Temperature-sensitive Mutants of Sindbis Virus: Biochemical Correlates of Complementation

(1)

Temperature-sensitive

Mutants

of Sindbis

Virus:

Biochemical

Correlates of

Complementation

BOYCE W. BURGE' AND E. R. PFEFFERKORN2

Department of Bacteriology andImmunology, Harvard Medical School,Boston, Massachusetts 02115

Receivedfor publication5June1967

Temperature-sensitive mutants of Sindbis virus fail to grow at a temperature

that permits growth of the wild type, but when certain pairs of these mutants,

mixed together, infect cells at that temperature, viral growth (i.e., complementa-tion) occurs. Theyield from this complementation, however, is of thesameorder ofmagnitudeastheinfectivity in the inoculum. Since in animal virus infections the proteincomponentsof the virionprobably enterthecell with the viral nucleic acid, itwas necessary to demonstrate that the observed complementation required syn-thesis ofnewviral protein and nucleic acid rather thansomesortofrearrangement ofthe structuralcomponentsofthe inoculum. To demonstrate thatcomplementation does require new biosynthesis, three biochemical events of normal virus growth have been observed during complementation and correlated with the efficiency of viral growthseenincomplementation. Theseeventsinclude: (i) entranceof parental viral ribonucleic acid (RNA) intoa double-stranded form; (ii) subsequent synthesis ofviral RNA; and (iii) synthesis and subsequent incorporation of viral protein(s) intocell membranes where theyweredetected byhemadsorption. Although the in-fecting single-stranded RNA genome ofthe wild type was converted to a ribo-nuclease-resistant form, thegenomeofamutant(ts-11) incapable ofRNAsynthesis

at a nonpermissive temperature was not so converted. However, during comple-mentation with anothermutantalso defective in viral RNAsynthesis, someof the RNA ofmutant ts-1 was converted to a ribonuclease-resistant form, and total synthesis of virus-specific RNA wasmarkedly enhanced. The virus-specific altera-tion ofthe cell surface, detected byhemadsorption, was also extensively increased duringcomplementation. These observations support theview that complementa-tion between temperature-sensitive mutants and replication of wild-type virusare similarprocesses.

We have previously described complementa-tion between certain pairs of temperature-sensitive (ts) mutants ofSindbis virus (1, 2). In thosereports, complementationwasmeasured by comparing the yield produced by cells infected

by two mutants together, at a nonpermissive

temperature,withthesumoftheyields produced

by thesame twomutants grown separately. Under ideal circumstances, a complementing pair ofmutantsmightproduce 100 timesasmuch virus as either mutant grown separately, a complementation yield representing 1 to 10% of

the titer produced by wild-type virus grown under similar conditions. However, since a high multiplicity of infection was required for

suc-1Present address: Department of Biochemistry,

Albert EinsteinCollegeofMedicine,Bronx, N.Y. 2Present address: Department of Microbiology,

DartmouthMedicalSchool,Hanover,N.H. 03755.

cessful complementation (2), the inoculum used to produce the mixed infection was usually of the same order of magnitude as the comple-mentation yield.

Thus, it could be argued thatcomplementation between ts mutants occurred wholly or partly through a reshuffling of viral components

introduced into the cell by the inoculum. This explanation could theoretically be tested by determining whether labeled precursors were incorporated into the virus released as a result ofcomplementation. However, the lowyields of virus in complementation experiments would complicateitspurification.

We have therefore used several biochemical methods to examine the biosynthetic conse-quences of complementation. (As in the usual complementationtest, these biochemical methods were used to compare cells infected at a non-permissivetemperature by single ts mutants and

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COMPLEMENTATION IN MUTANTS OF SINDBIS VIRUS

by pairs of ts mutants.) Thefollowing important events in viral replication were studied: (i) entrance of the parental viral ribonucleic acid (RNA) into a double-stranded form; (ii) subse-quentsynthesis of viral RNA; and (iii) synthesis andsubsequent incorporationof viral protein(s) into the cellular membrane where they were detected by hemadsorption. In each case, the magnitude of these biochemical changes was compared withtheefficiencyofcomplementation asmeasured bytheusual viraltitrations.

MATERIALS AND METHODS

Tissueculture and viruses. Methods for thegrowth

andtitration of Sindbis virus inprimarychickembryo

fibroblast cultureshave beenalready described (10).

The ts mutants were isolatedand characterized as

described by Burge and Pfefferkorn (1, 2). These ts

mutants growwellat30C,apermissivetemperature,

butfailtogrowat40C,anonpermissivetemperature.

All ts mutants fall into one of twomajor categories:

(i) those defective in viral RNAsynthesisat

nonper-missive temperatures (RNA-), and (ii) those ableto makeviralRNA butdefectiveinsomeother matura-tional step at nonpermissive temperatures (RNA+). All experiments describedhere usedRNA- mutants.

When a comparison with the wild-type virus was

required, weusedthe HR strain ofSindbis virus (1) from which the ts mutants had been derived. This strain has notsmutations and willbe simplycalled

thewildtypeinthisreport.

Complementation.Thiswascarriedout asdescribed previously (2). Briefly, to test two mutants for the ability to complement, stocks of both mutants were

used to infect doubly a monolayer with about 20 plaque-forming units (PFU) of each per cell. This monolayer wasincubated at 40 C, and the yield of virus producedbetween the 4th andthe6thhrof in-fectionwascomparedwiththe sumoftheyields pro-duced by the two mutants in single infections. The ratios ofthesetwomeasurementsis calledthe comple-mentationlevel; forapair ofcomplementarymutants this level may be from three to 100, depending on several variables, including multiplicity ofinfection, incubation temperature, and the mutant pair under study (2).

Actinomycin D-resistant 3H-uridine incorporation.

Measurementwasin aconventionalway. Cellswere

exposed to 1 gg ofactinomycin D per ml (Merck, Sharp and Dohme Research Laboratories, Rahway, N.J.) throughout theinfection. At a specified time,

freshprewarmedmediumcontaining actinomycinand

3H-uridine (20 c/mmole, 0.03 mc/ml; Schwarz Bio

Research Inc., Orangeburg, N.Y.) was added; 2 hr

later the monolayers werechilled, rinsed twice with

phosphate-buffered saline (4) lacking calcium and

magnesiumandcontaining0.001 M

ethylenediamine-tetraacetic acid(EDTA),anddissolved at room

tem-perature in 2 mlof thesame solutioncontaining 1%

sodiumdodecyl sulfate (SDS).Asampleof this

solu-tion was precipitated with 0.3 Ntrichloroacetic acid

with 200 Ag of yeast RNA as carrier. The rest was

stored frozen at -60 C for later sedimentation analysis.

Sedimentationanalysis. It wascarriedout on15 to

30% lineargradientsof glyceroldissolved in a buffer

containing 0.1 M NaCl, 0.01 M tris(hydroxymethyl)-aminomethane, pH 7.5, 0.001 M EDTA, and 0.2%

SDS. A 0.24-ml sample was layered onto a 4.5-mi

gradientandcentrifugedin a Spinco SW-39 rotor at

38,000 rev/min for 2.75 hr at 25 C. Fractions were collected bypuncturingthetubeandcollecting drops.

All fractions were precipitated with 0.3 N

trichloro-aceticacidwith200 Ag ofyeast RNA ascarrier,

col-lected on membrane filters (Millipore Corp., Bed-ford,Mass.),rinsed withethyl alcohol,and dissolved in 2 ml ofpolyether scintillation solution for

deter-mination of radioactivity. When a sedimentation

marker wasrequired,asmallamount ofchick

fibro-blast monolayer previously labeled for 3 days with 14C-uridine and dissolved in SDS was added to the

sample. Acorrectionwas made for 14C counts in the

3Hchannel.

Fate of theinfecting viral RNA. This was traced by

use of virus labeled with 3H-uridine (20 c/mmole;

Schwarz Bio Research). To prepare this virus,

cul-tures wereinfected at 28 C in a medium containing

0.25 ugof actinomycin D per mland 125 Muc of 3H-uridine per ml. The low temperature allowed ts

mutantsand the wild type to be labeled under

identi-cal conditions. Thepresence ofactinomycin assured

that most of the 3H-uridine would be incorporated

intovirus-specificRNA.After 16 hr ofincubation,the

virus in the medium was partially purified by one

cycle of differential centrifugation. The resulting preparation was radiochemically homogeneous by several criteriaincluding sucrosegradient centrifuga-tion, resistance of the radioactivity to ribonuclease, andultracentrifugal homogeneity ofthe labeled RNA extracted from thevirus. Thevirus (about 3 X 10-8

countsper min perPFU) wassuspendedin medium

containing 100 ,ugofuridine per ml toeliminate in-corporation of any residual 3H-uridine from the growthmedium.

Thislabeled viruswasallowedtoadsorbto mono-layer culturesfor 1 hr at 4 C. Thecultureswere then rinsed andincubated at the desired temperature. At intervals,thesecultureswereexaminedforconversion

of the infecting viral RNA to a

ribonuclease-resistant form byuseof amodification ofthe method describedby Lodishand Zinder (8). Rinsed cultures weredissolvedin 3.5 ml of asolution containing0.07 MNaCl,0.001 MKCI,0.001 MEDTA,and0.1%SDS.

TheresultingSDS extract wascentrifugedat2,000X

g, and samples of the supernatant fluid, which

con-tainednearlyall of theradioactivity, were diluted

10-fold into water and buffer (8) containing 25 ,ug of

pancreatic ribonuclease perml. Thesesolutions were incubated at room temperature for 1 hr; then the ribonuclease-resistant material was precipitated with

0.3 N trichloroacetic acid, and its radioactivity was

determined inascintillation counter. In each case, the

ribonuclease-resistant radioactivity from the sample

diluted into water was subtracted from the value

ob-tained inbuffer as recommended by Lodish and Zinder

(5). Only a negligible fraction of the infecting viral 957

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BURGE AND PFEFFERKORN

RNA was ribonuclease-resistant immediately after

adsorption. Under conditions that permitted viral

replication, the infecting viral RNAwaspartially

con-vertedtoaform thatwas ribonuclease-resistant only

athigh salt concentrations and had the sedimentation

properties reported for double-stranded arbovirus

RNA (6, 11). [The characterization of the labeled

virus and details of the intracellular conversion ofits

RNA to a double-stranded form will be presented

elsewhere (Pfefferkorn, Burge, and Coady, Virology,

inpress).]

Hemadsorption. The capacity of monolayer cultures

toadsorb goosered blood cellswasdetermined bya

modification of the hemagglutinationtestfor Sindbis

virus (3). To allow quantitative estimation of

hemad-sorption,the red cells werelabeled with 51Cr asina

modification of the usual hematological procedure

(5). A 7.5% suspension of gander red cells in

bar-biturate buffer containing glucose and gelatin (3)

wasincubated with Na251CrO4 (0.125 Ac/ml) for 1 hr

atroomtemperature.The labeled red cellswerethen

washed twicewith the same buffer and once with a

1:1 mixture oftheborate andpyrophosphate buffers

(final pH 5.8) used in hemagglutination titration (3,

10).The cellswerefinally suspendedata3% concen-tration in the borate-pyrophosphate mixture and used within the next few hours.

Monolayerstobeassayedforhemadsorptionwere

chilledin anice bath,drained and rinsed twicewith

borate-pyrophosphatebuffer. A0.3-mlvolumeof the

6'Cr-labeled red cell suspension (about 7 X 10-4

counts permin per cell) was thenpipetted ontothe

monolayer, which was held at 4 C with occasional

rockingtoredistribute the red cells. After 45min,the

monolayersweredrained and rinsedgently four times

with 2.5-ml volumes of cold borate-pyrophosphate buffer.

The monolayer, together with any adsorbed red

cells,wasdissolved in 1 ml of 0.3NNH40H,added to

10 ml of polyether scintillation solution (7), and

counted inascintillation counter. Quenching bythe

dissolved monolayer was a constant factor in all

samples. Hemoglobin from cultures positive for

hemadsorption did not quench significantly. The

hemadsorption appeared to be a virus-specific

reac-tion, for itwasreducedtothecontrol levelbyprior

treatment of the infected monolayer with rabbit

anti-Sindbis virus serum that had been exhaustively

adsorbedwith uninfected cells.

RESULTS

Complementation between RNA- mutants.

Complementation between the RNA-ts mutant

ts-6 and other RNA- mutants is quite efficient,

in terms of both the complementation level achieved and the absolute titer of virus

pro-duced (9a). Hence, if complementation did

involve an increase in viral RNA and protein synthesis, these increases should be easily

detectable. Table 1 records the results ofa

com-plementationtestinvolvingmutants ts-6, -11 and

-24.In eachcase,thesingleinfectionsbymutants

produced only about 0.01 % the infectivity

TABLE 1. Complementation as measured by infectivitya

Virusproduced Complemen- Percent of Inoculum between 4 and 6 tation Pe eld

hr(PFU/ml) levelb wild-typeyield

Wild-type 1.9 X 1O9 100.0

ts-6 32.0 X 104 0.016

ts-11 2.0 X 104 0.001

ts-24 10.0 X 104 0.005

ts-6 X ts-11 12.0 X 107 353 6.3

ts-6 X ts-24 8.0 X 107 190 4.2

aComplementationwas carried out as described

in Materials and Methods. Data in Tables 1 and

2 were obtained in the same experiment.

bThe complementation level is the titer

pro-ducedby the double infection divided by the sum

ofthe titers produced by the two mutants in single

infections.

produced by the wild type. The complementing

pairs, however, produced about 5% of that yield. These results are similar to those

pre-sented elsewhere (2). With the exception of

the study of the fate ofmutantviral RNA, which

required the use of labeled parental virus, all

of the biochemical assays of complementation

were done with replicate cultures infected and

incubated with those described in Table 1.

Thus, the biochemical results can be directly

comparedwith this bioassay of complementation.

Fate of parental viral RNA during comple-mentation. The replication of a single-stranded

RNA virus probably requires the formation

of a virus-determined enzyme that directs

synthesis of new "minus" strands,

comple-mentary to the viral RNA "plus" strand (8, 9,

12). Newly synthesized "minus" strands are

found together with parental "plus" strands in

a double-stranded structure that can be

iden-tified by its resistance to ribonuclese.

The RNA of wild-type Sindbis virus labeled

with 3H-uridine was partially converted to a

ribonuclease-resistant form very

early

in the

course ofinfectionsat 30 orat 40 C. This form

had a sedimentation constant of about 20S and

several of the other properties expected for

double-stranded RNA (Pfefferkorn, Burge, and

Coady, in

press).

We attempted to correlate

complementation

with thefate of labeled viral RNA in

single

and

mixed infections. Figure 1 records the

con-version of labeled

input

RNA of mutant ts-11

to aribonuclease-resistantforminsingleinfection

at 30 C and in

single

and mixed infection with

mutant ts-6 at 40 C. In

single infections,

con-version of the RNA of mutant ts-11 to a

ribonuclease-resistant form was normal at 30 C

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2

HOURS OF INCUBATION

FIG. 1. Intracellular conversion oflabeledinfecting

viralRNA to a ribonuclease-resistant form early in

thecourseof inifection.Labeledviruswaspreparedand

thefate ofits RNA was traced as described in the

Materials and Methods.Symbols: A,3H-labeled

wild-type infection at 40 C; 0, 3H-labeled mutant ts-1l

infectionat40C;A,3H-labeledmutantts-1i infection

.at 30 C; 0, 3H-labeled mutant ts-11 and unlabeled

mutantts-6 mixedinfectionat40 C.

but completely defectiveat 40 C. Since the wild

typeshowednormal conversion of its RNA toa

ribonuclease-resistant form at 40 C, mutant

ts-11 appears to be defective in this initial step

of viral replication at the nonpermissive

tem-perature. This defect was partially relieved in

the mixed infection with unlabeled mutant ts-6,

another RNA- mutant. The efficiency of

con-version ofmutant viral RNAto a ribonuclease-resistant form, about 5% ofthe value obtained

forthemutant at 30 Cor the wild typeat40C, wascomparabletothe efficiencyof

complementa-tionbetween thesetwomutants(Table 1).

Viral RNA synthesis during complementation. We next sought to correlate complementation with net synthesis of viral RNA. Since actino-mycin D was used to suppress cellular RNA synthesis, incorporation of 3H-uridine was largely into viral-specific RNA. Table 2 shows

thatcultures infected byany of thethree

RNA-mutants at a nonpermissive temperature incor-porated no more 3H-uridine than did the

unin-fected control. This, ofcourse, is the definition

of an RNA- mutant. However, the cultures infected by the mutants together showed a

substantial increase in incorporation of 3H-uridine to values 8 to 10V of the wild-type level. These values are only slightly greater

[image:4.462.26.222.78.312.2]

than the complementation measured by virus production (Table 1).

TABLE 2. Complementation as measured by

hemad-sorptionand3H-uridinieincorporationa

Inoculum Hemadsorptionb 3Hiuridineb

Uninfected 103 4.9 X 103

Wild-type 12,481 325.0 X 103

ts-6 112 5.2 X 103

ts-11 99 5.1 X 103

ts-24 125 6.8 X 103

ts-6 X ts-1 1 5,419 25.5 X 103

ts-6 X ts-24 4,680 32.5X 103

aData in Tables 1 and 2 were obtained in the

samecomplementation experiment.

IMeasurement of hemadsorption and 3H-uri-dine incorporation is described inMaterials and Methods.Both areexpressed incountsperminute permonolayer.

Sedimentation properties of the RNA labeled

during complementation. Virus-specific RNA

produced during arbovirus infections is known

to sediment intwo major peaks, at 42S and 26S

(6, 11). Both RNA species are ribonuclease

sensitive. A third, ribonuclease-resistant peak,

sedimentingat20S, may be resolved by digesting

away the ribonuclease-sensitive species (6, 11).

Since the physiological significance of these

RNA's, particularly the 26S species, isnotcertain,

it was of interest to examine the sedimentation

properties of viral RNA produced during

com-plementation between ts RNA-mutants.

Samples of SDS-dissolved monolayers from

the

complementation

described in Tables 1 and 2 were analyzed on linear gradients of glycerol-SDS. Figure 2 shows the pattern seen in the wild-type infection at 40 C. No attempt

wasmadeto resolve theminor20S

ribonuclease-resistant fraction.

In Fig. 3, the 3H-uridine incorporated by the

RNA- mutants in single infections (in the presence of actinomycin D) can be compared

to the 3H-uridine incorporated by an uninfected

monolayer. In all samples, most of the labeled

RNA appeared in a slowly sedimenting band of

about 4S. A small fraction of the radioactivity sedimented more rapidly than the 16S marker, but there was no indication of any preferential synthesis of the virus-specific 42 and 265 RNA. Thus, the RNA- mutants in single infections at 40 C stimulate a negligible amount of

virus-specific RNAsynthesis.

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I

-r 111

w

z

w

a-C,

z I:2

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Z,w

z

300 LiJ

C,)

z

100 o

0

1 5

9

13 17

FRACTION -NUMBER

FIG. 2. Sedimentation analysis of 3H-uridine

in-corporated into a monolayer infected with wild-type

virus.A monolayerwasinfected withahigh multiplicity

ofvirus inthepresence of I jugof actinomycinD/ml.

At 4 hr after infection, 3H-uridine (0.03 mc/ml, 20

clmmole)

wasadded and aftera 2-hrlabeling period

themonolayerwasdissolvedinSDSbuffer. A 0.24-ml

sample with added 14C-labeled chick cell RNA for

sedimentation markers was centrifuged in a Spinco

SW-39 rotor at38,000 rev/min for 2.75 hr at 25 C.

Symbols: 0, 3H-labeled virus-specific RNA; 0,

cellularRNA. Thissample andthoseanalyzedinFig.

3 and 4 were obtainedfrom the complementation

ex-periment recorded in Tables I and 2.

Sedimentation patterns forlabeled RNA from cells infected by complementing pairs of

RNA-mutants appear in Fig. 4. These patterns were

similar to those seen in the wild-type infection; the additional radioactivity incorporated in mixed infections (Table 2) all appeared in the 26S and 42S regions. Proportionately more 42S RNAwas seeninthe mixedinfection ts-6 X

ts-24than inthewild-typeinfection. The signifi-canceof this observation is unknown.

Synthesis ofviralprotein during

complementa-tion. The synthesis ofthe viral protein active in

hemagglutination was measured by determining thecapacity ofthe infected cellstoadsorbgoose

red cells. To obtainquantitative results, weused 51Cr-labeled red cells and measured the radio-activity that could not be washed from the monolayer.

The ability of virus-infected monolayers to adsorb 65Cr-labeledred cellswas a verysensitive indicator ofviral growth. As Fig. 5 shows, this

alteration in cellsurface was detectable at least

1 hr before a linear rate of virus release was

w

z

w

(n z

0

100

50

100 50

100

50

100

50

ts-24

28s 16s

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UNINFECTED

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1 5 9 13 17 21

FRACTION NUMBER

FIG. 3. Sedimentation analysis of 3H-uridine

in-corporated into an uninfected monolayer and into

monolayers infected with single RNA- ts mutants.

Actinomycin D, I ,ug/ml, was present in all cultures.

Seelegendof Fig. 2 forconditions of sedimentation.

achieved. The number ofred

cells

adsorbed per

monolayercontinued to increase until the 4th hr

ofinfection. At this point, the ratio of labeled

red cells to monolayer cells was about 3, and

saturation of the available surface probably

prevented theadsorption of more red cells onto

monolayers incubated for longer times.

On the basis of this kinetic experiment, we

measured hemadsorption at 6 hr after infection

in complementation experiments. Table 2 shows

that cultures infected singly with the mutants

at the nonpermissive temperature had only the

minimal hemadsorbing capacity of uninfected

cultures. In contrast, mixed infection by

com-plementing

pairs of mutants induced a

hemad-sorbing capacity that was about

50%

that

induced by wild-type infection, indicating

ex-tensive synthesis of at least one viral protein

during

complementation.

DISCussIoN

In

complementation

between

bacteriophage

conditional-lethal mutants, the viral coat of the

infecting phage particle remains outside the cell

and could not

possibly

be used in the formation

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40(

w

204

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a

0 ts-6

x

fs-24

1

5

9

13

17

21 FRACTION

NUMBER

FiG. 4. Sedimentation analysis of 3H-uridine

in-corporated into monolayers treated with actinomycin

D,I ,ug/ml, and infectedinamixedmannerwith pairs

ofts mutants. Seelegend of Fig. 2for conditions of sedimentation.

of new infectious particles. In animal virus

infections, however, all viral components

prob-ably enter the cell, and might conceivably be used in theassemblyofnewparticles. Itis

there-fore important to demonstrate that

comple-mentation with ts mutants of an animal virus

involves new synthetic events and that these

events are similar to, or reconcilable with,

theevents observedin awild-type infection.

In this report, we have shown that

comple-mentation between ts mutants is accompanied

by three biochemical events: (i) an increase in the amount of mutant viral RNA that is con-verted to a ribonuclease-resistant form; (ii) an increase in the synthesis of virus-specific

RNA;and (iii) anincrease in theamountof the

viral hemagglutinating protein in the cell

mem-brane,reflectedbyanincrease inhemadsorption.

Each of these increases is commensurate with,

thoughnotpreciselyproportional to,theincrease

intiter of viralinfectivity.

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HOURS AFTER INFECTION

FIG. 5. Kinetics ofappearance of hemadsorption

capacity and viral infectivity. A series ofmonolayers

was infected with wild-type Sindbis virus (about 20

PFU/cell) and incubated at 36 C. At intervals, mono-layers were withdrawn; the infectivity in the medium

wasassayed(0) and thehemadsorbingcapacity of the

monolayer(0) was measured asdescribed in Materials

andMethods.

The only serious discrepancy lies in the measureofhemadsorption. Herethecellinfected by the mixed mutants showed about 50% of

the hemadsorbing capacity ofthe cells infected

by the wild-type virus, although the virus yield from mixed infections was only 5% of the

wild-type yield. It must be remembered, however,

that only a finite number of red cells can be adsorbed to a monolayer of fixed surface area, regardless of the concentration of hemadsorbing

protein in cell membranes. Once this saturation

point is reached, the number of red cells

ad-sorbed to the monolayer is no longer propor-tional to the amount of hemadsorbing protein

in the membrane (Fig. 1). In the wild-type

infection here compared to

complementation,

this point has been reached, and thus it is not

possible to determine the relative amounts of

hemadsorbing protein produced in the two

situations.

Using the three criteria listed

above,

we

con-clude that complementation requires new

bio-syntheticactivityand involves thesamesequence

of events thatcharacterizethegrowth of wild-type Sindbis virus.

ACKNOWLEDGMENTS

We aregratefultoJoanDaniels forsupplyinggoose

red blood cells andto ShelbyKashketfor advice on

labelingthemwith Na25CrO4. Helen M. Coady

con-tributed experttechnical assistance.

Thisinvestigationwassupported byPublic Health

Service research grantAI-04531-06from theNational

Institute ofAllergyandInfectious Diseases. Boyce W.

Burge is a Harold C. Ernst Fellow.

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LITERATURE CITED

1. BURGE, B. W., AND E. R. PFEFFERKORN. 1966.

Isolation and characterization of conditional

lethal mutants of Sindbis virus. Virology

30:204-213.

2. BURGE, B. W., AND E. R. PFEFFERKORN. 1966.

Complementation between

temperature-sensi-tive mutants of Sindbis virus.Virology

30:214-223.

3. CLARKE, D. A., ANDJ. CASALS. 1958.Techniques

for hemagglutination and

hemagglutination-inhibition with arthropod borne viruses. Am. J.

Trop. Med. Hygiene 7:561-573.

4. DULBECCO, R., AND M. VOGT. 1954. One step

growth curve of western encephalomyelitis

virus onchick embryocells grown in vitro and

analysis of virus yields from single cells. J. Exptl. Med. 99:183-189.

5. EBAUGH, F. G.,C. P. EMERSON, AND J. F. Ross.

1953. Use of radioactive chromium 51 as an

erythrocytetagging agent for the determination

of red cell survival in vivo. J. Clin. Invest.

32:1260-1276.

6. FRIEDMAN,R.M., H. B.LEVY,ANDW. B. CARTER.

1966. Replication of Semliki Forest virus:

three forms of viral RNAproduced during

in-fection.Proc. Nati. Acad. Sci. U.S. 56:440-446.

7. LAW, J. H., H. ZALKIN, AND T. KANESHIRO.

1963. Transmethylation reactions in bacterial

lipids. Biochim. Biophys. Acta 70:143-151.

8. LODISH,H. F.,ANDN.D. ZINDER. 1966.

Replica-tion of the RNA ofbacteriophage f2. Science

152:372-377.

9. MILLS, D. R., N. R. PACE, AND S. SPIEGELMAN.

1966. The in vitrosynthesis of a noninfectious

complex containing biologically active viral

RNA. Proc. Natl. Acad. Sci. U.S.

56:1778-1785.

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