Electron Microscopic Characterization of the Defectiveness of a Temperature-Sensitive Mutant of Moloney Murine Leukemia Virus Restricted in Assembly

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VIROLOGY, Oct. 1977,

Electron Microscopic Characterization of the

Defectiveness

of

a

Temperature-Sensitive

Mutant

of

Moloney

Murine

Leukemia

Virus

Restricted

in

Assembly

P. H. YUEN AND P. K. Y. WONG*

Department of Microbiology and School of Basic Medical Sciences, University of Illinois, Urbana, Illinois61801

Received for publication 6 May 1977

The effect of temperature shiftdown on the assembly of ts3 virions was

investigated by bothscanning (SEM)andtransmission (TEM) electron

micros-copy. Ts3 is a spontaneous temperature-sensitive mutant of Moloney murine leukemia virus (Mo-MuLV) which previous studiesindicatedtobe defectivein

assembly or release of the virions. Inthe present study, both SEM and TEM revealedthe following: (i) there were morecell-associated virionsints3-infected

cells grown at the nonpermissive temperature (390C) than either in cells grown

atthepermissivetemperature (340C) or in wild-type MuLV-infected cells grown

at

39QC;

(ii) there were morenormal single particles than multiploids (virions

with two or more pieces ofgenomic RNA) in ts3-infected cells grown at the

nonpermissive temperature; (iii) there were more multiploids in ts3-infected

cellsgrownatthenonpermissive temperature than either in cells grownatthe permissive temperatureor inwild-type MuLV-infectedcells grownatthe

non-permissivetemperature; (iv)upontemperature shift from 39to34WC,about90%

ofthecell-associatedvirionsdissociatedfromthe cell surface. TEM studiesalso

indicated that upon temperature shiftdown, virion assembly rapidly occurred. The above observations suggest that faulty assembly, which results in the production ofmultiploids, may not be the reason why ts3 virions accumulate on

the cell surfaceatthe nonpermissivetemperature.Therelativelyhigher

propor-tionofmultiploidsfoundints3-infected cells grownat39°Ccomparedwiththose

grown at34°C may be due tothe higher density ofbuddingvirions atthe cell

surfaceatthenonpermissivetemperature,which increases thepossibilityoftwo

or more particles assembling close to one another. The accumulation of ts3

virions in all stages ofassembly at the nonpermissive temperature, together

with the fact thatrapid assemblyand releaseofts3virionsoccurredon tempera-ture shiftdown, indicates that virion assembly is restricted after it has been initiated. Theprobablerole ofalteredglycoprotein(s)inrestrictingvirion

assem-blyisdiscussed.

One of the major eventsinthe replication of RNA tumor viruses is the assembly of the

viri-ons at the cell membrane. As yet the

mecha-nisminvolved ispoorly understood. It has been postulated (1, 4) that assembly of the virions is regulated by protein-protein interactions. One

approach toward understanding the processes

involvedinthe assembly of RNA tumor viruses is by defining the temperature-sensitive (ts) defects of viral mutants blocked in assembly. Recently, such mutants from both avian and mammalian RNAtumorviruses have been iso-latedand partially characterized (2, 3, 6-9, 11).

Ts3 is a spontaneous ts mutant of Moloney murineleukemia virus(Mo-MuLV) isolated by

Wong et al. (9). Previous studies (7, 8) have

indicated that the ts defect of ts3 may be dueto ablock either late intheassembly

proc-ess orintherelease ofthe virions. The present

investigation on the assembly of ts3 virions

further defines thets defect ofts3.

MATERIALS AND METHODS

Cells. Detailedproceduresfor thepropagationof TB cells, a fibroblastic cell line established from mixed cultures of bonemarrowandthymusCFW/D mouse cells, have beenpreviouslydescribed (7).

Virus. TB cellswere infectedin suspensionat a

multiplicity of infection of 5 with wild-type Mo-MuLV orts3,a ts mutantof MuLV. Infected cells(3 x 10') wereplated on each 60-mm plastic petri dish and incubatedatthe permissive (34°C) or nonper-missive (39°C) temperature for 48 h. Ts3-infected 222

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ts MUTANT OF MuLV RESTRICTED IN ASSEMBLY 223

cellswerealsoincubatedatthenonpermissive

tem-peraturefor48hand then shiftedtothepermissive

temperaturefor30or60min.

Transmission electron microscopy(TEM).Viral

pelletsandwild-typeMuLV-orts3;infectedTBcells

scraped offwitharubberpolicemanwerefixedin 2%

glutaraldehyde in sodiumcacodylatebufferor

phos-phate-buffered salineandthen postfixedin 2%osmic

acid overnightor inafreshly prepared mixtureof

0.12gof potassiumferrocyanidein 6mlof1%osmic

acid. After dehydrationthroughgraded ethyl alco-hol, the cellswereembedded in Spurr'splastic.

Sec-tionswere mounted directlyon300-meshgrids and

stainedinuranylacetate at70°Cinawaterbathand

inReynoldslead citrate. Specimenswereviewed in

a Siemens 102 electron microscope at 80kV. For

quantitationofvisions inthin sections,cellsections

werechosenatrandom,and the typeofvirionand

thestageinassembly of thevisionswerescoredby

viewingatamagnificationof x80,000.

SEM. The details for preparing infectedcellsfor

scanning electronmicroscopy (SEM)and

quantita-tion of the cell-associated virions have been

de-scribed(8).

RESULTS

Relative proportion of normal C-type particlestovirionaggregatesints3-or

MuLV-infected cellsasdetermined by SEM.We

rea-soned thatif mostofthets3particlesproduced

at the nonpermissive temperature (39°C) are

"polymorphic" as reported by Wong and

Mc-Carter (7), in contrasttotheproduction of

nor-mal C-type particlesatthepermissive

tempera-ture (34°C), faulty assembly of the virion at

39°C may beindicated. Ts3-infected cellswere

therefore grown at 34 or 39°C for 48hor 39°C

for48 h and then shifted to 34°C for 15 or 60

min; MuLV-infected cells were grown at 39°C

for 48 h and preparedfor SEMasdescribed in

Materials andMethods.

Figure 1A shows arepresentative portion of

the cell surface of a ts3-infected cell at the nonpermissive temperature 48hpostinfection. Most of the particles present were normal

C-type spherical forms. Scattered among them wereduplexes(black arrow) and short

microvil-lus-like structures (white arrow), which may

consist oftwoor morevirions alignedin

tan-dem(Fig. 1B). Triplexes (Fig. 1C and D), quad-ruplexes (Fig. 1E), and largeraggregates (Fig.

1F and G) were also observed in ts3-infected

cells grownat the nonpermissive temperature orgrownatthenonpermissivetemperaturefor 48hand thenshiftedtothepermissive temper-ature for 15min.

In ts3-infected cells grown at 34°C and in

MuLV-infected cellsgrownat39°C, the number

of particles observed on the cell surface was

greatly reduced as compared withts3-infected

cellsgrownatthe nonpermissivetemperature.

Duplexes and occasionally triplexes were also

observed in the former cultures; however,

larger virion aggregates were not found.

The relative proportions of normal C-type

particlestoaggregatesinthe different cultures

examined are shown in Table 1. The figures presented in Table 1 may be only rough esti-mates,because onlyvirions inthe latestagesof

assembly could be identified with certainty.

The total number of aggregates may also be underestimated, since rod-shaped virions may be mistaken for microvilli. Atypically shaped or

unusually large spherical particles were not

included in the counts recorded in Table 1.

Since the same types of errors were

encoun-tered inall the samples examined, the figures

presented in Table 1 should be anindicationof

real differencesamongthe samples studied.

The resultspresented in Table 1 may be

sum-marizedasfollows: (i) there were more virions

onthe cell surface of ts3-infectedcells grown at

390C thanonts3-infected cellsgrown at34WC or

onMuLV-infected cells grown at39"C;(ii) more

than90%ofthe ts3 particles that had

accumu-lated on the cell surface at 390C disappeared

from the cell surfacewithin 1hafter

tempera-ture shiftdown; (iii) there were more normal

single particles than aggregates inthe

ts3-in-fected cellsgrown at390C; (iv)there were more

aggregates in the ts3-infected cells grown at

390C thanineither ts3-infectedcellsgrown at

34WC orMuLV-infected cells grown at390C. Theabovefindingssuggestthat formationof

morphologically aberrant particles may not be

thereasonwhyts3 virionsfail tocomplete

as-sembly and release from the cell surfaceatthe

nonpermissive temperature. The relatively

higher proportion of aggregates found in the ts3-infected cells grown at390C couldbe due to

thehigherdensity ofbuddingvirions atthe cell

surface, which increases theprobabilityoftwo

or more particles assembling close to one an-other. Furthermore, the accumulation of viri-ons atthe nonpermissive temperature may be attributed eithertoablockinsomeprocesses)

in virion assembly or to a ts defect in the release of the virion per se.This latter possibil-ity would be indicated ifmost of the particles werecompletelyassembled, but failed to

disso-ciatefrom the cell surfaceatthenonpermissive

temperature.

TEMstudies.Toinvestigatetheabove possi-bilities, TEM studies were conducted on

ts3-infected cellstodetermine thestageinassembly

at which the assembly of ts3 is blocked at the

nonpermissive temperature, and whether fur-ther assembly of these ts3 virions can take

place ontemperature shiftdown.

Ts3-infected TBcells grown at 34 or 39°C for

48h, and ts3-infectedTBcellsgrownat39°Cfor

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YUEN AND WONG

r,

-FIG. 1. (A) SEMofaportion ofa ts3-infected TB cell surface grown at the nonpermissive temperature showing normalspherical virionsaswellasduplexes (blackarrow) and short microvilli that may be short rod-shapedvirions(white arrow). Scale=500 nm.(B-G)Scale=250nm. (B) Shortrod-shaped virions; (C andD) triplexes;(E) quadruplexes; (FandG)largevirionaggregates.

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48 hand thenshifted to34TC for 30 or60min,

wereprepared for TEMasdescribed in Materi-als and Methods.

Ts3 virons. Thin-section electron microscopy ofts3-infectedcells grownatthenonpermissive temperature revealed the presence of both

nor-mal C-type particles andmultiploids (particles

with more than one ribonucleoprotein compo-nent) in all stages ofassembly. The structure

and assembly of both types of particles have been reportedrecently (12).

Effect of temperature shiftdownon the

as-sembly of ts3 virions. A composite view of

ts3-TABLE 1. Relative proportionsofnormalC-type particles to aggregatesa ints3-orMuLV-infected

cultures determinedby SEM

Total cell Total no. % of ag-surfaeof art'- gregates Type ofculture srfaex of 1 tototal

area exam- cles/100- no. of par-ined (Am') Am2area

tiles

ts3,390C(48h) 705 250 18

ts3, 39 -- 34"C 326 228 18

(15min)

ts3,

39-34"C 285 11 3

(60min)

ts3,340C (48 h) 367 38 8

MuLV, 39"C 825 29 8

(48h)

aDuplexes, triplexes, etc.

infected cells grown atthe nonpermissive

tem-perature for 48 h is shown in Fig. 2. TEM indicates that although both normal particles andmultiploidsindifferent stagesinassembly were present ints3-infected cells grown at the nonpermissive temperature, less than 20% of these particles werecompletely assembled (Fig. 2d) and on the verge of dissociating from the cellsurface. This finding is consistent with the infrequent observation of extracellular

imma-tureC-type particles and the virtual absence of mature C-type particles in the intercellular spacesof ts3-infected cells grown at the nonper-missive temperature (Fig. 3A). The above ob-servations suggest that at the nonpermissive temperature, virion assembly is initiated but probably restricted. It was further reasoned that if ts3 is temperature sensitive in some

factor (function) restricting assembly, on

tem-peratureshiftdown,oneshould be able to detect rapidvirion assembly andrelease. This would be reflected ina change in the composition of virions in the different stagesofassembly. In anattemptto verify the abovehypothesis, the relative proportions of virions in the various stagesofassembly were comparedinthe follow-ing cultures: ts3 grown at 390C for 48 h, ts3 grownat39°Cfor48 h and thenshiftedto 34°C for 30 or 60min, andts3grown at34°Cfor48h. The normal budding C-type particles were

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FIG. 2. Composite electron micrograph ofa ts3-infected TB cell grown at the nonpermissive temperature for 48h.Scale =100nm.(a)Normal virion at an early stage in assembly with anarc-shapedribonucleoprotein

(RNP).(b)Normal virionat anintermediate stageinassembly with a semicircular RNP. (c) Spherical duplex

atalate stage in assemblyshowing a break in the RNP (arrow) and a larger size and broader linkage to the cellcompared with (d). (d) Normal virion at a very late stage in assembly showing complete assembly of the

RNP,narrowlinkagetothecell, and resealing of the cellular membrane. (e) Normal virion at a very late stage inassembly sectioned through the core shell. (p) Multiploid virion at an early stage inassembly showing breaksinthe RNP and broadlinkage to the cell. (g) Multiploid during the final stages in assembly showing similarcharacteristics as pointed out for the normal virion shown in (d).

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FIG. 3. (A)Uncommon viewshowinganaccumulationofbuddingvirions(a), completeassembled normal

virions (b), andmultiploids (c) in the intercellular spaceofts3-infected cellsgrownatthe nonpermissive

temperature.Scale=200nm.Not all thecompletelyassembled virionsarenecessarily extracellular,since the

section could havebeencutthroughthetransverseaxisofsomeofthe virions. (B)Accumulationofmature

particles in the intercellularspaceof ts3-infectedcellsgrownat thepermissivetemperatureshowingvariation in theshapeand sizeofthecoresofthe virions. Scale=200nm.Virion witharegular pentagonalcore(single

black arrow); virion with an elongated core (double black arrow); spherical virion with twocores (white

arrow).

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trarily separated into four groups according to their stage inassembly: early (Fig. 2a), inter-mediate (Fig. 2b), late (Fig. 4b), and very late (Fig. 2d and4c).

The results of this investigation are summa-rized in Table 2. In thets3-infected cells grown atthe permissive temperature, almost 90% of the particles observed were non-cellassociated,

and more than 90% of these were mature parti-cles with electron-dense nucleoid (Fig. 3B). Only 2% of the cell-associated virions were mul-tiploids. The normal C-typecell-associated par-ticlesappeared to be evenly distributed among the four stages of assembly.

Some marked differences were observed be-tween ts3 virions from cells grown at the per-missive temperature as compared with those grown at the nonpermissive temperature. There were 22 cell-associated virions per cell section in ts3-infected cells grown at 390C as compared with 0.5 virion per cell section in ts3-infected cells grown at 34WC. In contrast to the virions found in cells grown at the permissive temperature, 96%of the virions observed in the nonpermissive culture were cellassociated. The

few extracellularvirionsobservedwere

imma-ture (Fig. 3A). Maturevirions may have been

present insuchlownumbersthattheywere not

detected. In the culture grown at the

nonper-missive temperature, 29% of the total virions

observedweremultiploids, whereasonly2%of

thevirions inthe culturegrownatthe permis-sive temperature were multiploids. Fifty

per-cent of the normal C-type virions observed in

the culture grown at the nonpermissive

tem-perature were

early-budding

virions, whereas

less than20%of them were in a verylatestage

inassembly.

Acomposite viewof ts3-infected cellsgrown

at390C for48hand then shiftedto34WCfor30

minisshowninFig.4.Severalmarkedchanges

were observed. The number of cell-associated

virions decreased to about 2 virions per cell

section,whereas theratioofmultiploidsto

nor-malparticles remainedthe same. The number

of extracellularparticles increased fromabout

4%toalmost50%of the total number of

parti-clesobserved, andmorethan80%of thesewere

immatureparticles. Amongthe cell-associated

normal C-type virions, over30%were late-bud-ding particles, and almost60%wereinthe final stagesofassembly.

As compared with a ts3-infected culture

shifted to the permissive temperature for 30

min, alonger shiftdown period (60 min) further reduced thenumber of cell-associated virions to about 0.5 virion per cell section. There was a

further increase in the proportion of non-cell-associatedvirions tocell-associated virions and

areduction in theproportion of multiploids to

total virions. Among the normal C-type parti-cles, there appeared to be a slight increase in the proportion of early-budding virions.

The above findings may be summarized as follows: multiploids were present in ts3-in-fected cells grown at both the permissive and nonpermissive temperature. Consistent with ourSEMfindings, the proportion of multiploids

to normal virions was higher in ts3-infected

cells grown at the nonpermissive than at the

permissive temperature. In spite of the fact

that 50% of the normal particles in the very

earlystages inassembly couldnotbe identified

inthe SEMstudies,ahigherproportionof mul-tiploids was observed in the TEM studies. This suggests that the duplexes, triplexes, etc.,

ob-served bySEM wereonlyaportionof the total

multiploids present. The proportion of

multi-ploids calculated from the TEMstudies didnot

exceed30%of the totalvirionsobservedin

ts3-infectedcells grown at the nonpermissive

tem-perature. Virionassembly rapidlyoccurred on

temperatureshiftdown with both normal parti-cles andmultiploids dissociatingfrom the cell

surface.

DISCUSSION

Our SEM and TEM studies confirmed the findings of Wong and MacLeod (8) that there were substantially more virions on the cell

surface ofts3-infected TB cells grown at the

nonpermissive temperature(39°C) than of cells grown atthepermissive temperature(34°C) or

ofMuLV-infected TB cells grown at39°C. Our

SEMstudiesagreewiththeir observationsthat

morethan 90% of the virions that had

accumu-latedatthe cell surface at 39°C dissociated from

the cell surface during the first 60 min after

temperature shiftdown. However, our TEM

studiesindicatethatwhen the accumulation of

virions on the cell surface is maximal, the total

cellsurfaceareathat can be occupied by virus

particles is greaterthan the 10% estimated by

Wong andMacLeod (8), since neither virions in

theveryearlystagesof assembly (about 50% of

the total number of normal cell-associated

C-type particles) nor many of the multiploids

(suchastherod-shapedvirions,the abnormally

large spherical forms, or the atypically shaped virions) were identified by SEM.

Ourpresentfindingssuggest thatthe

produc-tionofatypically shapedparticles

(multiploids)

atthenonpermissive temperature may not be a direct result of the ts defectof ts3. Less than

30% of the virionsobserved by TEMfrom

ts3-infected cells grownatthenonpermissive

tem-perature were multiploids. Moreover, multi-ploids, primarily duplexes, were also

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permissivetemperaturefor30minshowingextracellularimmaturenormal virions(d)andmultiploids(e-g)

andnormalvirions(bandc)andmultiploids (a)at a lateorverylatestageinassembly.Scale =100nm.

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ts MUTANT OF MuLV RESTRICTED IN ASSEMBLY 229 TABLE 2. Relative proportionsofnormal virionstomultiploidsand virions in variousstages ofassembly in

differentts3-infectedculturesdeterminedbyTEM

%Mem- %Freeim- % of different development stages of Totalno. No. ofcell- brane-as- % Multi- mature membrane-associated normal

parti-ofparti- associated sociated ploidsof

particles

of

clesa

Tyofculture clesob- virions/ particles totalpar-

non-mem-served cellsection of total tides

brane-as-

Inter- Late Ver particles sociated Early mediate late

ts3,340C 548 0.5 11 2 8 18 34 28 20

ts3, 390C 622 22 96 29 100b 50 13 18 19

ts3, 39- 34WC 559 2 53 28 82 3 7 31 59

(30min)

ts3, 39 . 340C 644 0.5 36 16 90 16 9 44 31

(60 min)

a SeelegendtoFig. 2and4.

bMatureparticles wereprobablypresent insuch low numbers thattheywerenotdetectable.

ently observed, althoughinlowernumbers, in

ts3-infected cells grown at 340C, in

MuLV-in-fected cellsgrownat 390C (12), andin a

Molo-ney murine sarcomavirus (Mo-MuSV) cellline

(unpublished data). The higher proportion of

multiploids to total particles found in

ts3-in-fectedcells grownatthenonpermissive

temper-ature maybeduetothe greater chance oftwo

or more particles assembling injuxtaposition becauseoftheaccumulationofbuddingviruses at the cell surface. The above suggestion is consistent with the observed decrease in the proportion ofmultiploids to total particles in

interferon-treated cells infected with ts3

(un-published data). Itwasshowninanotherstudy

(10) that the total number of ts3 virions

de-creasedbytwo- tothreefoldininterferon-treated

cells as compared with the controls. The

pro-duction ofatypically shaped virions in Friend

leukemia virus-infected cells after dimethyl

sulfoxide treatment has also been reported by

Sato et al. (5). In these dimethyl

sulfoxide-treated cells,therewas asignificantincreasein

the number of budding virions as compared

withthe controls.

The ts defect of ts3 appears to be due to some restriction in virion assembly. At the nonper-missive temperature, both normal and multi-ploid virions in all stages of assembly were observed. Only about 20%ofthenormal

parti-cles observed were at a very late stage in

as-sembly (Fig.2d).Four percentof the total

parti-clesobservedwereextracellular, noneof which were mature virions. However, aftera 30-min

shifttopermissive conditions, about 90% of the normal C-type virions observed were

late-bud-dingvirions(Fig. 4b), with almost60%of these

at avery late stageinassembly (Fig. 4d). This rapid increaseintheproportion of budding

viri-ons inthe finalstages of assembly on

tempera-ture shiftdown, inconjunctionwith a simulta-neousdecrease inthe number ofcell-associated

virions and an increase in the number of extra-cellular immature virions, suggests that rapid

assembly and release of the virions occurred

duringlte 30 min after shift to the permissive

temperature. On temperatureshiftdown,rapid

assembly and release of the multiploid virions

alsoseem tohave occurred. In the390C-infected

culture, mostof the multiploids observed were

broadly linked tothe cell at their bases (Fig.

2c). On temperature shiftdown, most of them

showed completeassembly of viral components,

resealing of the cellular membrane, and

shrinkingof the cytoplasmic linkage to the cell

(Fig. 2g and 4a). The large number of incom-pletely assembled ts3 virions in all stages of

assembly at the nonpermissive temperature

suggests that restriction in assembly only

oc-cursafterithas been initiated and that the ts

lesionis a step common to the whole assembly

process.

We have as yet no conclusive evidence as to how virion assembly is restricted at the nonper-missive temperature. Previous findings (7)

in-dicate that ts3 virions partially assembled on

the cell membrane of cultures held at

390C

be-fore the addition of actinomycin D or cyclohexi-mide are able to complete assembly and disso-ciatefrom the cell membrane on temperature

shiftdownto 34C inthe presence of the

inhibi-tors.Thissuggests that the ts defect liesbeyond events associated with the synthesis of viral

DNA, DNA-dependent RNA, and protein. The

ts defecthasalso been demonstrated (7) to be

reversible, because even in an established in-fection a shift from 34 to

390C

turns offvirus production almost immediately. In the same study, the ability of ts3 to rescue MuSV from an MuSV-infected nonproducer cell line was also examined. Whereas ts3 was found to rescue MuSV at

340C,

a shift to

390C

immediately blocked releaseofboth viruses. A subsequent shift from39 to

340C

again resultedinthe

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230 YUEN AND WONG

nomycin D- and cycloheximide-insensitive

release of both ts3 and MuSVduring the first hour aftershiftdown. Recently, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (J. A. Ptak and P. K. Y. Wong, manuscript in

preparation) indicated that the mobilityof the glycoproteinfraction of ts3 virions grown atthe

permissive temperature isdifferent fromthat of wild-type MuLV. In addition, the mobility of viral proteins precipitated by anti-MuLV gp7l from ts3-infected cells grown atthe nonpermis-sive temperature was also different from that precipitated from wild-type MuLV-infected

cells. Furthermore, the proteins precipitated

fromts3-infectedcells grown at the

nonpermis-sive temperature showed an increase in the amount of the 85,000-molecular-weight

glyco-proteinsand a concomitantdecreaseingp69/71.

All the above findings indicate that the de-fect of ts3 mayresideineitherthe processingor

conformation of the viral envelope

compo-nents(s). Ithasbeensuggestedthat the

assem-blyofenvelopedviruses isregulated by

protein-protein interactions (4). The conversion of a membrane area into a budding patch may be

brought aboutbyinsertionofglycoprotein

mol-ecules into the lipidbilayer (1). Itwasfurther

suggested that the shape and rigidity of the

viral envelope is maintainedby aviral struc-tural componentlaid downinnumerical

corre-spondencetotheenvelope

glycoprotein(s).

It is

conceivable that other structural proteins,

in-cludingtheribonucleoprotein,maybesimilarly

assembled in their correct order. We suggest

that if the glycoprotein(s) of ts3 is unable to

assume its correctconformationatthe nonper-missive temperature, thenthe assemblyof the rest of theviral components may behampered

by either an inefficiency or inaccuracy in the protein-protein interactions. However, once

permissive conditions are restored, the

defec-tive conformation of the glycoprotein is

cor-rected andnormalassemblycanbe resumed.

Among the late ts mutants ofRauscher

leu-kemiavirusisolatedby Stephenson and

Aaron-son (6), ts24 exhibits characteristics similar tothose of ts3. Recentmicroscopic studies (11) suggest that ts24is defective either inthe late

stages of assembly of the particles or in the release of the virus particles from the cell sur-face. Ts24 resemblests3 inthatthereare more

cell-associated virions at the nonpermissive than at the permissive temperature and that virions are rapidly released on temperature

shiftdown. However,unlike ts3, there appearto

be far fewer cell-associated virions in

ts24-in-fectedcellsgrown for6daysatthe nonpermis-sivetemperature (4 to 5virions/cell section)as

compared with ts3-infected cells grown for48h

at 39°C (22 virions/cell section). Furthermore,

allthebudding ts24 virions observed in infected cells grown atthe nonpermissive temperature werealmost completely assembled, normal vir-ions. By contrast, both normal particles and multiploids in all stages of assembly were ob-served ints3-infected cells grown at the nonper-missive temperature.

Itis possible that although ts3 and ts24 have certaincharacteristics in common, they are in fact temperature sensitive in different func-tions. Alternatively, they may be defective in the same function to varying degrees. An ap-proach in resolving this possibility may be achievedby means ofcomplementationand re-combination tests between thesemutants.

ACKNOWLEDGMENTS

Wewish to thank H. C. Chen and L. Jones for their skillfulassistance and M.Weber for critically reviewing the manuscript.

This investigation was supported by Public Health Serv-iceresearch grant CA-17695 awarded by the National Can-cerInstitute to P.K.Y.W.

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http://jvi.asm.org/