jvirol00090-0376.pdf

Copyright (C 1988, American Society forMicrobiology

Interactions between Coronavirus Nucleocapsid Protein

and

Viral

RNAs: Implications for Viral Transcription

RALPH S. BARIC,' GARY W. NELSON,2 JOHN0. FLEMING,2ROBERT J. DEANS,2 JAMES G. KECK,2

NANCY CASTEEL,3tAND STEPHEN A. STOHLMAN23*

Department ofParasitology and Laboratory Practice, University of North Carolina School of Public Health, Chapel Hill, North Carolina 27514,1 and theDepartmentsof Microbiology2 and Neurology,3 Universityof Southern

CaliforniaSchool ofMedicine, Los Angeles, California 90033 Received 28April1988/Accepted 1August1988

Theinteractionof themousehepatitis virus (MHV) nucleocapsid protein (N) and viral RNAwasexamined.

Monoclonalantibody specific for N protein coimmunoprecipitated MHV genomic RNAaswellasallsixMHV subgenomic mRNAsfound in MHV-infected cells. Incontrast, monoclonalantibodies tothe MHV E2orEl

envelope glycoproteins, an anti-I-A monoclonal antibody, and serum samples from lupus patients did not

immunoprecipitatetheMHV mRNAs.Moreover, the anti-N monoclonal antibody didnotcoimmunoprecipitate vesicular stomatitis virus RNAorhost cell RNA underconditionswhichimmunoprecipitated all MHV RNAs.

These data suggest a specific interaction between the N protein and the virus-specific mRNAs. Both the

membrane-bound and cytosolic small MHV leader-specific RNAs ofgreater than 65 nucleotides long were

immunoprecipitated only byanti-N monoclonalantibody. These datasuggestthatanNbinding site ispresent within the leader RNAsequencesata siteatleast 65 nucleotides from the5'endof genomic RNA and all six subgenomic mRNAs.Thelarger leader-containing RNAs originating frommRNA 1 and mRNA 6, aswell as

the MHV negative-stranded RNA, werealso immunoprecipitated by theanti-N monoclonalantibody. These

dataindicatethat the MHV Nproteinisassociated withMHV-specificRNAs and RNAintermediatesandmay

playan importantfunctionalroleduring MHV transcription and replication.

Mouse hepatitis virus (MHV) is a member of the corona-virus group of animal corona-viruses. MHV is an enveloped corona-virus

containing a helical nucleocapsid structure composed of a

plus-sensedRNAmolecule (5.5 x 106daltons) and a

nucleo-capsid protein (N) of 60,000 daltons (37). The envelope

containsa 180,000-dalton N-linkedglycoprotein(designated E2) which functions in cell attachment and fusion and an

0-linked matrix glycoprotein of 23,000 daltons (designated E1)(38).

Duringinfection, virionRNAisinitiallytranscribedbyan early polymerase activity into a genome-sized

negative-stranded RNA (9). In turn, the negative-stranded RNA is transcribed by a late polymerase activity into a full-length genomic RNA that is both bound to polysomes (9) and

detected in EDTA-resistant nucleocapsid structures (34). Viral transcription is associated mainly with membrane

fractions (9, 10). Six species of subgenomic mRNAs are

synthesized during infection.They arearranged in the form ofanested setwith common sequences derivedfrom the 3' end of thegenomic RNAthat extend in the 5' direction for variousdistances (21, 34, 38). Therefore, each mRNA con-tains its own unique sequences plus all the sequences contained within the next smallermRNA species. In addi-tion, all viralmRNAscontainanidentical leader sequence of

approximately 72 nucleotides attheir 5' end (21, 22).

Anal-ysis of MHV-infected cells has revealed the presence of small leader RNAs 65 to 84 nucleotides long which could serve a primer function by binding to specific sequences conserved at the intergenic regions in the negative-strand template. These leader sequences are derived from the 5' end of the genomic RNA and are probably joined to the

* Correspondingauthor.

tPresentaddress:DepartmentofMicrobiology, Chicago College

ofOsteopathic Medicine, 1122E.53rd St.,Chicago, IL60615.

individual mRNAs bya unique leader-primed transcription mechanism (4-6, 21, 25, 32).

Theaminoacid sequence ofN, deduced from thenucleic acid sequence, reveals that this protein is455 amino acids long and highly basic (1, 33). Immediately after synthesis, the N protein is phosphorylated exclusively on serine resi-dues. Phosphorylated N is associated with cellular mem-branes via a strong noncovalent interaction, presumably through association with the El protein (36, 39). A 140,000-daltontrimer ofN has been described in nonreduced prep-arations ofpurified virions byusinganRNAprotein-overlay blot assay,suggestingthat Nprotein mayexist as a disulfide-linked multimer in the mature virion (29). The N protein is also theonly RNA-binding proteininvirionsand is amajor RNA-binding proteinfound in infectedcells (29).

The N protein has a variety of functions in addition to servingas astructuralcomponentof the helicalnucleocapsid (38). It also plays important roles in viralpathogenesis and replication,since anti-N monoclonal antibodiesprotectmice from lethal infection (27) and inhibit viral transcription in vitro(12);however, the function ofN inviraltranscriptionis unclear. In thisreport,we have examined the associationof theNprotein with MHV RNAby usingananti-N monoclo-nal antibody to immunoprecipitate the N-RNA complexes. In the accompanying article, we have examined the speci-ficityof N-RNA interactions bya secondtechnique usinga direct RNA-protein binding assay (8, 15, 29, 35). Together, thesedata suggest that Nprotein-MHV RNAinteractionis mediated by binding signals contained within MHV leader RNA sequences and suggest that this interaction is impor-tant inviral transcription.

MATERIALS AND METHODS

cells, a murine astrocytoma cell line, or in L2 cells as

previously described (4, 22, 23). Cellsweregrownin 150-or

100-mm-diameter petri plates and infectedatamultiplicity of

infection of 1 to5. For experiments using

32Pi

toradiolabel MHV-specific mRNAs, infected cellsweretreated with2,ug ofactinomycin D per ml at 2.5 h post-infection (p.i.) and labeled with 500 pLCi of 32p; (ICN Pharmaceuticals Inc., Irvine, Calif.) at 4.5 h p.i. Samples were harvested as

described below at 7.5 to 8.5 hp.i. [3H]uridine (ICN) was

added to actinomycin D-treated cells at 15 ,uCi/ml at 4.5 h p.i. For coinfection studies, the Indiana strain of vesicular stomatitis virus(VSV)wasusedatamultiplicity of infection

of 5. Plates were infected with A59 as described above, incubated for 30 minat 37°C, and subsequently coinfected with VSV for 1 hat37°C. Radiolabeled purified virionswere

preparedas previously described (23).

Celllysates.At 7.5to8.5 hp.i. the plateswerechilledinan

ice-water bath, the medium was removed, and the plates werewashed twice with ice-cold phosphate-buffered saline.

Cellswerelysed by the addition of 3 ml of 10 mM Tris buffer

(pH 7.4) containing 150 mM NaCl per plate, 0.5% Nonidet

P-40, and 200 ,ug of phenylmethylsulfonyl fluoride per ml. After 10 to 15 min the cells were removed with a rubber policeman and the nucleiandlarge celldebriswerepelleted

by centrifugation at 1,500 x g for 5 min at 4°C. The

supernatantwasthenadjustedto10 mM EDTA and200 mM ammonium acetate. After an additional centrifugation at

15,000 x gfor 10min, the supernatantwasadjustedto0.2% sodium dodecyl sulfate (SDS) before immunoprecipitation.

Preparation of P100orS100 subcellularextracts. Prepara-tions of subcellular fracPrepara-tions were as previously described

(6). Briefly, cellswere swollenin0.5x reticulocytestandard

buffer (1x reticulocyte standard buffer is 10 mMTris [pH 7.4], 10 mM NaCl, 1.5 mM MgCl2) for 30 min at 4°C. The cells weredisrupted by Dounce homogenization and

centri-fuged at 1,000 x g for 5 min. The nuclear pellet was

discarded, and the membrane fractions (P100) were

sepa-rated from the cytosol fraction (S100) by centrifugation at

100,000 x gfor90min. Under these conditionsover90% of

the (Na+K-)-ATPase and 5'-nucleotidase were present in theP100 fraction(6). Membrane fractionsweresuspendedin

0.5x reticulocyte standard buffer before

immunoprecipita-tion. RNA in the immunoprecipitates was extracted and

separated on 8 to 12% polyacrylamide gels before electro-transferto Zeta-Probe paper(5).

Immunoprecipitation. Immunoprecipitations were carried

out as previously described (14, 16, 36). In these

experi-ments, anti-A59 monoclonal antibodies A.1.10, specific for N; A.3.10, specific for the E2 envelope glycoprotein; A.1.8, specific for the El glycoprotein; and 7.16.17, specific for I-AP, were used (14, 16, 18). Briefly, antibodieswere

incu-bated with the cell lysates for 30 min on ice, and then

Formalin-fixed StaphylococcusaureusCowan 1 wasadded.

This mixturewas incubated 45 minonice and then washed

twice in10mMTris(pH 7.4) containing 150mMNaCl, 0.5% NonidetP-40, 0.2% SDS, and 200 ,ug of phenylmethylsulfo-nyl fluoride per ml. The final pellets were suspended in 10

mMTris(pH 7.4) containing 60mMNaCl,1 mMEDTA, and 1.0% SDS. The RNA was extracted with phenol and

chlo-roform as previously described (4, 22) and precipitated

overnight with ethanol at -20°C. RNA was treated with

glyoxal and analyzed by electrophoresis on 1%agarosegels as previously described (4, 22). Radiolabeled nucleic acids were visualized by either fluorography or autoradiography

(4, 22).

Immunoprecipitation ofthe MHV leader RNAs.Cultures of

infected and uninfected cells werelysedand incubated with avariety ofantisera as described above. Inaddition, antise-rum samples from systemic lupus erythematosis patients specific for the La,Rho, Sm, andribonucleoprotein (RNP) complexes (kindly provided by Jack Keene, Duke

Univer-sity, Durham,

N.C.)

were used. After

immunoprecipitation

as described by Kurilla and Keene (20), the RNA was

extracted, precipitated, and separated on 8 or 12% poly-acrylamide gels containingurea (4). The RNAwas

electro-transferredto Zeta-Probepaper,

prehybridized,

andprobed

with anick-translatedleader cDNAprobe

(E1-L)

orinternal El sequences (El-500). The El-L cDNA probe encodes nucleotides1 to 84ofmRNA6, including the72-nucleotide leader sequence, while E1-500 encodes the internal nucleo-tides 90 to 609 of mRNA 6 (4). Theblotswerewashed and

exposed to XAR-5 film in the presence ofan intensifying

screen at

-70°C (4).

Northern(RNA)blots. ForNorthern

analysis,

RNA

sam-ples were

electrophoresed

on 0.8%

formaldehyde-agarose

gelsand blottedby capillarydiffusiontonitrocellulose(40).

Bound RNAwashybridizedto a32P-labeled1,360-base-pair fragment ofahuman gammaactingene

probe,

which

cross-hybridizes

to mouseactinsequences(11).The

hybridization

probewas prepared toa

specific activity

of109

cpm/,ug by

the methodof

Feinberg

and

Vogelstein

(13).

In vitro transcription of MHV strand-specific probes. Recombinant

plasmid

pT7-2 11,

containing

sequences

de-rived fromMHVgene F(nucleotides 1to760), and

plasmid

pT7F82P (nucleotides 5 to 1149) (4, 32),

containing

se-quences derived from MHV gene A, are oriented in the

plus-sense

configuration

sothat T7

polymerase-derived

tran-scriptsareidenticaltomRNAandcapable of

annealing

with

MHV

negative-stranded

but not

plus-stranded

RNA.

Tran-scription

wascarriedoutina

100-,ul

reaction

containing

2,ug

oflinearized

plasmid

DNA; 40 mMTris

hydrochloride (pH

7.5);6 mM

MgCI2;

2 mM

spermidine;

10 mM

NaCl;

10 mM

dithiothreitol; 1 mM each ATP, CTP, and GTP; 0.01 mM

UTP; 100

pRCi

of

[a-32P]UTP;

10 UofT7 RNA

polymerase;

and 40UofRNasin. Reactionswereincubatedat

37°C

for1

h, and the radiolabeled products were separated from free nucleotides by chromatography through P-60 columns.

Ra-diolabeled RNAswere mixed with

hybridization

bufferand

incubatedwith the filter at65°C for24 to36 h(5, 6).

Detection of MHV negative-stranded RNA. Intracellular

RNAfrom MHV-infected cells was

immunoprecipitated

as

described

above,

denatured

by dimethyl sulfoxide-glyoxal

or

heat,and

applied

tonitrocellulosepaper

by

usingadotblot

unit

(Bio-Rad Laboratories, Richmond, Calif.).

The paper

was bakedat80°Cfor 2h, prewashedand prehybridizedas

previously described (4, 6), and then probed with

plus-stranded RNA

probes

(2 x 107

cpm/ml)

capableof

annealing

with MHVnegative-strandedbut notplus-strandedRNA or

purified genomic

RNA. Thefilters were hybridized at65°C

for 24to36 hinasolution

containing

50%

formamide,

10 mM sodium

phosphate

(pH 7.0), 1Ox Denhardt

solution,

1.83 mM EDTA,6x SSC

(lx

SSCis 0.15 MNaCl plus0.015M

sodiumcitrate),83

jig

oftRNAperml,and 125,ug of salmon sperm DNA per ml. After hybridization, the filters were washed with several changes of 2x SSC containing 0.01%

SDS and with 0.2x SSC

containing

0.01%SDSat65to70°C. Finally, blotsweretreated with 0.5 ,ugofRNaseAper mlat room temperature in 2x SSC for 5 to 15 min. Filters were

exposed to XAR-5 film in the presence ofan

intensifying

B C D E _{A B C} D E F

."W

2

3

4 5 6

FIG. 2. Immunoprecipitation of RNA from MHV-infectedcells. Lanes: A and F, controls showing the virus-specific RNA extracted from infected cells; D, allsevenvirus-specified RNAs

immunopre-cipitated by the anti-N monoclonal antibody; B, C, and E, immu-noprecipitates obtained by using anti-El, anti-E2, and anti-I-AP monoclonalantibodies, respectively. MHV mRNAs 1 through 7are

indicatedonthe left.

FIG. 1. Immunoprecipitation of RNA from pelleted MHV vi-rions. Lanes: A, genomic-length RNA extracted from the pelleted virions with somecontamination from mRNAs 5, 6,and 7; B, no

RNA recovered in the absence of antibody; C, RNA of genomic lengthimmunoprecipitated by using anti-N monoclonal antibody; D and E, no RNA recovered with anti-El or anti-E2 monoclonal

antibodies.

RESULTS

Association of N protein with viral RNA. Monoclonal antibodies specific for the N protein, the El envelope glycoprotein, and the E2 envelope glycoproteins of the A59 strain of MHV (16) were tested to determine whether a

specific interaction between a particular viral structural

proteinandtheMHVgenomicRNAcould bedemonstrated bycoimmunoprecipitation. Antibodies wereincubated with concentrated 32P-labeled detergent-disrupted A59 virions. TheRNAwasextracted fromtheimmunoprecipitated

com-plexes and analyzed by electrophoresis on denaturing aga-rose gels. Anti-N monoclonal antibody immunoprecipitated MHVgenomic RNAas partofan N protein-RNP complex (Fig. 1). No RNA was detected in immunoprecipitates

formed by usingtheanti-El, anti-E2,orirrelevantanti-major histocompatibility complex class II I-AP monoclonal

anti-bodies. These data supportprevious studies indicating that N proteininteracts withthegenomic RNAtoform ahelical

nucleocapsid structure (38) and demonstrate the feasibility of examining RNA-protein interactions by immunoprecipi-tation.

We next analyzed the RNA species immunoprecipitated withanti-Nmonoclonalantibody fromlysatesof32P-labeled actinomycin D-treated MHV-infected cells. Anti-N

mono-clonalantibody immunoprecipitated notonly genomicRNA (RNA 1) but, surprisingly, the other sixvirus-specific

intra-cellular RNAs as well (Fig. 2). Also, no virus-specific

mRNAswereimmunoprecipitatedwithmonoclonal

antibod-ies specific foreitherthe El or E2 protein(Fig. 2).

Immu-noprecipitation of individual El or E2 mRNA from

poly-somes was not detected because of the dissociation ofthe complexes with EDTA before immunoprecipitation (see Materials andMethods). Inaddition, novirus-specific RNA was immunoprecipitated with the antibody specific for the

I-APprotein. These datasuggestthat allsevenviral

intracel-lular mRNAs exist asRNPcomplexesduring infection.

Thespecificityof theimmunoprecipitation of the

intracel-lular virus-specific mRNAs byanti-N monoclonal antibody

was further examined in experiments designed to clearly demonstrate specificity for MHV RNAs. First, cells were

coinfected with VSV and MHV in the presence of

actino-mycin D to determine whether the MHV N protein was

associated with VSV RNAduring coinfectionaspreviously

suggested (29). These experiments are critical, since VSV RNAsprobablywould beimmunoprecipitatedifnonspecific interactions occur during sample preparation. Coinfected cells contained both MHV and VSV RNA (Fig. 3). The relative amounts ofVSV and MHV RNA in the coinfected cells variedslightlyfromexperimenttoexperiment. Figure3 shows the results ofanexperimentinwhichrelatively equal

amountswerepresent.Moreimportantly,theMHV-specific

anti-N monoclonal antibodies immunoprecipitated only MHV-specific mRNAs butnotVSVRNA from the coinfec-ted cells.

Cells were also prelabeled overnight with [3H]uridine to label host cell RNAs. Label was removed, the cells were

infected and treated withactinomycin D,and the intracellu-lar RNAs were immunoprecipitated as described above. Control cellswereinfected,treated withactinomycin D,and radiolabeled with[3H]uridine duringinfection. Cells labeled during infection yielded only virus-specific mRNAs after

A

a'.

A B C D l 2 3

1

2 3

4 S 6

7

FIG. 3. Immunoprecipitation ofMHV-specific RNA fromcells coinfected with VSV. Lanes: A, pattern ofRNA extracted from MHV-infected cells;B, RNAextractedfromVSV-infected cells;C, RNA extracted from cells coinfected with both VSV and MHV.

Notethat mostMHVmRNAs(numbered1through7 ontheleft)can be detected. LaneD,RNAimmunoprecipitated from cells coinfec-tedwithVSV and MHV, demonstrating thatonlyMHV mRNA is recovered by using the anti-N monoclonal antibody.

immunoprecipitation with anti-N monoclonal antibody.

RNAimmunoprecipitatedfrom cells labeled before infection

contained nohostcell RNAspecies; however,a verysmall

quantityofvirus-specific mRNA, presumablyderived from the turnover of [3H]uridine during the infection, was de-tected (data not shown). Finally, RNA from infected and

uninfected cells wasimmunoprecipitatedwith anti-N

mono-clonalantibody and examinedfor actin mRNAby Northern blotanalysis. Similaramountsofactin mRNA were detected in infected and uninfected control cells; however, no actin RNA was detected in the immunoprecipitates from either infected or uninfected cells (data not shown). These data

indicatethat the MHV N protein is notassociated witheither host cell or VSV mRNA molecules and suggest that a

specific

interaction

between

the MHV N protein and viral

RNA occursduring infection.

Mapping the Nbindingsite toalocation within theleader RNA. Immunoprecipitation of all the MHV genomic and

subgenomicmRNAssuggested thatthe N binding sitemust reside within sequences commontoeach RNA. The5' and 3' endsof all seven MHV RNAsareconservedbecause of the presence ofa common leader RNA at the 5' end and the nested-set structureat-the 3' end (21, 22). Previous results from our laboratories have also demonstrated that several

small

leader-containing RNAs of 65 to 84 nucleotides in

lengthare derived from the 5' endof the viral genome and may function as primers in mRNA synthesis (4, 6). To

FIG. 4. Immunoprecipitation of MHV small leader-containing RNAs. Cultures of infected cells were lysed and immunoprecipi-tated with a variety of antisera directed against MHV structural proteins or antiserum obtained from lupus patients. The RNA proteincomplexes wereextracted, and the nucleic acidwas

sepa-rated on 8% polyacrylamide gels. After electrotransfer to Zeta-Probepaper,the blotswerehybridizedwithaleader-specificcDNA. Lanes: 1,RNAimmunoprecipitated with anti-N; 2, immunoprecip-itation with anti-E2; 3, total RNA extracted from MHV-infected cells. Nucleotide numbersareshownontheright.

determine whetherthe small leader RNAsofMHVbind N,

we performed immunoprecipitationsusing monoclonal

anti-bodies

specific

forthe MHV Nand E2

proteins

andanumber of serum samples from systemic

lupus

erythematosis

pa-tients containing antibodies specific for the La, Sm,

RNP,

and Rhoproteins.Theseserumsamplesweretested because

previousdata havedemonstrated that the VSVleader RNA is bound to the host La protein (20). Immunoprecipitated

RNA from infected cells was separated on 8% polyacryl-amidegels, transferred to Zeta-Probepaperand hybridized with a nick-translated leader-specific cDNA probe

(E1-L).

Anti-N monoclonal antibody immunoprecipitated the small MHV leader RNAs of65nucleotides orlonger, butnotthe 47- or 50-nucleotide RNAs. It is unclear whether the 57-nucleotide leader RNA isprecipitatedby N(Fig;4). Aslight

sizedifference (2 to 5 nucleotides) was detected in the 74-, 77-, and 84-nucleotide RNAs between the

immunoprecipi-tated andnonprecipitated

RNA.

Although the

exact reason for this is unclear, itwas probablycausedbyendogenousor exonuclease activities, orboth, present

during

immunopre-cipitation. Antibodies with specificities for the MHV

El,

W

:, ...

Ku.

.Ull,

r...

n 2 f:

4.1":..,.":.

'fl :..

Rho, Sm, RNP, and La proteins did not immunoprecipitate the small leader RNAs of MHV (data not shown).

In addition to the small containing RNAs, leader-containing RNAs of 100 nucleotides and larger were also detected (Fig. 4). We have previously demonstrated that specific, highly reproducible subsets of these small RNAs originate during the transcription (4). Furthermore, se-quence analysis suggests that these RNAs are synthesized by transcriptional pausing in AU-rich regions or regions of secondary structure (4). To determine whether the N protein is also bound to these larger leader-containing RNAs, we separated immunoprecipitated RNA from MHV-infected cells under conditions which resolved RNAs 100 to 300 nucleotides long (4). These RNAs were hybridized with a radiolabeled cDNA specific for nucleotides 90 to 609 in the mRNA 6 (gene F) sequence. Transcripts of identical length were present in the anti-N precipitated and control RNA preparations (Fig. 5). These RNAs are identical in size to those previously reported on the replicative-intermediate (RI) RNA during mRNA 6 transcription (4). Similar results were also obtained with a gene A-specific cDNA probe (data not shown). No RNA was detected after immunoprecipita-tion with the anti-E2 monoclonal antibody. Since the only 5' sequences common to mRNA 6 and mRNA 1 are within the leader RNA, these data suggest that the N protein binds to sequences contained within or adjacent to the leader RNA sequences.

Subcellular locations of N-bound leader-containing RNAs. MHV leader RNA synthesis is discontinuous from mRNA synthesis, and the leader RNA(s) probably act in trans to prime viral transcription (4-6, 25, 32). The data described above suggest that the small leader RNAs are bound to N in an RNP complex. However, it is not clear whether a leader-containing RNP complex or a naked RNA molecule is released from the template to act in trans as a primer for subgenomic mRNA synthesis. Previous data have demon-strated that MHV leader RNAs are associated with the cellular membranes and cytoplasmic fractions in roughly equal molar ratios (6). Todetermine whether N is associated with the free leader RNAs, the subcellular location of the leader-RNP complexes was determined by separating the membrane(P100) and cytosolic (S100) fractions by ultracen-trifugation as previously described (6). Subcellular fractions were immunoprecipitated with anti-N or anti-I-A monoclo-nal antibodies, and the small RNAs were separated by polyacrylamide gel electrophoresis. After transfer to Zeta-Probe paper, the blots were hybridized with a nick-trans-lated leader-specific cDNA (E1-L) probe. Identically sized leader RNAs were present in immunoprecipitates and con-trol preparations (Fig. 6), indicating that the small leader RNAs of MHV areassociated with the N protein in both the membrane andcytosolic fractions. These data are consistent with previous data indicating that MHV leader-containing RNAs and the N protein are associated with the membranes of infected cells (6, 36).

Analysis ofN-functioninviraltranscription. Anti-N mono-clonal antibody inhibits the in vitro transcription of viral RNA, suggesting that the N protein is tightly associated with thetranscription complex (12). To further clarify the role of N in viral transcription, we examined the interaction of N with the RI RNA. RI RNA consists of a genome-length negative-stranded RNA hydrogen bonded to nascent plus-strandsrepresenting newly transcribed RNA (5). Since little, if any, free negative-stranded RNA is present in infected cells (31), the presence of negative-stranded RNA in the immunoprecipitates would suggest that N was bound to the

1 2 3 4

164

IS#

I5S

143445

127 123 116

FIG. 5. N binding byleaderRNAs originating duringmRNA 6

transcription. Cultures of cells werelysedand immunoprecipitated

withanti-N oranti-E2 monoclonal antibodiesasdescribedin Mate-rialsandMethods.The complexeswereextractedandseparatedon

8%polyacrylamide gelsunder conditionswhichresolve small RNAs 100 to300nucleotides long. The RNAwas electroblottedto Zeta-Probe paper and hybridized with a nick-translated cDNA clone spanningnucleotides90to609inthe gene F sequence(6).Lanes: 1, anti-E2-immunoprecipitated RNAs from infected cells; 2,

anti-N-immunoprecipitated RNAsfrominfectedcells;3,totalinfected-cell intracellularRNA; 4, total RNA from uninfectedcells. Nucleotide numbers are shownonthe right.

RNAof the RI complex. Therefore, lysates ofinfected and uninfected cellswereincubated with either anti-Noranti-E2 monoclonalantibodies, immunoprecipitated, andprobedfor the presence of

negative-stranded

RNA. Studies in our

laboratory

and others indicate that

strand-specific

RNA

probes

are

capable

of

detecting

as little as i~t 0

infectious units of virus and that in vitro-transcribed

plus-sense RNAsdonotbindto

purified

genomic

RNA

(41).

The

:

: : e :

1 2 3 4

1 2 3 UNI-DBT

ANT-E2 A59-DBt 3-.

ANTI-N _

84

77

74 65

57

FIG. 6. Subcellular location of leader RNA-N protein RNPs. Cultures of infected cells were lysed and separated into S100 and P100 fractions (5). The fractions were immunoprecipitated with anti-N or anti-I-AP monoclonal antibodies and separated on 12% polyacrylamide gels containing urea. After electrotransfer, the fil-ters werehybridized withaleader-specificcDNAprobe (6),washed, and exposedto XAR-5 film at -70°C. Lanes: 1, S100anti-I-A; 2, P100 anti-I-A; 3, P100 anti-N; 4, S100 anti-N; 5, infected-cell control; 6, uninfected-cell control. Nucleotide numbers are shown ontheright.

RNAwasprobedfor the presence of negative-stranded RNA

with in vitro-transcribed plus-sense viral RNAs. Negative-stranded RNA wasimmunoprecipitated withanti-N but not

anti-E2 monoclonal antibody (Fig. 7), consistent with the

suggestion that the N protein is associated with the

tran-scription complex (12). These data cannot distinguish be-tween N binding to negative-stranded RNA and the coim-munoprecipitation of the negative-stranded RNA as a component ofthe RI complex via N interactions with the

leader-containing nascent plus-strands on the RI RNA.

While it is possible that negative-stranded RNA associates with plus-strand RNA during extraction and

immunoprecip-itation, Sawicki and Sawicki (31) have shown that little, if any,freenegative-strandedRNAispresent ininfectedcells. This suggests that the negative-stranded RNA is

coimmuno-precipitated aspart of the RI complex.

The P100 and S100 subcellularfractions were also exam-ined for the presence of N protein associated with negative-stranded RNA. Virtually all of the N-bound negative-stranded RNA (90%) was associated with membrane

fractionsby densitometric scanning of slot blots (Fig. 8). The small amount of negative-stranded RNA detected in the

cytosol probably represents contaminating membrane

FIG. 7. Analysis of N-protein negative-stranded RNA interac-tion. IntracellularRNA wasextractedfrom MHV-infected cells and coimmunoprecipitated with monoclonal antibodiesdirectedagainst the MHV N or E2 structural proteins. The RNA was denatured, bound to nitrocellulose filters, and hybridized with radiolabeled plus-strandedRNAtranscribed in vitro with theT7 RNA polymer-ase. Lanes 1 through 3, 10-fold dilutions of intracellular RNA. UNI-DBT, UninfectedDBTcells.

present during separation. These data support previous findings suggesting that free leaderRNA-N complexeswere present in the cytosol and were dissociated from the nega-tive-stranded template. These data furthersupport the con-tention that theN protein is associated with the membrane-boundtranscription complex (9, 10).

DISCUSSION

In this report,wehavedemonstratedaspecific interaction between the MHV N protein and MHV viral mRNAs. Neither prelabeled host cell RNA, actin mRNA, nor VSV RNAs are associated with the MHV N protein. Previous studies inourlaboratory have suggested that the leader RNA ofMHV acts in trans as a primerfor subgenomic mRNA synthesis, on the basis both offindings that leader RNA reassortment occurs at highfrequency during MHV infec-tion andofthe presence of small leader RNAs in infected-cellextracts (4-6, 25, 32). Although the exact leader RNA which functions in virus transcription has not been

identi-A

1 m

B C

2

3

4

5

fied, several small leader RNAs 65 to 84 nucleotides long which could serve a primerfunction have been identified (4, 5). The data presented in this article indicate that an N binding site is present within the MHV leader RNA se-quence and suggest that thisinteraction may be important in viral transcription.

The N proteins of a variety of helical negative-stranded RNA viruses are essential forvirustranscription. For exam-ple, the N protein of VSV is believed to regulate the shift from transcription to replication (2, 3). At least two distinct

formsof the VSV N protein have been identified by mono-clonal antibodies in cytoplasmic pools of infected cells (2). Thesestudies imply that theavailability of the unbound VSV N protein controls the rate ofgenomic RNA replication. The Nproteins of VSV and La Crosse virus are also bound to the viral RNAs in an RNA complex (17, 28, 30); however, the function of these complexes in virus replication is unclear. Coronaviruses represent the only plus-polarity RNA animal viruses with helical nucleocapsids (38). Compton et al. (12) have clearly shown that the N protein of MHV functions in viral RNA synthesis, since anti-N monoclonal antibody inhibits in vitro transcription. Similar results have been demonstrated in ourlaboratory (P. R. Brayton et al., unpub-lished observation). However, the nature of the interaction between N and the transcriptional complex is unclear. Our findings suggest that N is bound to the cytosol- and mem-brane-bound small leader RNAs 65 to 84nucleotides long as well as to the transcription complex. Although the exact leader RNA which functions in viral transcription has not been identified, it is clear that trans-acting leader RNAs function during MHV subgenomic RNA synthesis and that the minimum size of this RNA is approximately 65 nucleo-tides (25, 32). The datapresented in this paper suggest that such an RNA will contain a functional N binding site, suggesting that a trans-acting leader RNA-N complex may function duringsubgenomic mRNA synthesis. Immunopre-cipitation of thenegative-stranded RNA, as well as the small leader RNAsoriginating during mRNA 6transcription, also suggests that N isassociated with the transcriptioncomplex. Since thenascent plus-strands on the RI RNAcontain leader RNA (4, 5), it seems likely that Nproteinbinding ismediated atleast in part via these sequences. These data suggest that Nmay play an important structural role in the transcription complex; however, our data cannot exclude the possibility that the Nprotein iscomplexed withnegative-strandedRNA orwith other viral proteins which are directly boundto the leader RNA.

VSV leader RNA isassociated with the La protein but not the Rho, RNP, or SM proteins (20). We were able to immunoprecipitate the MHV leader RNA or mRNA species with any of theserum samples from SLEpatientscontaining antibodies to the La, Rho, RNP, or Sm cellular proteins.

This is not surprising, since the physiological roles of the VSV and MHV leader RNAs are quitedifferent. In the case ofVSV, the leader RNA istransported to the nucleus, where it inhibits host transcription, presumably by binding to cellular La protein (3, 20). Conversely, MHV probably inhibits the host cellmacromolecular synthesis at the level of translation (19), while leader RNA probably functions as a primer fortranscription of thesubgenomic mRNAs(4-6,24). The VSV leader RNA is also bound to the nucleocapsid protein in an RNP complex (3, 7). Since the leader RNA sequences of VSV are alsopresent in genomic RNA, but not mRNA, it is thought that these sequences regulate encapsi-dation of genome into RNP structures. Interestingly, the VSV mRNAs are also detected in RNP complexes but are

not

packaged (17).

In the case ofthe MHV leader

RNA,

these sequencesarepresentonallviral mRNAs,

suggesting

that otherfactors are involved in regulating the

encapsida-tion of the

genomic

RNA, but not mRNA, into infectious

virions. Encapsidation ofviralgenomicRNAs isacomplex

processfrequently requiring oneor more binding sites

(26).

Theexactroleof the leadersequenceinencapsidationof the MHVgenome iscurrently under study.

Presently, the exact nucleotides within the leader RNA sequencewhichbindNhavenotbeen

determined;

however,

the datapresentedheresuggestthatthesitemayreside 3' of

nucleotide

57,

sincewe wereunabletodemonstratethat the small leader RNAs of 47, 50, and 57 nucleotides were presentininfectedcellsasRNPcomplexes. Alternatively,N may require a minimum RNA length for high-affinity

bind-ing. In the accompanying articlewedemonstratethat MHV leader RNA sequences,transcribed invitro bythe T7 RNA

polymerase, bindtoNinaspecificandhigh-affinity

fashion,

while internalMHV sequencesingeneA (genomeRNA)and gene F(mRNA 6) do not bindto N (35). Together with the

immunoprecipitationdata presented inthisstudy, these data suggestthat the leader RNA containsasequence-specific N

binding

site. More importantly, the accompanying article

describes a novel approach for determining the N

binding

site within the leader RNA sequences as well as other

regions

in the viral genome

(35).

ACKNOWLEDGMENTS

We thank J. Goodnight, K. Trass, P. Driscoll, and L. Pen for excellent technical assistance, Carol Flores for preparation ofthe

manuscript, and Michael M. C. Lai and Stanley Taharaforhelpful discussions.

This workwas supported by National Science Foundation grant DMB-87-17148, American Heart Association grant 40073,and Pub-lic HealthService grants Al 31386, Al 19244, NS 18146, NS 00796, and Al 23946 from the National Institutes of Health.

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