Vitro Reassembly of Vesicular Stomatitis Virus Skeletons
WILLIAM W.NEWCOMB, GREGORY J. TOBIN,JOHN J. McGOWAN, ANDJAYC. BROWN* Department of Microbiology, UniversityofVirginiaSchool of Medicine, Charlottesville, Virginia 22908
Received14August 1981/Accepted 19 October 1981
Vesicular stomatitis virus (VSV) has been disrupted with nonionic detergent plus 0.5 M NaCl under conditions which result in solubilization of the viral glycoprotein (G), matrix protein (M), and lipids, leaving the nucleocapsid in a highly extendedstate.Dialysis of these suspensionstoremoveNaClwasfoundto
result in reassociation ofnucleocapsids with M protein. Reassociated structures were highly condensed and similar in appearance to "native" VSV skeletons produced by extraction of virions with detergent at low ionic strength. For instance, electron microscopic analysis revealed that, like "native" skeletons, "reassembled" skeletonswerecylindrical in shape, with diameters in therangeof
51.0to55.0nmandcross-striations spaced approximately 6.0nmapartalong the length of thestructure. Likenative skeletons, reassembled skeletonswerefound
by sodium dodecylsulfate-polyacrylamide gel electrophoresistocontain the viral N and M proteins, but they lacked the glycoprotein entirely. Both native and reassembledskeletonswerefoundtobe capable of in vitroRNA-dependent RNA synthesis (transcription). In vitro skeleton assembly required the presence of M protein and nucleocapsids. No skeleton-likestructureswereformed by dialysis of nucleocapsids in the absence of M protein or of M protein in the absence of nucleocapsids. These resultsprovidestrong supportfor the view that the VSV M protein plays afunctional role in condensing the viral nucleocapsid in vitro and raise the possibility that itmayplaya similar role in vivo.
Recent experiments in our laboratory have
emphasized the fact that NaCl can profoundly affect the disruption of vesicular stomatitis virus (VSV)bynonionic detergents(14).Extraction of native virions with60 mMoctylglucosideor1% Triton X-100 at low ionic strength results in solubilization ofthe viral glycoprotein (G) and
lipids, leaving insolublestructurescalled "skele-tons" (1, 12). Skeletons contain the viral RNA
complexed withthe N andMproteins in highly condensed andveryregularstructuresthat have
the same overall cylindrical shape and striated
appearance as the native virus. Similar struc-tures are producedby extraction with digitonin (18) or withTween 80 plus diethylether (2). In contrast, detergent extraction in thepresenceof 0.5 M
NaClresults in solubilization of the G protein, theMprotein, and lipids,liberating the
viral nucleocapsid in an irregular and highly extended state. One molar KCl was found to have a similar effect on the extraction ofSendai virus with 2%TritonX-100 (15).
Thedramatic effectof NaClon the detergent-induced disruption of VSV has motivated us to ask whether its removal from detergent-high salt-solubilized virus might promote some
de-greeof reassociation of viralcomponents. Intact
vesicular stomatitisvirions were, therefore,
sol-ubilizedin nonionic detergent containing0.5 M
NaCl and then dialyzed against a
low-ionic-strength bufferto remove NaCl. The results of
this basic experiment, described below, show
that dialysis is accompanied by the reassembly of VSV nucleocapsids and M protein to form
condensed structures quite similar to "native" VSV skeletons.
Cell and virus growth. All experiments were per-formed with the Mudd-Summers strain of VSV
(Indi-ana), which was grown on monolayer cultures of BHK-21 cells. Cellswere propagatedat 37°C in 150-cm2 plastic tissue culture flasks containing 40ml of Dulbecco modified minimal essential medium
supple-mented with10% calf serum,10%tryptosephosphate
broth, 100 U of penicillin per ml, and 0.1 mg of
streptomycinperml.Virus stocksweregrownat alow
multiplicity of infection as previously described (5)
and were shownbyrate-velocityultracentrifugationto
befree from defectiveinterfering particles.Viral pro-teinswereradioactively labeledbyincluding5p.Ciof
L-[3H]leucine (60Ci/mmol;Amersham Corp.)per ml inthe virusgrowth medium. VSV was purifiedfrom the infected cell medium by a two-step procedure
involving rate-zonal followed byequilibrium
ultracen-trifugation,essentiallyasdescribedbyHuntand Wag-ner(9). Thepurified viruspreparationswerejudgedto
befree from cellular contaminationby sodiumdodecyl
sulfate (SDS)-polyacrylamide gel analysis which
re-vealedonly thefive VSV proteins (N,NS, M, G,and
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L); nocontaminating cellular proteins could be detect-ed.
Disruption of VSV. Freshly purified VSV to be extractedwith detergent was gently suspended in 0.01 MTris-hydrochloride buffer, pH 7.4, and centrifuged for 10 min at 1,000 x gto remove virus aggregates. Thesupernatant virus suspension was then extracted with either octylglucoside(1-O-n-octyl-P-D-glucopy-ranoside;Calbiochem, La Jolla, Calif.) or Triton X-100 in thepresence of 0.5 MNaCl. Octylglucoside extrac-tion was carried out byadjusting the virussuspension to60 mM octylglucoside, 0.5 M NaCI, 5 mM dithio-threitol,10% glycerol, and 10 mM Tris-hydrochloride buffer, pH 7.8 (octylglucoside-0.5 M NaCl dissocia-tion medium) at0°C, and a concentration of 0.1 to 0.2 mg of viral protein per ml. Similar conditions were employed for Triton X-100extraction except that 1% Triton X-100 was substituted for 60 mM octylgluco-side. In bothcases virussuspensions were thoroughly butgently mixed with detergent plusNaCl and allowed tostand on ice(0°C) for 30 min before further opera-tions were performed. The initially turbid virus sus-pensions were clarified immediately upon addition of detergent plus NaCI, and the suspensions remained clearduring the 30-minperiod allowed fordisruption. Detergent extractionofVSV in the absence of NaCI wascarried out as describedpreviously (14).
Reassembly conditions. VSV components were al-lowed to reassociate during dialysis against 0.01 M Tris-hydrochloride (pH7.4)-S5mMdithiothreitol (TB). Four-milliliter samples of VSVdisrupted in detergent plus 0.5 M NaCI as described above were dialyzed against TB for 16 h at4°C and foranadditional 12 hat
25°C.Ifdialyzedsolutionswerenotanalyzed immedi-ately, theywerestoredonice foramaximumof 4 h. Ultracentrifugation. Insoluble materialwaspelleted
fromsuspensions of dissociated VSVorfrom reassem-bly mixtures byultracentrifugation in 5-ml Beckman SW50.1 nitrocellulose tubes. Four-millilitersamples of dissociated or reassembled viral components were layered on topof a 0.5-ml "cushion" ofglycerol and werecentrifuged at 38,000 rpm (130,000xg) for 2 h at
4°C.The fluid above andincludingtheinterfaceofthe glycerol pad wasaspirated while the remainder of the pad, containing thepelleted material,wasmixed with 0.5 mlofTBanddialyzedovernightat4°Ctoremove glycerol. Dialyzed samples were used directly for electron microscopic and transcription analyses or werelyophilizedprior toSDS-polyacrylamide gel elec-trophoresis.
Both rate-velocity and sedimentation equilibrium sucrose gradient methods were employed to purify
subviralstructures fromdissociated and reassociated VSVcomponents. Rate-velocity analyses began with 0.5 ml ofsample whichwaslayeredonto thetopof10 to70%(wt/vol)linearsucrosegradientspreparedin 5-ml Beckman SW50.1 nitrocellulose tubes. Stock su-crosesolutionswereprepared bydissolvingultrapure sucrose(Schwarz/Mann)inTB.Gradientswere centri-fuged at38,000 rpm for 90min at4°C inanSW50.1 rotor. After centrifugation, the gradients were frac-tionated from the bottomby use ofaperistalticpump andanLKB-7000 fraction collector.Theradioactivity
present in each fractionwasdeterminedby dissolving
20-piusample in 0.5 ml of NCS tissue solubilizer
(AmershamCorp.)containing10ml oftoluene-based scintillation cocktail(Research ProductsInternational,
Grove Village, Ill.) and counting in a Packard model 3320 liquid scintillation spectrometer. Gradient frac-tions to beanalyzed further were dialyzed against TB overnight at 4°C to remove sucrose and then were stained directly for electron microscopy or lyophilized for SDS-polyacrylamide gel analysis. Equilibrium ul-tracentrifugation was carried out by the same method described for rate-velocity centrifugation except that centrifugation was for 16 h rather than 90 min. The density of each gradient fraction was determined by weighing a100-,ul sample on a Mettler H18 analytical balance.
In vitro RNA-dependent RNA synthesis and poly-acrylamide gelanalysis of RNA. Native and reassem-bled skeletons were tested for the presence of endoge-nousRNA-dependent RNA polymerase activity by the assay ordinarily employed for intact VSV (4). Assay mixtures contained 0.14 M NaCl,0.2% TritonX-100,
7.5 mM MgCl2, 0.01MTris-hydrochloride, pH 7.9, 1 mMdithiothreitol, 0.3 mM ATP, GTP, andCTP, and
[a-32P]UTP(0.45Ci/mmol) plus 25 to 35 ,g of
viral protein in a total volume of 30,lI. Incubations for up to 3 h werecarried out at 31°C, after which
5-RIsamplesof the reaction mixture were precipitated in 1 ml of5% trichloroacetic acid. Precipitated RNA was collected by filtration on 24-mm filters (Millipore Corp.), dried, and counted at an efficiency of 65% in toluene-based liquid scintillation cocktail. Results were expressed as nanomoles of[32P]UMP incorporat-ed per minute per milligram of protein.
Other methods. Electron microscopicanalysis was
carriedoutwith subviral structures negatively stained with2% phosphotungstic acid (pH 7.0) as described previously (14). Particle measurements were made on
positive enlargements of electron microscope nega-tives.SDS-polyacrylamide gel electrophoresis and gel staining with Coomassie blue were performed on 2-mm-thick slab gels as described by Nagpal and Brown (13).Seven-millimeter lanes were loaded with samples containing 50 to 100 ,ug of viral protein. All gels containedalane, labeled "std," for electrophoresis of solubilized VSV, andpositions of the five VSV pro-teins (L, G, N, NS, and M) areindicated. All protein concentrations were determined by the Lowry method (11).
Disruption ofVSV in nonionic detergentplus NaCl. Vesicular stomatitis virions were disrupt-ed innonionicdetergent
plus0.5 MNaCl under
conditions shown previously to solubilize the viral envelope components and release the nu-cleocapsid in an uncondensed form (14). The extent of detergent-NaCl-induced virus
by sedimentation velocity ultracentrifugation,
SDS-polyacrylamide gel electrophoresis.
Figure 1A shows a negatively stained
prepara-tion of insoluble structures
disrupted with 60 mM
NaCI bycentrifugationfor 2 hat130,000x gas described in Materials and Methods. Pelleted material was found to contain only extended
viral nucleocapsids (6); no intact VSV or
con-densed VSV skeletons could be detected. It
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REASSEMBLY OF VSV SKELETONS 1057
FIG. 1. Electron micrographs ofdetergent-extracted VSV and reassembled VSV skeletons. (A) Nucleocap-sids isolatedbycentrifugationof solutionsproducedbyextractingVSV with 60 mMoctylglucoside plus0.5 M NaCl. (B) Skeletons isolated by centrifugation of suspensions produced by extracting VSV with 60 mM octylglucoside. (C) Reassembled skeletons (arrows) identifiedby direct observation ofthesuspension resulting fromdialysis of VSV solubilized with60 mMoctylglucoside-0.5MNaCl. (D) Reassembled skeletons isolated by
centrifugationof thesuspension resulting fromdialysis of VSV solubilized with60 mMoctylglucoside-0.5 M
NaCl.(A) x105,000;(B,C, and D) x125,000. Bar= 0.2 ,.m.
must be, therefore, that all or nearly all intact virus particles were disassembled.
Figure 2A shows the results obtained when
VSV,grownin the presenceof[3H]leucine,was disrupted in 60 mM octylglucoside plus 0.5 M
NaCl and analyzed by rate-velocity
sedimenta-tionon10 to70%sucrosegradients. Two bands
of3H-labeled viralproteins were observed, one
(bandII) at the top of the gradient where soluble
proteins should occur and the other (band I) VOL. 41,1982
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0 X 10
FIG. 2. Rate-velocity ultracentrifugation of VSV solubilized with 60 mM octylglucoside-0.5 M NaCI
before (A) and after (B) dialysis against TB. VSV employed in these experiments was radioactively la-beledby growth in the presence of[3H]leucine.
fartherdown the gradient. Electronmicroscopic
analysisof bandIdemonstrated that it contained
extended viral nucleocapsids
indistinguishablefrom those shown in Fig. 1A.
SDS-polyacryl-amide gel analysis, as shownin Fig. 3, demon-strated that band I contained predominantly N
protein (lane 2) whereas bandIIcontained both G and M proteins (lane 1). Virtually identical results of bothelectronmicroscopicand sucrose
gradientanalyses wereobtainedwhen1% Triton
X-100was substituted for 60 mM octylglucoside
in the virus dissociation medium. We conclude
that disruption of VSV in nonionic detergent plus0.5 MNaCl results in solubilization of the G and Mproteins and release ofthe viral nucleo-capsidin an extended state.
Reassociation ofVSV components: electron mi-croscopic analysis.Reassociation of viral
compo-nents wasassayed after vesicular stomatitis viri-ons were disrupted in nonionic detergent containing 0.5 MNaCl and then dialyzedagainst
TB. Dialysis was expected to remove detergent
andNaCl in the case of virus disrupted in octyl-glucoside-0.5MNaCl and NaCl onlyin the case of Triton X-100-0.5 M NaCl disruption (8, 17). Dialysis was found to promote reappearance of turbidity in initially clear solutions of viral com-ponents. Turbidity increased gradually during the first 12 to 18 h of dialysis and did not change thereafter. Direct electron microscopicanalysis
ofdialyzed suspensions revealed the presenceof condensed structures, as shown in Fig. 1C.
Extended nucleocapsids were very rare or ab-sent altogether. Condensed structures all
ap-peared to be made up of a fine filament or thread. In some cases the thread or threads
appeared tobe clumped in an irregular way to form roughly spherical particles ("tangled
clumps"),asshowninthe upper left of Fig. 1C.
Quite often, however, condensed particleswere
cylindrical in shape with cross-striations over the central portion of the overall structure and
irregularly packagedthreads at the ends (arrows in Fig. 1C). The cylinder diameter and the
-FIG. 3. SDS-polyacrylamide gelanalysisof subvi-ralstructuresseparated byrate-velocity
ultracentrifu-gationof VSV solubilized with60 mM
octylglucoside-0.5 MNaCl before(lanes 1 and2) and after(lanes3 and4)dialysis againstTB. Lane 1: bandII fromFig.
2A. Lane 2:bandIfromFig.2A. Lane 3:bandIIfrom
Fig. 2B. Lane 4: bandI fromFig. 2B. Thegel was stained with Coomassie blue, and the positions of standardVSVproteinareindicated.
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spacing between cross-striations were found to
be similartothose observed for "native" VSV skeletons,asshownin Table1. Thelength ofthe
cross-striated region was variable but never greater thanthelength ofnative skeletons. The morphological similarity ofnative and reassem-bled skeletons was emphasized by electron mi-croscopic analysis of insoluble material pelleted from reassembly mixturesbyultracentrifugation
at 130,000 x gfor 2 h. Reassembled skeletons harvested in this way were indistinguishable
from native skeletons, as shown inFig. 1Dand B.
Control experiments showed that VSV
nu-cleocapsids and soluble components must be present together for striated skeletons to form
during dialysis.Theseexperiments involved dis-assembly of native virions in60 mM octylgluco-side plus 0.5 M NaCl as described above and
thencentrifugationto removethenucleocapsids from solution. The
supernatantcontaining solu-bilized viral G protein, M protein, and lipids (soluble components) was then dialyzed against
TB under the same conditions employed for reassembly of viral skeletons. Similarly, the isolated nucleocapsids were resuspended at a
concentration of 0.1 mg ofprotein per ml in octylglucoside-0.5MNaCldissociation medium and dialyzed against TB. Electron microscopic analysis ofthe two dialyzed fractions revealed
thatneither contained skeletonsorskeleton-like
structures. Isolated nucleocapsids gave images similartoFig. 1Abothbefore and afterdialysis. Dialysis of soluble components resulted in for-mation of a precipitate which
centrifugationat600 x gfor 5 min.
SDS-poly-acrylamide gel analysis showed that the
precipi-tatecontainedMprotein only (datanotshown); theglycoprotein remained in the supernatant.
Protein composition of reassembled skeletons.
The protein composition of reassembled
skele-tons wasdeterminedby SDS-polyacrylamide gel analysis ofstructures purified from reassembly mixtures by sucrose density gradient
ultracen-trifugation.Preparation of reassembled
skele-tons began with VSVgrown in thepresence of
[3H]leucine.Purified virions were disrupted in nonionic detergent plus 0.5 M NaCl, and the resulting solutions weredialyzed toreassemble skeletons as described above. Reassembled
skeletons were purified from the dialyzed
sus-pensions by rate-velocity ultracentrifugation in one set of experiments and by sedimentation equilibrium in another. Figure 2B shows the
results of rate-velocity purification performed with skeletons reassembled after disruption of VSV with 60 mM octylglucoside plus 0.5 M
3H-labeledviral components werefound
II)near the top of the gradient and the other (band
cen-TABLE 1. Dimensions of native and reassembled VSVskeletonsa
Spacingof Skeletons Length (nm) Diam(nm) striations
Native 204.4 ± 6.00 51.7 ± 3.00 5.7± 0.75
Variable'53.7 ± 2.70 6.2 ± 0.32 a Results are reported as the mean value + 1 stan-darddeviation for at least 20 measurementsof
repre-sentativeparticles.Datafornativeskeletonsarefrom Newcomb and Brown(14).
bThe length of the cylindrical, striated portion of
reassembled skeletons was variable but nevergreater than 200.0 nm.
analy-sis revealed the presence of reassembled skele-tons inband I, but not inband II (micrographs
notshown). SDS-polyacrylamide gel analysis of band I from Fig. 2B (reassembled skeletons) demonstratedthat it contained the viral M and N
proteins, as shown in Fig. 3, lane 4. No G
protein was detected in reassembled skeleton
preparations; G proteinwas foundinband II at
the top of the gradient (see Fig. 3, lane 3). Results essentially identical to those shown in
Fig. 2 and 3 were obtained when 1% Triton
X-100wassubstituted for 60mMoctylglucoside in
thevirus dissociation medium.
When dialyzed reassociation mixtures were
centrifuged to equilibrium, two bands of
3H-labeled viral components were observed, one (band I) with abuoyantdensity ofapproximately
1.26g/cm3and theother(band II)nearthetop of the gradient. Electron microscopic analysis demonstrated thepresenceof reassembled
skel-etonsin bandI;none werefound in band II.
SDS-polyacrylamide gel analysis of band I material showed that M and N were the predominant
protein components present (seeFig. 4, lanes 1 and 3). As inthe case ofreassembledskeletons
purified by rate-velocity centrifugation, little or no G protein could be detected in reassembled skeletons purified by equilibrium sedimentation. Instead, G protein was found in band II at the
topofequilibrium gradients,asshown inFig. 4,
lanes 2 and 4. The identity of the disrupting detergent did not substantially affect eitherthe buoyant density or the protein composition (comparelanes 1and3inFig.4)ofreassembled
skeletons. Both parameters were virtually the samein skeletonsreassembledfrom virions dis-rupted with octylglucoside-NaCl and with Tri-tonX-100-NaCl.
RNA-dependent RNA polymerase activity. In vitro assays showed that both native and
reas-sembled skeletons containedendogenous
RNA-dependentRNApolymeraseactivity. Their spe-cific polymerase activities were found to be 41,
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Std 1 2
d-FIG. 4. SDS-polyacrylami sembled skeletons and solubl by sedimentation equilibrit Lane 1: skeletons (band I) i
solubilized with60 mM octy Lane2:solublecomponents
reassembly of skeletonsfrom mM octylglucoside-0.5 M N (band I) reassembledfrom V Triton X-100-0.5 M NaCl. I nents (bandII) remaining aft tonsfromVSVsolubilized wi MNaCl. Gelswere stainedw
the positions of standard VSN
comparable to the activit nucleocapsids,asshown ir
tially lower than the spec
virions assayed under t] Polyacrylamide gel analys
patternofsimilarity in the sized by reassembled sk
tons, and native VSV. I RNA (3) and many
RNA species were syi
DISCUSS The results reported I
formation ofVSV skelet( reassembly took place dui of solutionsproducedbyd
ularstomatitis virions inn
taining 0.5 M NaCl. The sembled under these con
be virtually indistinguisi VSV skeletons isolated b ruswithnonionicdeterger (14). For example, both n, skeletonswere foundtob
ders, with diameters of5
tions spaced approximatt native andreassembled si viral N protein, M proteil
3 4 Std protein entirely.
tons, reassembled skeletons were found to be
capableof in vitro
syn-thesis. There can be little question, therefore,
thatreassembled skeletons arefaithful replicas
oftheir native counterparts.
It is most likely that removal of salt, not
removal of detergent, was responsible for
pro-G moting skeleton formation in vitro. Skeletons
oz1 i NS formedabundantly during dialysisof virus
de-tergent and NaCl were removed. They also formed normally from virus solubilized with
so_ Triton X-100-NaCl when only NaCl was
re-_ -111 M moved. The presence of detergent, therefore,
did not appear to affect skeleton formation in
vitro, but thepresenceof NaCl clearly did. Salt de gel analysis of reas- wasfoundtohaveasimilareffectontheability
ecomponents separated of Semliki Forest virus glycoproteins to
asso-reassembledntfrfoumga iSnVciatewithSemliki Forest virus nucleocapsids in eglucoside-0.5 M NaCl. vitro. Glycoprotein-nucleocapsid interaction
(band II) remainingafter was observed in 24 mM octylglucoside-10 mM
VSV solubilized with60 sodiumphosphate buffer, pH 6.8,but not in the laCl. Lane 3: skeletons same solutioncontaining 0.1 M NaCl atpH 8.0
'SV solubilized with1% (7).
Lane 4: soluble compo- Althoughmatureskeletonswerethe
predomi-terreassembly of skele- nant structuresformedduringdialysis of
solubi-ith 1%Triton X-100-0.5 lized VSV, other related structures were also
ithCoomassieblue,and observedatlower frequency. These included the
Vproteinsareindicated. "tangled clumps" described above andtangled
clumpscontaining central cross-striations. Tan-ty observed for VSV gledclumps appearedtoconsist ofafinethread
iTable2, but substan- wound in an irregular way to form a roughly :ific activity of intact spherical particle. The thread diameter was he same conditions. smaller than the diameter of VSV nucleocap-is revealedan overall sids, and no nucleocapsids were found to
pro-RNAspecies synthe- trude from tangled clumps. Cross-striated
skele-n all cases "leader"
gher-molecular-weightnthesized (data not
here demonstrate the ons in vitro. Skeleton ring overnight dialysis
lissolvingnative vesic-onionicdetergent con-skeletons which reas-ditions were found to
hable from "native"
y extracting intact vi-at vi-atlowionicstrength ,ativeandreassembled
cylin-il to 55 nm and
stria-ely 6 nm apart. Both[image:6.4188.8.131.52.231.2]
keletons containedthe n, andRNA, but both
TABLE 2. RNA-dependent RNA synthesis by native skeletons, reassembled skeletons, and
Specific % of native Preparation polymerase
NativeVSV 1.78 100
NativeVSV skeletons 0.45 25
Nucleocapsids 0.44 25
Reassembled skeletons 0.38 21
Reassembled skeletons 0.89 50
a Native skeletons and nucleocapsids were prepared by disruption of VSV in 60 mM octylglucoside in the absence of salt and in the presence of 0.5 M NaCl, respectively, asdescribed previously (14). Reassem-bled skeletons were prepared by dialysis of VSV solubilized with Triton X-100-NaCl or octylglucoside-NaClasdescribed in Materials and Methods.
bExpressedasnanomolesof[32P]UMP incorporat-edper minute permilligram of protein.
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REASSEMBLY OF VSV SKELETONS 1061
gled clumps were similar in appearance to
tangled clumps except that they contained a central cylindrical region with regularly spaced striationsandtangledclumplikematerialatboth ends. The centralcylindricalregionwasof
vari-ouslengths indifferentparticles, but its diameter and striation spacing were the same as those found inmatureskeletons. Particles of thistype can be seen in Fig. 1C (arrows). We presume
that theminor components (tangled clumps and cross-striated tangled clumps) found in reassem-bly mixtures are intermediates in the skeleton self-assemblyprocess.Their structural
relation-shiptomature skeletons suggeststhat skeleton
reassembly invitro took place inthe following way. Nucleocapsids and M protein aggregated
to form tangled clumps in which condensed nucleocapsids weresurroundedbyafinethread
composed of M protein. Cross-striations were
then formed by subsequent
internalrearrange-ments in the tangledclump structure. This
pro-posed pathwayforskeleton reassembly is shown
schematicallyin Fig. 5. Further experimental
studies will be requiredtosubstantiate its role in skeleton reassembly.
The results described in this paper indicate that Mprotein musthave avery strong affinity for VSV nucleocapsids. Thiscanbeappreciated from thefact that veryfew,ifany,free nucleo-capsids remained after dialysis of virions solubi-lized with detergent-0.5 M NaCl;
were complexed with M protein in condensed
structures(see Fig. 2B).
protein became associated with nucleocapsids. Very little remained in the soluble fraction, as
shown inFig. 3,lane3, orFig.4, lanes2and4.
This contrasts tothe behavior of the glycopro-teinwhichwaspresentduringdialysis of solubi-lized VSV, butwas notincorporated into
reas-sembled skeletons. The interaction of nucleocapsids with M protein must, therefore, bevery strongandatleast somewhatspecific.
The abundantformation of VSV skeletons in vitro raises the possibility that similarstructures may be formed in vivo. Calculations based on the data ofSimonsen et al. (16) show that the molar concentrations of nucleocapsids and M
compa-rableto or greater than their concentrations in
thereassemblyexperiments described here.
De-pending on the values assumed for the cell
volumeand the time after infection, the
concen-tration of negative-strand nucleocapsids in
fallsinthe range of2 x
10-9to 4 x
proteinisapproximately 2 x
10-6to 5 x 10- M. The concentrations employed forinvitro skeleton
experiments were 1 x
10-9to 2 x 10- M for
nucleocapsids and 1.2 x
10-6to 2.4 x
nucleocap-a. b. C. d.
FIG. 5. Proposed pathwayfor invitroreassembly
ofVSV skeletons.(a) Tangled clumps, (bandc)
cross-striated tangled clumps, and (d) reassembled
sids and Mprotein are, therefore, high enoughto support skeletonformation in infected cells.
Experimentalresults ofKnipeetal. (10)also
suggest thatMprotein may associatewith VSV
nucleocapsids invivo. These authors examined CHO cells infected with a strain of
tsM601, which carries a temperature-sensitive
lesion in the N protein. An unusuallylow level ofVSV nucleocapsids accumulated in cells
in-fected with tsM601 at the nonpermissive
andlittlefull-length (42S)viral RNA was
synthe-sized. Incontrast,G proteinwasmade innormal amountsand itmigrated normallytothe
plasmamembrane. M protein was also synthesized in normal amounts, but it accumulated in soluble form in the cytoplasm rather than becoming attachedtomembranesasitdoesincells infect-ed with wild-type VSV. This is the result that
would be expected ifMprotein could associate
with membranes only after it had bound to
We thankNancySalomonskyandMargiePhippsfor techni-calassistance.
This workwas supported byNational Institutes of Health contractN01-NS-8-2391 and by Public Health Service grant HD-06390from the National Institutes of Health.
1. Cartwright, B., C. J. Smale,and F.Brown.1970. Dissec-tion of vesicularstomatitis virus into the infective ribonu-cleoprotein andimmunizingcomponents.J. Gen. Virol. 7:19-32.
2. Cartwright, B., P. Talbot, and F. Brown. 1970. The proteins ofbiologically active sub-units of vesicular sto-matitis virus. J. Gen. Virol. 7:267-272.
3. Colonno,R.J.,andA. K.Banerjee. 1976.AuniqueRNA speciesinvolved ininitiationof vesicular stomatitis virus RNAtranscription in vitro. Cell 8:197-204.
4. Emerson, S. U., and R. R. Wagner. 1972.Dissociation and reconstitution of the transcriptase and template activities of vesicular stomatitis B andTvirions. J.Virol. 10:297-309.
5. Fong,B.S.,R.C.Hunt, and J. C. Brown. 1976. Asymmet-ricdistribution ofphosphatidylethanolamine in the mem-braneofvesicular stomatitis virus. J.Virol. 20:658-663. 6. Heggeness, M.H., A. Scheid, and P. W. Choppin. 1980.
Conformation of the helicalnucleocapsids of paramyxo-viruses and vesicular stomatitis virus: reversible coiling anduncoiling induced by changes insaltconcentration. Proc. Natl.Acad. Sci.U.S.A. 77:2631-2635.
7. Helenius, A., and J. Kartenbeck. 1980. The effects of octylglucoside on the Semliki Forest Virusmembrane. Eur.J. Biochem. 106:613-618.
on November 10, 2019 by guest
8. Helenius, A., and K. Simons. 1975. Solubilization of membranes by detergents. Biochim. Biophys. Acta 415:29-79.
9. Hunt, D. M., and R. R. Wagner. 1975. Inhibition by aurintricarboxylic acid and polyethylene sulfonate of RNAtranscription of vesicular stomatitisvirus.J.Virol. 16:1146-1153.
10. Knipe, D. M., D. Baltimore, and H. F. Lodish. 1977. Maturation of viral proteins in cells infected with tempera-ture-sensitive mutants of vesicular stomatitis virus. J. Virol. 21:1149-1158.
11. Lowry, 0.H., N. J. Rosebrough, A. L. Farr, and R. J.
Randall.1951. Proteinmeasurementwith the Folin phenol
12. Miller,D. K., B. I. Fever, R. Vanderoef, and J. Lenard. 1980.Reconstituted G protein-lipid vesiclesfrom vesicu-lar stomatitis virus and their inhibition of VSV infection. J. Cell Biol.84:421-429.
13. Nagpal, M. L., and J. C. Brown. 1980. Protein and glycoprotein componentsof phagosome membranes
de-rivedfrommouseLcells. Int. J. Biochem. 11:127-138.
14. Newcomb,W. W., and J. C. Brown. 1981. Role of the vesicular stomatitis virus matrix protein in maintaining the viral nucleocapsid inthe condensed form found in nativevirions. J. Virol. 39:295-299.
15. Shimizu, K., and N. Ishida.1975. Thesmallest protein of Sendai virus: its candidate function of binding nucleocap-sidtoenvelope. Virology 67:427-437.
16. Simonsen, C.C., S. Batt-Humphries, and D. F.Summers. 1979.RNAsynthesis of vesicular stomatitis virus-infected cells: in vivoregulationofreplication.J. Virol. 31:124-132.
17. Stubbs, G.W.,H.G. Smith, Jr.,andB.J.Litman.1976. Alkylglucosidesaseffective solubilizingagentsforbovine rhodopsin: a comparison with several commonly used detergents. Biochim. Biophys. Acta 426:46-56. 18. Wagner,R. R., andC.A.Schnaitman. 1970. Proteins of
vesicular stomatitis virus,p.655-671.In R. D.Barryand B. W.J. Mahy (ed.),ThebiologyoflargeRNA viruses. AcademicPress, Inc.,New York.