro

transcription-translation

was performed in TnTlysates

(Promega),

using T7 RNA

poly-merase

trans-[35S]methionine

(ICN) and 1 p,g of

plasmid

DNA.

Immunofluorescence. BHKcellswere grown at low den-sityoncoverslipsand infectedwith VSVathigh

multiplicity.

At 5.5 h p.i., cells were washed with

phosphate-buffered

saline(PBS)andfixed in3% formaldehydefor 20 min. After fixation,cellswerewashedthree timeswith PBS

containing

0.2% Tween 20

(PBST)

and then

permeabilized

with 1% Nonidet P-40 for 10 min. Forblocking,the cellsweretreated withnormalgoatserumfor 1 hand washedwith PBST.The primary antibodywas added at a 1/50dilution in 10% goat

seruminPBSTandplacedat4°Cfor 1 h. The cellswerethen washed three timeswithPBST andincubatedfor 1hat4°C with the secondary antibody, fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (Sigma) di-luted 1/100 in 10%goatserumin PBST.Cellswerewashed againthreetimeswithPBST,and thenmultiplefinal washes

weredonewith PBStoremove any tracesof the detergent. Thecellsweremountedonmicroscope slides and viewed by using a Bio-Rad MRC-600 laser scanning confocal micro-scope with a Nikon inverted microscope attached. Cells were viewed in the form of 0.5-p,m-thick scans, which facilitated differentiation of nuclear and cytoplasmic stain-ing. Photographic imageswere generated bymultiple-layer composites ofthe entire cell.

RESULTS

PredictedP-gene ORF2. Bases 11to834 of the virus P gene encode thepolymerase-associated phosphoprotein.Close to the 5' end,atbases51 to 248there is a potential ORF2 (Fig. 1). It is unclear how this ORF could be utilized, given Kozak's rules for ribosomal scanning (22, 23). These rules

state that leaky scanning through an upstream AUG can

occur only when the upstream AUG is in weak context. However, in our case, the upstream AUG for the P protein is inastrong context(A at position -3, G at +4). Only one exceptiontothis rule has been accepted by Kozak, which is thesynthesisof the NAprotein from the NB/NA mRNA of influenza Bviruses (22, 35). If leaky scanning through the first strong AUG were also the case here, then the first

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CN a) GL)

0- 0.

a.-L c a

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Ca Cm 0.

pptr ed

B

-0T 0

0-co LD

E U = a < 0-0

C C

-5 c .-

r,

r- 0-a

.-:: rZ C: -1D D D

>> > D .C < < l<

G _ N -_

P.- .

M-i

14.3-jUm4usg_.

6.2

-6 5 4 3 2 1 1 2 3 4 5 6 7 8

FIG. 2. Identification of ORF2 proteinsin VSV NJ-infected cells. BHK cellswereinfectedwithVSVNJ(Ogdenisolate) and labeledat

5.0 h p.i. for 45 min with [35S]methionine. Allofthe immunoprecipitation reactionswere done with the anti-Pl antibody, unless stated

otherwise, asdescribed in Materialsand Methods. (A) Lanes: 1 and 2, infected celllysatesimmunoprecipitated withP1 and P2 preimmune

sera,respectively; 3 and 4, infected cell lysates immunoprecipitatedwithanti-Plandanti-P2sera,respectively;5 and 6,immunoprecipitation

reactions specifically blocked by usingP1and P2 peptides, respectively. (B) Lanes: 1, purified VSV virion marker;2,purifiedVSVvirion

immunoprecipitated with theanti-Plantibody;3,uninfectedcells immunoprecipitated; 4, infected cell lysates immunoprecipitated;5,product

of invitro transcription-translation of the AUG78/GUG51construct,6;product of in vitro transcription-translation of the AUG78/AUG51 construct; 7; product of the in vitro transcription-translation of AUG78/GUG51 construct immunoprecipitated; 8, product of in vitro transcription-translation of the pBs+vectoralone. Sizesareindicatedinkilodaltons.

downstream AUGencountered would be the AUGat posi-tion 51. However, this AUG is in weak context, but it is closely followed by an AUG at position 78 which is in a strong context. Use ofboth of these AUGs would lead to

synthesisoftwoN-terminallynestedproteinsof Mr 7,900

and6,600, respectively. The predicted amino acid sequence

startingfromthe firstAUG is shown in Fig. 1. The predicted protein(s) isstronglybasic, withanapproximate pI of 11.5,

and unusually richinarginine.

PresenceoftheORF2 productsinVSVNJ-infected cells. On

the basis ofhydrophobicity analysis (20) of the predicted amino acid sequence of the potentialORF2protein(s), two shortdomainswerechosenforpeptide synthesistoproduce

antisera with whichtosearchfortheprotein(s).Antigenicity prediction methods (ANTIGEN program, PCGENE;

Intel-ligenetics, Mountain View, Calif.) suggested that peptideP1 waslikelytobethemostantigenic,followedby peptideP2. Theaffinity-purified antipeptide sera wereused in

immuno-precipitation experiments with lysates of

[35S]methionine-labeled VSV NJ-infected BHK cells.

An identical major protein band of Mr = 6,500 was

precipitated bybothantipeptidesera(Fig. 2A,lanes 3 and4).

In addition, a minor second protein bandof slightly larger

size was observed with use of both antibodies (the minor band barelyvisible in lane 4 is clearly visible on a longer

exposure). Neither protein were precipitated with

preim-mune sera(Fig. 2A, lanes 1 and2),and neitherwaspresent inuninfected BHKcellextracts (Fig. 2B, lane3).

Quantita-tion of the relative amounts of the minor andmajor ORF2 bands precipitated by either antipeptide serum (Fig. 2A, lanes 3 and4), usingthe MolecularDynamics

PhosphorIm-ager, indicatedaratioofapproximately 1 to7,respectively, correcting for methioninecontent. From the estimated pro-teinsizes, themajorbandwasthoughttocorrespondtothe ORF2 initiating at the second AUG (in strong context at

position 78), while the minor band was thought to

corre-spondtothelargerORF2initiatingatthe firstAUG(inweak

context at position 51). The reactivity of the P1 and P2 antiseracorrespondedwellwith the predictedantigenicity of

thepeptides,withthe P1 antiserum usefulata10-fold-higher dilution than the P2 antiserum. For this reason, the P1 antiserumwasused in allsubsequent experiments.

Use of invitro-synthesizedproteinstoconfirmORF2protein

identities. To confirm the identities ofthe major and minor

ORF2 protein bands, authentic ORF2 proteins initiating at the two different AUGs were synthesized in vitro from

P-gene fragments cloned into the pBs+ vector. The first

insertwassynthesizedbyusingPCRprimerswhich resulted inamplificationofnucleotides 16to 273. Thecorrect orien-tation andsequenceof theinsertwere confirmedby primer

extension dideoxy sequencing analysis. With use of T7

polymerase and the TnTlysate kit(Promega) with [35S]me-thionine, thisconstructwould havethepotential to synthe-size thelongerORF2initiatingatthe weakAUGatposition

51. Gel analysis revealed two bands (Fig. 2B, lane6), one

apparentlycorrespondingtothelongerORF2 and the other

corresponding to the shorter ORF2 (initiating at the strong AUG downstream at position 78). These bandscomigrated with, respectively, the minor and major ORF2 proteins

detected in VSV NJ-infected cells(Fig. 2B, lane4).

To further confirm these identifications, a second

con-struct was synthesized by using an altered PCR primer

sequenceto mutatethe first AUGtoaGUG,which wouldbe

predictedtopreventsynthesisof thelargerORF2butleave the smallerORF2 AUG(at position 78)intact(23). Following

in vitro transcription and translation, the protein product was analyzed by gel electrophoresis, and only the single

smaller ORF2proteinwasvisualized,aspredicted (Fig. 2B, lane5). This bandcomigratedwith the majorORF2protein

band detected in VSV NJ-infected cells (Fig. 2B, lane 4), againconfirmingtheidentityof theprotein. Inaddition,the in vitro transcription-translation control using pBs+ alone

gave noproduct (lane 8).

These results indicatethatduringORF2synthesisinVSV

A

V 0

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18.4--5

c 0 0 a)

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NJ-infected

cells,

there is limited initiation at the first downstream AUG

(position 51,

in poor

context),

with the

majority

of initiation

occurring

at the second downstream AUG

(position

78,

instrong

context).

The

specificity

of the

antipeptide

antibodieswasconfirmed

by

the

complete

block-age of the

reactivity

ofanti-Pl and anti-P2 with the ORF2

protein by preincubation

with the

corresponding peptides

(Fig.

2A,

lanes 5 and

6).

This resultwasfurtherconfirmed

by

specific immunoprecipitation

of the invitro-translated

major

ORF2

protein

with the anti-PI serum

(Fig. 2B,

lane

7).

Lack ofmodification of the ORF2

protein.

The

comigration

of the in vivo- and in

vitro-synthesized

ORF2

protein

sug-gested

the absence of extensive

protein

modificationinvivo. A

potential N-glycosylation

site does exist at amino acid

asparagine

at

position

21

(Fig.

1). However,

treatment of cells with

tunicamycin (an

inhibitorof N

glycosylation)

prior

to and

during

virus infection gave ORF2s identical in

gel

mobility

to ORF2s from untreated cells.

Also,

endoglycosi-dase Hor

N-glycosidase

F

(New England Biolabs)

enzyme treatmentof ORF2

proteins

failedtoaltertheir

gel

mobilities

(data

not

shown).

These resultssuggestthat the

N-glycosy-lation siteat

position

21 isnot

utilized,

at leastunderthese virus

growth

conditions. In

addition, gel analysis

of

immu-noprecipitated

ORF2

protein

from VSV NJ-infected cells grown in the presence of 1 mCi of

32p;

perml revealed no

band, indicating

that ORF2 is also not

phosphorylated

in vivo

(data

not

shown).

ORF2

proteins

are not detectable in virions.

Analysis

of

heavily [35S]methionine

labeled, purified

VSV NJ virion

proteins

failed to reveal the presence of ORF2

proteins

in virus

particles (Fig. 2B,

lane

1).

This observation did not

change

evenafter gross overexposure of the

autoradiogram.

In

addition,

immunoprecipitation

of

35S-labeled purified

vir-ions with the anti-Pl serum also failedto reveal any ORF2

protein (Fig.

2B,

lane

2). Again, analysis

of an excess amount of

immunoprecipitated samples

did not alter this

result,

consistent with the ORF2

proteins being

nonstruc-tural.

Pulse-labeling experiments

for

30-min

intervals

(in

the presenceof

[35S]methionine)

throughout

thecourseof infec-tion indicated that the timecourseof

synthesis

of theORF2 nonstructural

protein

is similar to that of the other VSV structural

proteins,

with

peak synthesis occurring

5 to 6 h

p.i. (data

not

shown).

Intracellular localization of the ORF2

protein.

Analysis

of theVSV NJ ORF2 amino acid sequence for the presence of known functional motifs identified a

eukaryote

proteasome nucleartranslocation

signal

site

(RKKNR)

ataminoacids40 to 45 from thefirst ORF2 AUG

(36).

The

potential

nuclear localization of the ORF2

protein

wasexamined

by

indirect immunofluorescence studies. VSV NJ-infected BHK cells

were stained 5.5 h

p.i., using

the

affinity-purified

anti-Pl

rabbit serum and

fluorescein-conjugated

goat anti-rabbit

immunoglobulin

G. Stained slideswere

analyzed by using

a Bio-Rad MRC-600 confocal laser

microscope.

In

parallel,

a

phase-contrast

image

wasobtained in orderto

clearly

define cell

shapes

and structures.The results

(summarized

inFig.

3)

show that the ORF2

protein

is localized in the cell

cytoplasmic

compartment andnotin the nucleus. Most cells exhibited a diffuse

cytoplasmic staining

pattern (Fig. 3B);

however,

a substantial

proportion

exhibited a perinuclear

staining

pattern

(Fig.

3C).

The

specificity

of the reactionwas

demonstrated

by

the lack of

staining

of theuninfected cell control

(Fig.

3D).

In

addition,

toshow that the

staining

isnot

due to some artifact created

by

nonspecific

virus

infection,

an additional

negative

control of cells infected with VSV

Indiana

(IN)

serotype was included

(Fig. 3E).

Further, the

positive immunofluorescence staining patterns could be completely abolishedby preincubation of the

anti-Pl

serum for 1 h with excess P1 peptide(Fig. 3F).

Evolutionary conservation ofORF2-like proteins. The nu-cleotide sequences of the P genes of 18 diverse VSV NJ isolates have been determined previously (6). Examination of theirpotential to encode ORF2 proteins reveals that the deduced ORF2 amino acid sequence is highly conserved amongviruses of the VSV NJ serotype (Fig. 4A). Immuno-precipitationexperiments confirm the presence of the ORF2 proteins in several of these different isolates (Fig. 5). The minor differences ingel mobility among the ORF2proteins of the different isolates is to be expected, as some have slightly different ORF2 amino acid sequences. In fact, this observationprovidesfurther evidence that theseproteinsare

virus-encoded and not virus-induced cellular proteins. Ex-amination of theP-genesequencesofsevendiverse VSV IN serotypeviruses (8, 16, 21, 39) reveals that they each also possessahighly conserved ORF2 protein (encoded by bases 84to 284) ofapproximately thesame size(67 amino acids) which is also very basic(pI 11.5) and arginine rich (Fig.4B). Depending on the actual AUG used forinitiation, the

en-codedproteins could have an Mr of =7,900 or6,700. Examination of theP-gene sequence of the moredistantly related vesiculovirusChandipura virus (27) reveals a similar ORF2protein (encoded by bases 93 to 335) that is 80 amino acids inlengthandhighly basic,withapI of 12. Analysis of the recently published sequence of the P gene of another distantly related vesiculovirus, Piry virus (3), also reveals thepotential for an ORF2 protein (encoded by bases 77to

170), althoughthisproteinsomewhatshorter, being only 32 amino acids inlength,butagain being highly basic, with pl of 11.6. These results suggest that the encoding of a short, highlybasic ORF2proteinwithin the5'-proximal end of the P mRNA may bea commonpropertyof the vesiculoviruses. However, there is little direct sequencehomology between the ORF2s of VSV NJ, VSV IN, Chandipura, and Piry viruses, despitethe similaroverall properties.

Short highly basic ORF2s can be found in the P-gene sequences of some other rhabdoviruses, including rabies virus and infectious hematopoietic necrosis virus, but are

morevariable in theirpositionswithin the P gene (28, 38). However, this does open the possibility of the existence of functionalshort, highlybasicORF2proteinsencoded in the P genes of the animalrhabdoviruses in general. No evidence exists for theencodingofashort, highlybasic ORF2 in the P-gene equivalent (M2 gene) ofthe plant rhabdovirus son-chusyellow net virus (18). Potential to encode ORF2-like

proteins

can be identified in the P-gene equivalent of two

well-characterized filoviruses, Marburgvirus and Ebola

vi-rus

(15).

These viruses are thought to be evolutionarily

relatedtotheparamyxovirusesand rhabdoviruses. DISCUSSION

Earlierevolutionary analysishad suggested the existence

ofa

potentially

functional 65-amino-acid ORF2 protein

en-coded inoverlappingframe within thePgeneof VSV NJ (6).

In this report, weprovided direct evidence that the ORF2

protein

exists in the cytoplasm of VSV NJ-infected cells in

the form of two N-terminally nested proteins, 55 and 65 aminoacids in length. The encoding of these small, highly

basic,

and

arginine-rich

proteinsinoverlapping frame close to the 5' terminus of the P mRNA is reminiscent of the C

proteins

found in severalparamyxoviruses (4, 13, 25). These

proteins

arealso small, highly basic, and arginine rich and

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FIG. 3. Indirect fluorescent stainingof VSVNJ-infected cells. BHK cells were infected for 5.5 h before fixation,permeabilization, and indirectimmunofluorescent labeling.(A) Phase-contrast image of VSV NJ-infected cells. (B) The same cells stained with theanti-Plantibody andanalyzed by confocal laserscanningmicroscopy. A diffuse cytoplasmic staining pattern was predominantly observed. In addition, a substantialnumberof cellsexhibitedperinuclearstaining by 7 h p.i. (C). The image in panel C isamplified threefold relative to that in panel B.Negative controls includeuninfected cells (D), cells infected with VSV IN (San Juan strain) (E), and the same cells as in panel B but with preincubation of the anti-Plantibody with excessP1peptide (F).Exposures of panels D to F are adjusted to be equivalent to exposures of panelsBand C.

arefound intwoN-terminally nested forms, C and C'. They

are also encoded in overlapping frame close to the 5' terminus of the P mRNA. We have no direct evidence to suggestthat theseproteinsarefunctionallyequivalent; how-ever,inanattempttostandardizenomenclature,wesuggest that the VSV major and minor ORF2 protein forms be tentativelytermed CandC', respectively.Although polycis-tronic messages with overlapping readingframes have been described inseveralothernegative-senseRNAviruses, this is the firstexampleofafunctionalORF2inamember ofthe rhabdovirusfamily.Inaddition,we have shown that similar small, highly basic ORF2 proteins are potentially encoded within the P genes of other vesiculoviruses and possibly otheranimal rhabdoviruses.

Theprecisemechanismby which the VSV NJ Cproteins

aresynthesizediscurrentlyunknown. Invitro

transcription-translation experiments indicate that the major form of ORF2 (C) initiates at the AUG at position 78 and that the minor form (C') initiates at the AUG at

position

51. On speculating as to the potential mechanism of synthesis of

theseproteins,we first examine thepossibilityofsynthesis by leakyribosome scanning(22,23).Thiscommonlyoccurs

when the first AUG lies in an unfavorable context for initiation. However, in the P mRNA molecule, the start

codon for the P protein (at position 10) is in excellent

context. Theonly exceptiontothis ruleaccepted byKozak is thesynthesisof the NBproteinfrom theNB/NAmRNAof influenzaBvirus, in which the NAprotein initiationcodonis downstream of the NB initiation codon which is in strong

context (35, 41). If VSV C

proteins

represent another exception, then theobservedresultswould fit the

predicted

outcome well, that is, much less synthesis of C proteins relativeto

P,

aminoramountof C'synthesizedfromthe first AUG (in weak context at position 51), and much greater relative amountof C

synthesized

from thesecond AUG

(in

strongcontext atposition 78).Wehavenodirectevidenceto favor this model over the alternative

possibilities, i.e.,

synthesisof the C

proteins (i)

byinternal ribosomeinitiation directly at the position 51 and 78 AUGs or

(ii)

from a

separateminor mRNA

transcript(s) starting

after the PAUG

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MIIWILPCRMQMNLKREERINISRTSSSKIKEINQLRRIIRRKNRQIQILTIMLNILRCCRQMRE

K S PRK

P T S K

R S K

T KK

H K

R H K

R K H K

R H K

H K

I

RK K HR K RK RK RK

S RK

I RK

RK Y RK

B

Mudd-Sunmers MRSKHNELKSPIMSCSKRTETKSILGPLIPRQQMILTQNLNQKLKTIKACMYQIRKLSKLKALYRGL

../56NMB V H Q

..

/42C0E L R

..

/870AB

..

/87VCB

01/85GMB L GR N DR S L R

09/82HDB L GR N DR S L R

FIG. 4. Predicted amino acidsequencesfor theORF2proteins of 18diverseVSVNJisolates(A)and 7diverseVSV Indiana isolates(B), including VSV NJ isolates../49UTB2(Ogden) and ../52GAP (Hazelhurst) and VSV IN isolates ../56NMB (San Juan)and..42COE (Glasgow).

Aminoaciddifferencesarerelativetothemostancestral isolate(10/82CRB)forthe VSVNJ viruses and the Mudd-Summers isolate(thought

tobederivedfromtheoriginal1925 VSVINisolate) fortheVSVINviruses.

codon. Elucidation of the exactmechanism ofsynthesis of the C proteinswill require further experimentation. How-ever, it is interesting that internal ribosome initiation has

recently been suggested to occur with the P/C mRNA of measles virus(1). Inaddition, some years ago,Herman had

suggestedthatanadditional smallprotein (7K), representing the carboxy-terminal 62 amino acids of the P protein, was

synthesizedfrom the VSV IN P mRNAbyinternal ribosome

initiation (19).

The correspondence ofthe predicted size of VSV NJ C

H- < I D

43- 29-

18.4-14.3

6.2- -dU

3-FIG. 5. Immunoprecipitation ofORF2 proteins from cells

in-fected withdifferentVSV NJisolates. BHK cellswereinfected with

differentisolatesof NJviruses andlabeled for 45 minat5.0 h p.i.

with [35S]methionine. The cell lysates were immunoprecipitated

with the anti-Pl serum as described in the text. Theviruses are

../49UTB2 (Ogden isolate), ../52GAP (Hazelhurst isolate), 10/ 85HDB, and 10/84GMP. Sizesareindicated in kilodaltons.

proteins and their actual size on polyacrylamide gels

sug-geststhat these overlappingframesare notbeing linkedto otherORFs within the P gene by RNAediting or splicing mechanisms as has been seen in some paramyxovirus and influenza virusgenes(7, 24, 37).Itis, however,curious that of all thenonsegmented negative-sensevirusgenes,it isonly

the Pgenesofsome paramyxoviruses and now some rhab-doviruses that have been showntoencodemultipleproteins by use of overlapping reading frames. This finding would suggest that the primary amino acid sequence required for the functionalancestralgeneproductwassufficientlyflexible to allow sequence alteration to open up other functional

reading frames and the virus to gain additional functions.

The possibility exists that the evolutionary constraints on

proteinsotherthan the P-gene ancestral productswere too tighttoallowadditional framedevelopmentwithin theother

virusgenes.

Thehigh degreeofconservation of the VSV NJC-protein

sequence amongdiverse members ofthe VSVNJ serotype andof theVSVINC-proteinsequenceamongdiverse VSV INserotypememberssuggeststheC proteinsmustperform

functionscritical for the virus lifecycle.Todate,thereisno direct evidence indicatingwhatthe C-protein functionmay be.Thedemonstrationthat theregionof theVSV NJ Pgene

encodingCcanbedeleted andreplaced bythatencoding the

1-tubulinproteinamino terminus andyetstillsupportvirion transcription in vitro (10) suggests that C does not play a vitalrole invirusRNAtranscription. This would be consis-tentwithourinabilitytodetectCproteins in virions. These

observations are also consistent with detailed analysis of VSV temperature-sensitive mutants possessing defects in

the Pgene. Both VSV NJ and INserotypeP-gene mutants exhibitmultiple phenotypes, including defects in transcrip-tion, replicatranscrip-tion, or postreplication development (32).

Nu-A

10/82CRB 01/85PNB *./60PNB 07/83NCP 12/82HDB 10/85HDB *./76ECM 10/84GMP 09/82HDB ../49UTB2 11/84HDB 11/82VCB2 07/840AB 01/84SNP 11/83CAB 06/85NME

../52GAP

07/83GAP

p p p p p

RKD

RKGQ

RKD

KKD K

K K K K K

K K K K K K K K K K K

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cleotide sequence analysis of the VSV NJ mutants revealed that they each possessed point mutations located in the

region encoding the C protein (33). None of the viruses

defective in transcription had nucleotide changes which would alter the C-protein amino acid sequence. However,

eachof the mutants with defective replication or

postrepli-cationphenotypes had nucleotide changes which would alter theC amino acid sequence in additionto that of P. At this

point, no direct evidence exists for the involvement of the C proteins in replication. However, these observations to-gether with the small, highly basic, arginine-rich nature of these proteins make it tempting to speculate that these proteins could function in virus RNA replication.

Experi-ments testing the ability of C to bind to virus nucleocapsid

arecurrently being initiated. The recently developed ability

to perform reverse genetic experiments with VSV (29, 30)

shouldalso providea means toevaluate the role, ifany, of C

inVSV replicationsteps in vivo.

ACKNOWLEDGMENTS

We thank Bonita Beigalke, Carol Condit, Ardythe McCracken, and PatrickO'Hara for helpful discussions. We also thank Karen McCoyforhelp with the confocal microscopy, Elmer Otteson for assistance with antibody purification, and Anthony Sanchez for assistance with artwork.

Thiswork wassupported by theU.S.DepartmentofAgriculture Animal Molecular Biology, NationalResearch Initiative (90-37266-5473).

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