Unusual transcripts in La Crosse virus-infected cells and the site for nucleocapsid assembly.

0022-538X/87/030667-06$02.00/0

CopyrightC)1987, American Society forMicrobiology

Unusual Transcripts in La Crosse Virus-Infected Cells and the Site

for Nucleocapsid

Assembly

RAMASWAMYRAJU ANDDANIEL KOLAKOFSKY*

Department of Microbiology, University of Geneva Medical School, 1211

Geneva,

Switzerland

Received25August 1986/Accepted17November 1986

The LaCrosse virus S genome segment is known to code for two plus-strand transcripts, free S-mRNA (nucleotide

[nt]

-15 to 886) and encapsidated antigenome RNA (nt 1 to 983). Early in infection only these plus-strand transcripts could be detected, but at later times plus-strand RNAs representing nt 1 to 886 in an encapsidated form and nt -15 to 983 as a free RNA could also be seen, as well as S-mRNA in an encapsidated form.TheencapsidatedS-mRNA became relatively important at later times because the free S-mRNA turned overrapidly after 5 h postinfection. The existence of these unusual RNAs and their presence as either free or

encapsidatedspecies suggests that the sitefor nucleocapsid assemblyislocatedat the 5' ends of the genome and antigenome chains.

La Crosse virus (LAC) is a member of the California

encephalitis serogroup of the insect-transmitted bunyavirus family (13). The genome of these viruses consists of three segments of single-strandedRNAofnegative polarity, each

contained within a separate nucleocapsid (NC), which are

labeled small(S), medium (M), and large (L). The viral NCs have helical symmetry and often appear in the electron

microscope ascircularandsupercoiled structures (9).

The mechanism by which bunyavirusesinitiate transcrip-tion of their mRNAs is remarkably similarto that of influ-enza viruses (8). Bunyavirus S-mRNAs in vivo contain 5'

nontemplated extensions ofca. 15 nucleotides (nt) in length

whichareheterogeneous in sequence(2, 12),presumably the

result ofahost cell primer usedto initiate transcription. In

vitro, purified LAC virions were found to contain a

tran-scriptase which is stimulated by natural mRNAs such as

alfalfa mosaic virus (AlMV)RNA4,whichwasshown to be

actingas aprimer. LAC virionswerealso foundtocontaina

methylated cap-dependent endonuclease which cleaves AIMV RNA 4 at thepositions expected from the length of the 5'extensions foundon thetranscriptsmade invitro(11).

Bunyaviruses, like influenza viruses,thusapparentlysnatch

capped

oligonucleotides

from host cell mRNAs to prime their mRNA synthesis and to provide it with a cap group

required for efficient translation. However,unlike influenza viruses, LAC transcription takes place in the host cell cytoplasm rather than in the nucleus (16), in which ituses a

stable pool ofmRNAs as substratesforprimers, consistent withtheknownimmunityofbunyavirusreplication todrugs

which disrupthostcell mRNAsynthesis (10). The 3' endof

theLACS-mRNA, which isapparentlynot

polyadenylated,

hasbeenmappedtoposition 886,ca.100 ntbeforethe endof the negativegenome template (12).

Since the S-mRNA is only some 80 nt shorter than the

antigenome RNA and does not selectively bind to olig(dT) cellulose, we have taken advantage of the fact that

antigenomes, like genomes, are almost

exclusively

found intracellularly in nucleocapsid structures as the basis for

their separation from mRNAs. Intracellular mRNAs were routinelyseparatedfromgenomes and

antigenomes

by

cen-*Correspondingauthor.

trifuging cytoplasmicextractstoequilibriumonCsCl density gradients under conditions in which the unencapsidated

mRNAs pellet, whereas the nucleocapsids are found as a

sharp bandattheirbuoyant density of1.31g/ml.Whenthese separated fractionswereexaminedbyNorthernblotanalysis byusingstrand-specific riboprobes,thebandedmaterialwas

found to contain all the intracellulargenomes and the vast

majority ofthe antigenomes, whereas the pelleted material contained all the mRNA (14). More recently, however, we havefoundthatunder someconditions thebanded material unexpectedly contained asizableproportion ofthe

intracel-lularS-mRNA(Fig. 1). Thisunusualfinding formsthebasis ofthe presentcommunication.

MATERIALS AND METHODS

Isolation of cytoplasmic RNAs. Confluent cultures of

BHK-21 cells in 10-cm-diameterpetrie disheswereinfected with 20 to 50 PFU of LAC virions per cell. At the times indicated, the cells were harvested by being scraped into

phosphate-buffered

saline

and were recovered by centrifu-gation.Cytoplasmicextractswereprepared byvortexing

107

cells in0.2 ml of 0.5% Nonidet P-40-0.15 MNaCl-10 mM Tris hydrochloride (pH

7.4)-i

mM EDTA (except as indi-cated forthe

experiment

in

Fig.

2) followed

by

centrifugation

for4 minat4,000 x g. When

denaturing

agents were used (see Fig. 4), the

cytoplasmic supernatant

was

immediately

mixed with 6 M

guanidinium-thiocyanate-1%

sarcosyl-0.2

M

P-mercaptoethanol

(5).

Alternatively,

either 1 mg of heparinper ml or 2 mMvanadyl ribonucleoside

complex

was added to limit

degradation

when

denaturing

agents were omitted. The extracts were then

centrifuged

on20 to 40% preformed CsCl density

gradients

for 16 h at

45,000

rpm in the SW60 rotor. In the absence of

denaturing

agents, the

NCs formedasharp visible band in thecenterof the

gradient

(1.31 g/ml) and were removed with a

hypodermic

syringe

fromthesideofthe tube.This CsClsolutionwasthenmixed with 2volumesoftheabove

denaturing

agents

plus

40 ,ug of carrieruninfected BHK cell

RNA,

and the NC RNAswere recovered by

centrifugation

through

aCsCl

gradient.

Northernblotanalysis.The RNA

samples

wereheated for 2minat

900C

in80% formamideand0.1%

xylene

cyanol

FF and

electrophoresed

on either a 4.0or

2.8%,

1.5-mm-thick 667

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Pst 764 Pst

196

4...e

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4..

P

F. ...

...

Alu 896

4....0

...

4.. 5'

-l5 + oligomer 51

1

m

5'S::

-15

3'

lWw Yo3

mRNA

886

u)

983antigenome .Q

983~ ~

1-886

886~~ c

8-)15-983 _

983 Jo

983 gernome

-FIG. 1. Schematic diagram of LAC S-RNAs. The LACS-genomesegmentis shown inthemiddle,withopen bars above thelineindicating theoverlapping N and NS coding regionsandthe numbers belowindicating nucleotide positions.Aboveareshown the variousriboprobes used, with arrowheads showing the direction of transcription from their SP6plasmids andkey restriction sites used in theirgeneration.Below areshown theS-genome segmenttranscripts found in infected cells. Thick andnarrowlines indicate abundantandrarespecies,respectively, with the numbers at the left showing the positions of their5'and 3'ends. The numbers below the lines show the nucleotidepositionsof the ends of the chains. The closed circle followedbyanopen bar represents thenontemplatedcapped primer.Thethickarrowlabeledoligomer shows theposition of theoligodeoxynucleotide primer usedtolocate the positions ofthe 5'ends(Fig.5B).

polyacrylamide gel containing 8 M urea (17). The gel was

then soaked for 10 min in TAE (10 mMTris, 5 mM sodium acetate, 0.5 mM EDTA, pH 7.8) and electroblotted onto zeta-probe membranes in the same buffer. The membrane was dried, prehybridized with 5x SSC (lx SSC is0.15 M

NaCl plus 0.015 M sodium citrate)-5x Denhardt solution-50% formamide-200 ,ug of yeast RNA per mlfor4 h,andthenhybridized inthe samesolutioncontainingca. 5 x 106 cpm of

[a-32P]UTP

(400Ci/mmol)-labeled riboprobes

for8 to 12h. Thefinalstringent washwasfor1h at70°Cin 0.1 x SSC.

Primer extension analysis. The oligonucleotide

represent-ingnt38 to50 asminus-strandDNAwas 5' end labeled with

[_y-32P]ATP

and T4 polynucleotide kinase, separated on an

18% sequencing gel, eluted, and ethanol precipitated with the template RNAs. The pellets were suspended in 6 ,ul of

water, heated at 90°C for 2 min, and quick chilled. The

reaction conditionswereadjustedto 50 mMTris

hydrochlo-ride (pH 8.3)-8 mM

MgCl2-80

mM KCl-20 U of murine

leukemiavirus reverse transcriptase in atotal volume of 20

,ul and incubated at 38°C for 45 min. The reaction mixture was then phenol extracted, ethanol precipitated, and

ana-lyzed on a9%sequencing gel.

RESULTS

As described in the Introduction, when intracellular

nu-cleocapsid RNA isolated from CsCl density gradients is

examinedbyNorthernblotanalysis with S genome negative-strandriboprobes,only the S antigenome RNA can normally bedetected. However, in some cases,S-mRNA-sized mate-rial can also bedetected (Fig. 2). Since there is little or no

precedent among minus-strand RNA viruses for the

encapsidation ofmRNAs, we first suspectedthat the pres-ence ofmRNA in the nucleocapsid fraction was due to a

nonspecific contamination, e.g., free mRNA which was

sticking

tothe NCs or sticking to other cytoplasmic material

whoseaggregate buoyant density was similar to that of viral NCs. In anattempt to limit suchcontamination,cytoplasmic

extracts were prepared from replicate samples of infected cells in the presence of increasing concentrations of salt which mightpreventformation of such complexes. Varying

the NaCl concentrationfrom 0to0.75 M in the presence of 1 mMEDTA, however, had no effect on the relative amount of mRNA in the banded material(Fig. 2, lanes 1 to 3). The presenceof EDTA in the extraction buffers should also have eliminated complexformation dueto Mgsaltbridges.

Simi-larly, the use oflysolecithin rather than Nonidet P-40 to

solubilize membranes during sample preparation had no

effect(data not shown).

We also argued that if the mRNA wassimply stuck to the NC, the buoyant density of the complexes containing addi-tionalcomplements of RNA should besufficiently higher to allow thesecomplexestobe separated from free NCsduring

centrifugation. For example, assuming buoyant densities in CsCl of 2.1 and 1.28 g/ml for RNA and the NC protein,

respectively (3),andthat, like Sendai virus NCs or tobacco

1

2

3

4

5

'I 'W

-0--MRNA

FIG. 2. Effect ofpreparation conditionson amountofS-mRNA in CsCl-banded NCs. Cytoplasmic extractsof LAC-infected BHK cellsat8 hp.i. wereprepared with Nonidet P-40extraction buffer containing either 0, 0.15,or0.75 MNaCl (lanes1to 3,respectively) andtheir NCRNAswereisolatedandanalyzed onNorthern blots of a4%polyacrylamide gelwitha negativeriboprobe representing nt 1 to196(seeFig. 1).In aseparateexperiment, half of the NCs isolated from cells at 20 hp.i. werecentrifuged to equilibrium on a second CsCl density gradient. Lanes 4 and 5 show the Northern blot analysis as above for the once- and twice-banded NC RNAs, respectively.

I. _Sam 4

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pellet

(p)

_band(NC)

5 8 12 16 20 3 5 _{8 12 16 20 P}

_qlom

0

a)

(U)

4)

l._

81

2-51

mRNA( P)

I

151

mRNA(NC)

4 12

** a1-wMRNA

-"Kl-886

20 4 12

[image:3.612.136.474.67.366.2]

harvest

time(hrs)

FIG. 3. Kinetics of encapsidated and unencapsidatedplus-strand S-RNAaccumulation inLAC-infectedcells. Fourpetrie dishes eachof LAC-infected BHKcellswereharvestedatthe timesindicated, and 5% of their CsCl pelleted (P) and15%of their banded (NC)RNAswere

analyzedonNorthernblots ofa2.8%polyacrylamide gel by usinganegativeriboprobe representingnt1%to764 (seeFig. 1). Lanepshows aduplicate of the 3-h-pelletedRNAas amarker. Below is shown thedensitometricanalysisof theautoradiogram normalized forthedifferent

amounts of pelleted andbanded material used. Note that the ordinate ofthe right-handgraph has been amplified 16-fold relativeto the left-handgraph. The closed circles andopentrianglesatthe baseline of thelower right-hand graph markedPrefertotheantigenome and1

to886RNAs, respectively, found in the CsClpellet.

mosaic virus, LAC NCs are composed of 96% protein and

4% RNAby weight, free LAC NCs would have a buoyant

density of 1.31, whereas an NC containing only one addi-tional complement of RNA would bandat 1.35 g/ml. Since these large structures form extremely sharp bands even in relatively shallowCsCl density gradients,aseparatebandat

1.35 g/ml should have been visible, but no satellite band below the free NC band could be detected from cells harvested at any time postinfection (p.i.) (see below). In addition, when the hypersharp band from the preparative gradient was carefully removed and subjected to a second

cycle ofCsCl density gradient centrifugation andthe RNAs weresimilarly examined, the second cycle ofcentrifugation

was found not toeliminate any of the mRNA found in the banded material (Fig. 2,lanes 4 and 5). Since onlythe viral

N protein can be detected by dye staining of the banded material run on protein gels (H. Lindsey-Regnary, Ph.D. thesis, Emory University, Atlanta, Ga., 1983; M. Hewlitt,

personal communication; results not shown), it would

ap-pearthat under someconditions, significant amountsof the

intracellular S-mRNA canbefound in bona fide NC struc-tures.Unfortunately, since, unlike Sendai virusorvesicular

stomatitis virus NCs, the RNA within LAC NCs is highly

sensitive to RNase A (9), this criterion cannot be used to further characterize the S-mRNA-containing structures

which cobandwith viralNCs inCsCldensity gradients.

As mentionedabove, mRNAs of other minus-strand RNA viruses are not known to be encapsidated. In the case of

vesicular stomatitis virus, interestingly, two groups have

neverthelessdetected mRNAs associated with the Nprotein late in infection (7, 15). However, unlike the encapsidated LAC S-mRNA described above, these vesicular stomatitis virus complexes could clearly be separated from genome NCsonCsCl density gradients, since they containedalesser

amountofprotein relativetoRNA.

Kinetics of S-RNA accumulation in LAC-infected celis.

Replicate samples ofcellswere harvestedatdifferent times after infection, and their cytoplasmic extracts were again

separated into CsCl-pelleted and -banded material. This

material was then examined by Northern blot analysis as above, exceptthat 2.8% polyacrylamide gels were used to

increasetheresolutionbetween theS-antigenomeRNA and

the mRNA. When the pelleted material was examined,

S-mRNA was clearlydetectableby 3 hp.i. and reached its

maximumat5 hp.i. However,after 5 hp.i.thesteady-state

concentration of the S-mRNAunexpectedlydecreased such

that itwas atthe limit of detection by12 h p.i. Fig. 3also

showsthat the S-mRNA wasthe onlyband detectedinthe

pelleted material by the probe. When the banded material

from the same infection was examined, at 3 h p.i. only antigenomeRNA could be seen.At 5 hp.i., someS-mRNA

could nowalso be detected. Curiously, by 8h p.i., aband

hrs(peij):

3

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196-764 (+)

' 3 5 8 12 16 20 24'

to4amp

genome

* I ~antigenomet

. .

/mRNA

+

A 1-886

4 12 20

harvest time(hrs)

FIG. 4. Kinetics of total S-RNA accumulation in LAC-infected

cells. One-fourth of the

cytoplasmic

extractsfrom the experiment

shown in

Fig.

3 were denatured directly with

guanidinium-thiocyanate,

and the total RNAs were pelleted through a CsCl

density gradient. Duplicate samples

of eachpelletedRNA

(amount-ingto0.2ofa

petrie

dish) werethen

analyzed

byNorthern blotting ofa2.8%

polyacrylamide

gel.

The

replicate

blotsthusobtainedwere

probed

with either

positive

(+) or

negative

(-) riboprobes

repre-senting

nt196to764,asindicated. The densitometric analysis of the

autoradiogramis shown below.

just

belowthe S-mRNAwasalso nowdetectable, andthese

three bands increased

slowly throughout

the remainder of the infection.

Inthe above

experiment,

the RNA in the bandedmaterial

from the CsCl

gradient

was recovered by mixing the CsCl

solution with

guanidinium-thiocyanate

todissociate the NC

and

repelleting

the RNA in the presence of carrier RNA

through

asecondCsCl

gradient.

This second

ultracentrifuga-tion

step

may have led to

unequal

recoveries ofthe RNAs

between

samples,

since it

appeared

unlikelythatantigenome

RNA,

for

example,

would continue to accumulate after8 h

p.i.

when the S-mRNA level was barely detectable (the

S-mRNA codes for the NC

protein).

To control for this

possibility,

total RNA was also recovered from the same infection

by

mixing

the

cytoplasmic

extracts directly with

denaturing

agents before

centrifugation,

such that all the

viral RNAs are now recovered in the

pellet

in a single step

(14).

In this

experiment,

the Northern blots were also

examined with

riboprobes

for both

plus

and minus strand S-RNAs. As shown in

Fig.

4,

the

steady-state

concentration

of both genomes and

antigenomes

reached amaximum at 5

to 8 h

p.i.

and then increased

only

very slowly throughout

the remainder of the infection. The continuous increase in

the accumulation of

antigenomes

found in the Fig. 3 would

thusappeartobe duetoincreasedrecoveriesoftheRNAsat

later times

p.i.

for reasons whichare notclear. The pattern

of

S-mRNA,

on the other

hand,

did not change from the

previous experiment.

The S-mRNA reachedits maximumat

5h

p.i.

and then

again

decreased dramatically from thenon. The band

just

below the S-mRNAis alsodetectablehere,but its ratio relative to the S-mRNA is lower since in this

experiment

both the

pelleted

and banded material are

com-bined,

and the band

just

below the mRNA cannot be

detected in the

pelleted

material at any time p.i.

The above

experiments

demonstrate that the S-mRNA-sizedmaterial whichbands in CsCl

density

gradients

can be

resolved into two RNA

species,

one which migrates

pre-cisely

with S-mRNA

(Fig.

3,

lane

p)

and onewhich

migrates

slightly

faster,

called 1 to 886

(see

below).

Neither ofthese

species

can be detected

early

ininfection. At 5 h p.i. when

free

(i.e.,

unencapsidated)

S-mRNAaccumulation and viral

protein synthesis

are

maximal,

the

encapsidated

S-mRNA and the 1 to 886 RNA are

extremely

minor species,

repre-senting

less than 2% of the

unencapsidated

S-mRNA. At

later times

p.i.,

however,

sincethe

unencapsidated

S-mRNA has turned over almost

completely,

the encapsidated

S-mRNA and 1 to 886 RNA become the predominant

S-mRNA-sized material intracellularly.

Mapping

the 5' and 3' ends of the encapsidatedRNAs. To

further characterize the

plus-strand

S-RNAs which were

encapsidated,

NC RNAsisolated from cells at 20 hp.i. were

recovered and

electrophoresed

on a 2.8% acrylamide gel

along

with radiolabeled S-mRNA made

(1)

asamarker. The

antigenome

RNA was isolated from the

gel

separately, but no

attempt

was made to

separate

the S-mRNA from the band

just

below it because of their

closeness,

and both the bands

were isolated

together.

Unencapsidated

(i.e.,

CsCl pellet) S-mRNA was

similarly

isolated fromthe same gel to serve as

a marker. To determine the

position

of the 5' ends of these

plus-strand

RNAs,

a radiolabeled

oligodeoxynucleotide

rep-resenting

nt38 to 50 as minus-strand DNAwas extended on these

templates

with reverse

transcriptase,

as well as on tRNA as a control. As shown in

Fig.

5B,

the primer

extended on the

antigenome

RNAto a

single

band (position +

1)

and on the

pelleted

mRNA to a ladder of bands at

positions

-12 to

-18,

as

previously

described (11). When

the

primer

was extended on the

encapsidated

S-mRNA and

the RNA

just

below

it,

both

a band at

position

+ 1 and the

ladder of bands

at

positions

-12

to -18

were found. The

most

straightforward

interpretation

of these

results is that

the RNA in the banded material which comigrates with

authentic

(i.e.,

unencapsidated)

mRNA contains a

nontem-plated primer

12 to 18 nt in

length

at its 5' end like the

pelleted

S-mRNA,

whereas the band

just

belowthe mRNA

starts at

position

+1 like the

antigenome

RNA. The

differ-ence in

migration

of these two bands is alsoconsistent with

adifference of 12 to 18 nt.

The 3' ends of the

encapsidated

RNAs wereexamined by

Northern blot

analysis

with two

riboprobes

representing nt

764

to 983 and 896 to 983 as

minus-strand

RNA(the 3' end of

the S-mRNA

maps

at

position

886,

see

Fig.

1).

As shown in

Fig.

5 the

riboprobe representing

nt 764 to

983 annealed to

the

antigenome

RNA,

the

S-mRNA,

and the bandjust below

it,

whereas the

riboprobe representing

nt

896

to

983annealed

only

to the

antigenome

RNA.

These results indicate

that the

encapsidated

S-mRNA and

the

band

just

below

it terminate

before

position

896. Taken

together,

the above

evidence

suggests

that

(i)

a small

fraction of

the bona

fide S-mRNA

can

be

encapsidated

and

(ii)

the

encapsidated

RNA which

migrates just

ahead of

the S-mRNA

represents

a positive

transcript starting

at

position

+1

and

most

probably

ending

at

position

886.

Unusual

LAC S

positive

transcripts.

The

RNA which

begins

at

position

1

and

ends at 886 is

presumably

neither an

probe: 196-764 (-)

hrsp.i.i: 3 5 8 12

1-983-

-mnRNAR

1

-888= , r 4m 4

-16 20 24

6

4

C')

c

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[image:4.612.57.300.62.327.2]

mRNA (it would not be expected to have a cap group nor

could it be translated as an encapsidated species) nor an

antigenome (it is incomplete) or a defective interfering genome (its complement as minus-strand RNA cannot be

detected [Fig.

SA]).

This RNApresumably resultsfrom the polymerase starting the chain as an antigenome but ending it as an mRNA. We therefore examined whether a molecule

representing theinverse parallel, namely a transcript which starts like an mRNA (position ca. -15) but ends like an

antigenome (position983) could also be detected in infected cells. Indeed, acandidate forthis molecule could be found

butonly in the pelleted material. As shown in Fig. 6, when

theNorthern blotofthe pelleted material from two separate

infectionsisoverexposed, adoubletband above the mRNA canclearly beseen,the lower band of which comigrates with

antigenomeRNA, and the upper band migrating at a position

consistentwithit containing ca. 15 extra nucleotides. Both ofthese bands reachtheir maximumswith themRNA or just

after it and like the mRNA in the pelleted material are

unstablewith time p.i. Noattempt was made to characterize

themfurtherdue totheir extremely lowabundance.

DISCUSSION

The work reported here has described two unusual S genome positive-strand transcripts in LAC-infected cells (see Fig. 1). One transcript starts like an antigenome at

position +1but ends likethe S-mRNA atposition 886 (called 1 to886).Theothertranscriptstarts on a primer of ca. 15 nt in length likethe mRNA but like theantigenome continues on to the end ofthe genome template (called -15 to 983).

The -15to983transcript is foundonly asan unencapsidated

species and never represents more than a smallfraction of

1% of theS-mRNA at anytimeduringtheinfection. Like the

unencapsidated S-mRNA, the -15 to 983 transcript is

un-stable withtime and is drasticallyreduced later ininfection.

The 1 to 886 species, on the other hand, is foundonly as an

encapsidated form and like the minor form of encapsidated S-mRNA appears to be stableatlater times ofinfection.

The most plausible explanation forthese findings is that

both the 1 to 886 and -15 to 983 RNAs are made at

extremely lowfrequencies relativetothe S-mRNA and that the relative steady-state concentrations of the various S

positive transcripts laterininfectionsimply reflectsthe fact that the encapsidated species are stable, whereas the

unencapsidated RNAs turn over rapidly with time. In the case of the

encapsidated

form ofthe mRNA, ifan exceed-inglysmallfraction of the freepopulation becomes

encapsid-ated at a constant rate whereas the remaining

unencapsid-ated molecules turned over rapidly with time, this would

explainwhy late ininfectionthere isactuallymore

encapsid-ated thanunencapsidatedmRNAinthe cell. In this

view,

the -15 to 983 and the 1 to 886 transcripts are the result of polymerase errors, whereas the encapsidated mRNA is a

result ofan encapsidation error, all of which occur

infre-quently. Theynevertheless indicatethatatleastthis part of theviralreplication machinery does notfunctionwith abso-lute specificity.

The existence ofthe 1 to 886

transcript

and its relative

predominance late in infection may also

explain

the unex-pected results of

Bouloy

et al. (4)

working

withGermiston virus, another member of the bunyamwera virus genus. These workers reportedtheexistence ofan S-mRNA-sized

transcript which couldbe

capped

in vitroand thuscontained a di- or

triphosphate

at its 5' end and

suggested

that this

transcriptmight functionas amRNA. Inthese

experiments,

PROBE: (-) (-) (+)

764-983 896-983 764-983

SAMPLE: P NC P NC P NC

1-983---& s

H-15-886

t-86

2

z

I

0E

CL

4 4)

z

.o

E c

z

-z

E

4

z M

-18[

-121

f

+1-FIG. 5. (A) Mapping the 3' ends of the encapsidated S-RNAs. Triplicate samples of CsCl pelleted (P) and banded(NC)RNAfrom cells harvested at 5 and 20 h p.i., respectively were analyzed on

Northern blots ofa2.8%polyacrylamide gel by using the riboprobes indicated. (B) Mapping the5' ends of the encapsidated S-RNAs. CsClpelleted and bandedRNAsfromcells harvestedat5and 20h p.i., respectively, were separated on a 2.8% polyacrylamide gel along with radiolabeled S-mRNA made in vitro (1)as amarker.The positions correspondingtothe SantigenomeRNAand theS-mRNA

(including the band just below it)wereexcised from thegel, andthe RNA waselectroeluted and recovered byethanol precipitation in

the presence of carrier tRNA. A 5'-end-labeled oligonucleotide

representingnt38to50asminus-strandDNA(seeFig. 1)wasthen primer extendedontheseRNAsalong withtRNAas acontrol.The products were separated on a 9% sequencing gel. The genome

positionsof the 5' endsof thetemplateRNAsareindicatedonthe

left.

S-mRNA which contained 5'

nontemplated primers

would not have been scored, and so the relative amounts of the mRNA-sized

transcripts

with different 5' ends could notbe determined. Morerecently, work from the samelaboratory (6) has shown that this material can also be

separated

into

twobands, theuppermost of whichappearedtocontain ca. 15 nontemplated nucleotides at its 5'

end,

whereas the bottomband didnot. Itthus seems reasonableto conclude that the Germiston

transcript

which contains a 5' triphos-phateis the

equivalent

of the LAC 1to886

transcript.

In this

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[image:5.612.354.527.69.443.2]

hrs

(pDi):

(-)15-983 o-1-.983

(-)15-886-_

a

2

5

8

12

b

2

4

6

8

12

- .U ~P-

-.:

FIG. 6. Detection of a -15 to 983 positive S transcript in LAC-infected cells. Twopetriedishes each of LAC-infected BHK cellswere harvested at thetimes indicated, and 10% of the

CsCl-pelletedRNAsfrom twoseparateinfections(aandb)wereanalyzed onNorthernblotson2.8%polyacrylamide gels by usinganegative

riboproberepresentingnt 1 to195.

case, it would not be functional as a mRNA whether it is

capped or uncapped, if indeed this transcript were also

encapsidated.

Regardless oftheprecise mechanism oftheirgeneration,

theexistenceofthe -15to983 and1 to 886transcriptsand their presence intracellularly as unencapsidated and

encapsidated species, respectively, offers some insight on

thelocationof thosesequenceswhichdetermine

encapsida-tion. Since the vast majorityofthe mRNA is not

encapsid-ated when mRNA accumulation is maximal, whereas

antigenomesare encapsidated,the encapsidationsite of the

antigenome could have beenlocated betweenpositions 886 and 983. However since the 1 to 886 transcript is also

encapsidated, this would appear to eliminate these

se-quences nearthe 3' end of theantigenome. The next most

likely site is therefore the 5' end, since both genomes and

antigenomes are encapsidated and only at the two ends of thechainsaretheirsequenceshighlyconserved. Inthiscase,

one explanationforwhy the vastmajority of the mRNA is notencapsidated when mRNA accumulation is maximal is that this recognition sequence at the 5' end has now been

placed out of context by the addition of the ca. 15 nt

transcription primer, and the Nproteincannot now initiate

encapsidation herefor this reason. Alternatively, the pres-enceofacap groupincloseproximitytothissequencecould

bind a cytoplasmic cap-binding factor, for example, and

therebymasktheinitiation site.The presenceof theprimer, however, does not appearto confer complete immunity to

encapsidation, but it is relatively effective; when mRNA accumulationis atits maximum,less than2%of the mRNA has beenencapsidated (Fig. 3).Moredirectexperimentswill

be required, however, to determine whetherthe conserved

sequences at the 5' ends of the antigenome and genome

chains do indeed contain the site for the initiation of NC

assembly.

Finally, sincethe Northernblots ofFig. 4 wereanalyzed

with riboprobes made with [32P]UTP of identical specific activity, the relative specific activities of the positive and negative probes is then dependent only on their uridine

composition. From the data in Fig. 4, we can therefore

calculate the ratioofgenomesto antigenome intracellularly

to be 5.8. Curiously, this number does notagree wellwith

the 60% self-annealing ofS-sized NC RNA previously

de-termined by solution hybridization (16), which suggests a

ratio of only 2.3. Further, the ratio of 5.8 may be an

underestimate, since thegenomebands mayhave exceeded

thelinearrangeofthe filmontheexposureshown in Fig. 4.

Onepossible explanationforthisdiscrepancy is that the NC

RNA used in the solution hybridization experiment came

from cellsharvestedat8 hp.i. andwasseparated into

S, M,

and L RNAs by velocity sedimentation which would not

have separated S-mRNA, 1 to 886, or antigenome RNA. Inspectionofthe datain

Fig.

3 showsthatat8h

p.i.,

the NC

bandmaterial containsasmuch S-mRNA and 1to886RNA as antigenome RNA, all of which would anneal to the negative genomeRNAandtherebydouble the

self-annealing

value. Our more recent data would then indicate that

genomesare atleast 5.8timesasabundantas

antigenomes

in

LAC-infected BHK cells. Further, since bothgenomes and

antigenomes appear stable throughout the infection, their accumulation would then reflecttheirrate of

synthesis.

LITERATURE CITED

1. Bellocq, C., R. Raju, J. Patterson, and D. Kolakofsky. 1987. TranslationalrequirementofLaCrosse virus S-mRNA

synthe-sis: Invitrostudies. J. Virol. 61:87-95.

2. Bishop, D. H.L.,M. E.Gay,and Y.Matsuoko. 1983. Nonviral heterogeneous sequences are present at the 5' ends ofone

species ofsnowshoe harebunyavirus S complementary RNA. Nucleic Acids Res. 11:6409-6418.

3. Blumberg,B.M., C. Giorgi, K. Rose, and D. Kolakofsky.1984. Preparation andanalysis of the nucleocapsidproteins of vesic-ular stomatitisvirus and Sendaivirus, and analysisof theSendai virusleader-NPgene region. J. Gen Virol. 65:769-779. 4. Bouloy, M., P. Vialat, M. Girard, and N. Pardigon. 1984. A

transcriptfrom the S segmentof the Germiston bunyavirus is uncappedand codes for the nucleoprotein and anonstructural protein.J. Virol. 49:717-723.

5. Chirgwin, J.M., A. E. Przybyla,R.J. MacDonald, and W. J. Rutter. 1979. Isolation of biologically active ribonucleic acid from sourcesenriches in ribonuclease. Biochemistry 18:5294-5300.

6. Gerbaud, S.,N. Pardigon, P.Vialat, andM. Bouloy. 1986. InD. Kolakofsky and B. W. J. Mahy (ed.), Biology ofnegativestrand viruses. Elsevier BiomedicalPress, Amsterdam.

7. Grubman, M. J., and D. A. Shafritz. 1977. Identification and characterization of messenger ribonucleoprotein complexes from vesicular stomatitis virus-infected HeLa cells. Virology 81:1-16.

8. Krug, R. M.1981. Priming ofinfluenzaviral RNAtranscription bycapped heterologousRNAs.Curr. Top. Microbiol. Immunol. 93:125-149.

9. Obijeski, J. F., D. H. L. Bishop, E. L. Palmer, and F. A. Murphy. 1976. Segmented genome and nucleocapsid of La Crosse virus. J.Virol. 20:664-675.

10. ObiJeski,J. F., and F. A. Murphy. 1977. Bunyaviridae: recent biochemical developments. J. Gen. Virol.37:1-14.

11. Patterson, J. L., B. Holloway, and D. Kolakofsky. 1984. La Crosse virions contain a primer-stimulated RNA polymerase and a methylated cap-dependent endonuclease. J. Virol. 52: 215-222.

12. Patterson, J. L., and D. Kolakofsky. 1984. Characterization of La Crossevirus small-genome transcripts. J. Virol. 49:680-685. 13. Porterfield, J. S., J. Casals, M. P. Chumakov, S. Y. Gaidamovich, C. Hannoun, I. Holmes, M. C. Horzinek, M. Mussgay, N. Oker-Blom, and P. K. Russel. 1975-1976. Bunyaviruses and bunyaviridae. Intervirology6:13-14. 14. Raju,R.,and D.Kolakofsky.1986. Inhibitors of protein

synthe-sis inhibit both La Crosse virus S-mRNA and S genome syntheses in vivo. VirusRes5:1-9.

15. Rosen, C. A., H. L.Ennis,and P.S.Cohen. 1982. Translational control ofvesicular stomatitis virus protein synthesis: isolation ofanmRNA-sequestering particle. J. Virol.44:932-938. 16. Rossier, C., J. Patterson, and D. Kolakofsky. 1986. La Crosse

virus small genome mRNA is made in thecytoplasm. J. Virol. 58:647-650.

17. Sanger, F.,and A. R. Coulson. 1978. The use of thin acrylamide gels forDNA sequencing. FEBS Lett. 87:107-110.

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