Morphology of BHK-21 Cells Infected with Sindbis Virus Temperature-Sensitive Mutants in Complementation Groups D and E

JOURNAL OF VIROLOGY, May 1975, p. 1262-1266 Copyright 01975 American Society for Microbiology

Vol.15,No. 5 Printed inU.S.A.

NOTES

Morphology

of

BHK-21

Cells Infected with Sindbis Virus

Temperature-Sensitive

Mutants

in

Complementation Groups

DandE

DENNIS T. BROWN* AND JONATHAN F. SMITH

Institutfur Genetik der Universitdt zuKoln,5Koln41, Weyertal121, West Germany

Receivedforpublication 18December1974

BHK-21 cells infected with temperature-sensitive mutantsofSindbis virus in

complementationgroupsDand E differed in theirappearance under

nonpermis-sive conditions. Cells infectedatnonpermissivetemperaturewith virus defective incomplementationgroupE hadnucleocapsids attached in large numberstothe

inside surface of the host plasma membrane. Infection with a group D mutant

produced nucleocapsids that didnot attachtothe plasma membrane but rather

remained free in the cell cytoplasm.

The morphogenesis of the alpha-togaviruses invertebrate cellscanbe dividedintotwomajor

processes: (i) the assembly of the viral

nu-cleocapsid from the capsid protein and single-stranded RNA, and (ii)the envelopmentof the

completed nucleocapsid by the process of

bud-ding through the virus-modified

plasma

mem-brane. Little morphological information iF

available

regarding

the

assembly

of the viral

nucleocapsid sincenoobvious intermediates in

assemblycanbe detectedinthe electron micro-scope. On the otherhand, the buddingprocess attheplasma membrane hasbeendescribed

by

using various electron

microscopic

techniques

(1, 2).

The modified plasma membranesofSindbis

virus-infected cellscontainthreevirusproteins,

PE2, El, and E2 (7, 16). PE2 is cleaved toE2 during virus

morphogenesis,

and this

cleavage

seems tobe essentialforthe

budding

process

(7,

12-14, 16). Enzymatic radio-iodination studies

reveal that El and E2 are exposed on the cell surface, whereas PE2 cannot be iodinated and ispresumablyburied in the cellmembrane (16).

To characterize further the final stages of

Sindbis virus maturation, we studied cells

in-fected with temperature-sensitive mutants (3,

4) by electron microscopy. Those mutants

which produce complete nucleocapsids but no

mature virus were of particular interest, since these mutants are likely to be defective in the

budding process. The two complementation

groups that meet these conditions are D and E

(4), and we have chosen as representative of

these groups temperature-sensitive mutants ts-23 and ts-20 (obtained from E. R.

Pfeffer-korn, Dartmouth College). The known

charac-teristics ofthese mutants may be summarized

asfollows. (i)The intact ts-23 virion has

reversi-ble temperature-sensitive hemagglutinating

ca-pability. However, this virion can adsorb to

cellsatnonpermissive temperatures (4, 18). (ii)

The surfaces of cells infected with ts-20 at

nonpermissive temperatures hemadsorb (4) and

bind ferritin-conjugated antibodyagainst intact virus (Brown, unpublished data), whereas

ts-23-infected cells neither hemadsorb (4) nor

bind antivirus ferritin-antibody conjugates

(Brown, unpublished data). (iii) Cells infected

with either mutant produce complete

nu-cleocapsids (4). (iv) Cells infected with either mutant contain uncleaved PE2 but not E2 at nonpermissive temperatures (7; K. Jones,

per-sonal communication; Brown and Smith,

un-published data), and in ts-20-infected cells

some of this protein can be located in the

plasma membrane (7). It appears that the phenotypes of the two mutants under nonper-missiveconditions are similar in most respects,

although one might conclude from the above data that themembranes ofts-20-infectedcells contain inserted viral glycoproteins, whereas ts-23-infected cells may not.

For electron microscopy, BHK-21 cells

(ob-tained from P. Faulkner, Queens University,

Canada) infected withapproximately50PFU of

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-FIG. 1. Thinsection of BHK-21 cells infected with ts-23. BHK-21 cells were infected at room temperature

with approximately 50 PFU of ts-23 per cell for 1 h. The inoculum was removed and replaced with Eagle

medium (5) which had been prewarmed to 40 C. At the end of the incubation period, cells were fixed in situ with

1.5%glutaraldehyde in the phosphate buffer of Millonig (9). The cells were scraped from the monolayer and,

after postfixation with osmium tetroxide, dehydrated and embedded in epon (8). After sectioning, the

preparations were stained with uranyl acetate and lead hydroxide. (a) Incubated for 8 h after infection at

39.5C.Nucleocapsids (arrows)areseenrandomlydistributed in the cell cytoplasm. Magnification x40,000. (b)

Infected and incubated as described in (a) and then shifted to 28 C and incubated for an additional hour.

Magnification x20,000.

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1264

NOTES

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FIG. 2. Thinsection of BHK-21 cellsinfected with ts-20. Cells were infected, incubated, and prepared for

microscopyasdescribed for Fig. 1. (a)Incubation atnonpermissive temperature for 8 h. Nucleocapsids are seen

attached to the internal surface of the plasma membrane. The insert shows an area of this cell at higher

magnification. The bilayer nature of the modified membrane is evident, the nucleocapsids are intimately associated with its inner surface, and the outer surface appears thicker than normal in some areas (arrow).

Magnification x48,500 andx97,000 for insert. (b) Infected andincubated as described for (a) and then shifted

to 28C and incubatedforanadditional hour.Magnification x25,000.

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ts-20 or ts-23 percellwereincubated at

nonper-missive temperature (39.5

C)

for 8 h and

pre-pared for electron microscopy as previously described (2). Identical cultureswere shiftedto permissive temperatures (28C)for 1hafterthe initial incubation beforefixation.

Cells

infected with eithermutant at

nonper-missive temperature had large and

approxi-mately equal numbers ofnucleocapsids at 8 h

afterinfection;however, the intracellular distri-bution of the

nucleocapsids

was

considerably

different. ts-23 nucleocapsids were uniformly distributed in the cell

cytoplasm (Fig.

la), whereas ts-20 nucleocapsids were attached to

theinner surface oftheplasma membrane(Fig. 2a). Examination ofcells infected withts-20 at

earlier times (5 to 6h after infection) revealed

the presence of some nucleocapsids, but few

could be found in association with the

plasma

membrane (data not shown). The

observation

that cells infectedwithts-20readily hemadsorb

at this early time (4) suggests that membrane modification isquite extensivebeforethe

bind-ing of nucleocapsids. External to bound

nu-cleocapsids, the plasma membrane of ts-20-infected cells was often observed to be coated

with anamorphous materialapproximately12.0

nm thick (Fig. 2a, insert). This layer was not

found on uninfected cells or on cells infected with ts-23. Thesimilarity of the cross-sectional

thicknessofthis layertotheknownlength ofthe

spikes in the mature virion (1, 6, 17) suggests

that this layer contains the virus-specific

pro-tein(s) responsible for thehemadsorbing

capa-bility of the ts-20-infected cells (3, 18). The

tightassociation ofthe viral nucleocapsids with the modified plasma membrane in

ts-20-infected cells was also demonstrated by an

examination of membrane fragments obtained

fromgradientsofdisruptedinfected cells. Large

numbersof structuresresemblingnucleocapsids were found attached to these membrane

frag-FIG. 3. Negatively stained membrane fragment with associated nucleocapsids (arrows) obtained from

disruptedcellsinfected with ts-20 at nonpermissive temperatures. Cells were disrupted after 8 h of infection by

Douncehomogenization. An aliquot was layered onto a 10 to 45% (wt/wt)potassium tartrate gradient and

centrifuged at 30,000 rpm for 12 h in the SW-41 (Spinco) rotor. Membrane fragments of the type shown were

found at a density of 1.27gm/cm'. Thepreparation was stained with neutral 1.5%phosphotungstic acid.

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NOTES

ments (Fig. 3). No mature or budding virions

could be found in cells infected with either

mutant at nonpermissive temperatures. Both

intact and budding virions could be seen in

large numberswhen either mutant-infectedcell was returned to permissivetemperature(Fig.lb

and 2b). The processing oftheviral

nucleocap-sids produced at nonpermissive temperatures

into mature virion after the change to

permis-sive temperature demonstrates the reversible

nature of the mutant system with respect to virus production. Virus production after

tem-perature shift does not, however, mean that

membrane-associated viral proteins produced at nonpermissive temperature are actually chased into maturevirions.

It may be concluded from this work and

previous studies that ts-20-infected cells have

virus-specific proteins on the outer surface

which can hemadsorb and bind virus-specific

antiserum, and virus-specific alteration on the

inner surface of theplasmamembrane towhich

nucleocapsids attach. ts-23-infected cells have neither of these capabilities at nonpermissive

temperatures. Thus the

binding

of viral

nu-cleocapsids to the inner surface of the

plasma

membrane, like the

hemadsorbing

capability, is avirus-specific function thatmaybe attributed

to either or both PE2 and El present in the modified cell membrane (7). The inability to

attach

nucleocapsids

tothe

plasma

membrane and the absence of

hemabsorption

in

ts-23-infectedcellsmaybe duetothe lackofinsertion ofthe membrane proteinsincells infected with this mutant.

Alternatively,

if the viral

mem-brane proteins areinserted inthe ts-23-infected cells, the absence of

hemadsorption

and

ex-posed viral antigenssuggeststhat the insertion is less complete than ints-20-infected cells. In

eithercase,the defectin ts-23preventsboth the

binding

of the

nucleocapsids

and the proper

insertion oftheproteins. Either thesetwo func-tions are controlled

by

one protein or, if

sepa-rately, by different proteins, one function is

dependent upon the prior

completion

of the

other. Such a sequential relationship could be

invoked if the membrane proteins must be

installed in a particular arrangement before

nucleocapsidscouldbe attached. Suchseems to

bethecase, since ts-20-infected cells hemadsorb

before detectable quantities of membrane-associated nucleocapsids can be found (4; this

study). It is

possible, therefore,

that the

inabil-ity of the virus membrane proteins to bind

nucleocapsids in the ts-23-infected cells is a

result ofthe lack of an appropriate

configura-tion oftheprotein within the membrane rather

than a simple defect in some capsid-binding region of the protein. Investigations are in

progress to determine the organization of the

virus proteins inthe mutant systemsdescribed

and to elucidate the nature ofthe capsid-mem-braneinteraction in ts-20-infected cells.

The technical assistance of Doris Renz is gratefully ac-knowledged.

This research wassupportedby the Deutsche Forschungs-gemeinschaft, SFB 74.

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