cell killing by viruses iv. cell killing and protein synthesis inhibition by vesicular stomatitis...

VIROLOGY 7% 267-280 (1977)

Cell Killing by Viruses

IV. Cell Killing and Protein Synthesis Inhibition by Vesicular Stomatitis Virus Require the Same Gene Functions’

JACQUES L. ~RV~DI,* J. LUCAS-L~NARD, MARGARET J. SEKELLICK, AND PHILIP I. MARCUS3

Biological Sciences Group, University of Connecticut, Storrs, Connecticut 06268

Accepted February 14,1977

Temperature-se~itive fts) mutants of vesicular stomatitis fVS1 virus representing the five known eomplementation groups of the Indiana serotype were used to determine which viral gene functions were required for the expression of (i) cell-killing (CK) particle activity, and (ii) the inhibition of host cell protein synthesis. Cell-killing particle activity was quantitated by analyzing single-cell survival curves generated by infecting the Vero line of green monkey kidney cells at low multiplicities (m,r, 5 5) and at permissive (30”) and nonpermissive (40”) temperatures. Cellular protein synthesis inhibition was measured by difference analysis of ~lya~~lamide-Mel-electrophero~am patterns obtained from uninfected mouse L cells and cells infected at high multiplicity (mDtp = 100-500) at these two temperatures. Analyses of cell killing and protein synthesis inhibition by ts mutants at nonpermissive temperature demonstrate that both of these att,ributes require the same viral gene functions and that they are conjointly expressed or not expressed, ts~“‘~J~s” or t6’““-,““‘- , respectively. Complementation groups I and IV were subdivided into [email protected] and tsc~Y-~@8’- mutants, establishing that the viral proteins required for the expression of cell killing and protein synthesis inhibition need not be fully functional. Considering these and previous results [Virology 57: 321 (1974); 63: 176 (1975); 69: 378 (197611 we conclude that, although the expression of both cell killing and protein synthesis inhibition by VS virus does not require infectious virus, it does depend upon transcription of viral genes N and NS by virion-associated transcriptase and their translation into minimally functional proteins. We postulate that viral proteins N and NS then interact with minimally functional L protein to produce the putative proximate or ultimate factor(s) directly responsible for cell killing and protein synthesis inhibition by VS virus. The lack of cellular protein synthesis inhibition by several ts mutants following challenge at high multiplicities (mpf,, = 500) at 40” casts some doubt on the interpretation of experiments that appear to support assignment of these functions to preformed viral proteins.

I~RO~UCTION tion of cellular protein synthesis (Yama- Host cell infection with vesicular stoma- zaki and Wagner, 1970; Wertz and Young-

titis (VS) virus results in a rapid inhibi- ner 1970, 1972; McAllister and Wagner, 1976; Baxt and Bablanian, 1976) and the

* This research was supported by Grant No. 12144 development of a cytopathic effect which is and 20882, awarded by the National Cancer Insti- tute, DHEW, National Science Foundation Grant

expressed ultimately in cell killing (Mar-

No. PCM 76-00467, and the research benefited from cus and Sekellick, 1974, 1975, 1976). Cell

use of the Cell Culture Facility supported by Na- killing by this virus was shown to re-

tional Cancer Institute Grant No. 14733. Some as- quire (i> functional virion transcriptase,

pects of this paper were presented at the Third In- (ii> transcription of one-fifth of the viral ternational Colloquium on Rhabdoviruses on July 2, genome, (iii) translation of these tran- 1976, in Tiibingen, West Germany. scripts into minimally functional polypep-

2 On leave from Institut de Chimie Biologique, Universite de Provence, Place Victor Hugo, 13331 3 Author to whom requests for reprints should be Marseille, CEDEX 3, France. addressed.

267 Copyright 0 1977 by Academic press, Inc. All rights of reproduction in any form reserved. ISSN 00426822

268 MARVALDI ET AL.

tides which lead to (iv) formation of, and subsequent action by, a putative cell-killing factor (Marcus and Sekellick, 1974, 1975, 1976). These data, obtained by scor- ing the cell-killing (CK) particle capacity of several temperat~e-sensitive tts) mutants of VS virus and of uv-irradiated virions led us to postulate that certain viral polypeptides must be minimally functional for cell killing to be expressed and that the virion is not inherently toxic.

In this study we compare the cell-killing and protein synthesis-inhibiting propen- sity of ts mutants of VS virus, representing the five known complementation groups of the Indiana serotype, to determine whether these two cellular responses to infection require the same or different gene functions.

The capacity of each ts mutant to inhibit cellular protein synthesis was measured by difference analysis of polyacrylamide- gel-electropherogram patterns obtained from uninfected cells and cells infected at high multiplicities (mplrr = 100-500) at both permissive (30”) and nonpermissive (40”) temperatures. The mutants’ capacity to express CK particle activity at these two temperatures was determined by analyzing single-cell survival curves generated at low multiplicities (mpfp 9 5) (Marcus, 1959).

For these studies it is important to note that the genome of VS virus (Ind.) consists of a single negative strand of RNA which contains the genes for at least five polypeptides. The virion and the infected cell each contain all five proteins (Wagner, 1975); L, the transcriptase (Emerson and Wagner, 1973); G, a glycoprotein, and M, a matrix protein, both found in the virus envelope (Wagner et al., 1969); N, the nucleoprotein, and NS, a phosphorylated protein (Sokol and Clark, 1973), both found in the virus core. The genome RNA and proteins N, NS, and L constitute a tr~~ribing com- plex which produces primary transcripts coding for these five proteins (Bishop and Roy, 1971; Szilagyi and Pringle, 1972; Emerson and Yu, 1975). The gene order of transcription has been determined and oc- curs according to the sequence 3’-N-NS- M-G-L-5’ (Ball and White, 1976; Abra- ham and Banerjee, 1976). Temperature-

sensitive mutants of VS virus (Ind.) fall into five complementation groups (Wag- ner, 1975). The mutation affecting the members of group I is localized in the cis- tron coding for the transcriptase (L) protein (Szilagyi and Pringle, 1972; Hunt and Wagner, 1974). Mutants of group III and V are now considered to represent, respectively, the cistrons coding for proteins M and G (Lafay, 1974). Mutations affecting the viruses of group II and IV are probably localized in the cistrons coding for proteins NS and N, respectively (Wagner, 1975).

MATERIALS AND METHODS

Cells: growth conditions. Monolayers of mouse L cells were cultivated in Eagle’s minimum essential medium (MEM) supplemented with 9% fetal bovine serum. Confluent cell monolayers on 100- or 60- mm dishes (Falcon plastic) contained about 2 x lo7 or 4 x 106: cells, respectively. Monolayers of green monkey kidney (GMK) cells (Vero clone M3) were grown in NC1 medium (Marcus and Carver, 1965) supplemented with 6% calf serum that had been selected to give a high plating efficiency (Marcus and Sekellick, 1974). Con- fluent monolayers of Vero cells on 60-mm dishes contained about 2 x lO@j cells.

Viruses: stock preparation. The following wild types and mutants of vesicular stomatitis (VS) virus were used: wild-type HR-C, a heat-resistant strain of Indiana serotype (Holloway et al., 1970); ts mutants G114(1), Gil(I), G22(11), G31(111), G32(III), G411IV), and G44(IV) carrying the G (Glasgow) desi~ation (Pringle, 1970); WlO(IV) carrying the W (Winnipeg) designation (Holloway et al., 1970); T1026(1) and T54(111) from Toronto (T) (Farmilo and Stanners, 1972); 05(I), OlOO(IV), Olll(IV), 044(V), 045(V), and 0110(V) with the 0 (Orsay) designation (Flamand, 1970; Genty and Berreur, 1975; Lafay, 1974).

Confluent monolayers of mouse L cells in loo-mm dishes were infected with ts mutants at a multiplicity (m,f,) = 10e3. Virus was harvested after 36-48 hr at 30” when essentially all cells manifested a gross cytopathic effect. The wild type, HR- C, was grown under the same conditions except that incubation was carried out at

CELL KILLING BY VW 269

37” for 18-20 hr. Cell debris was removed from the virus-containing medium by cen- trifugation for 20 min at 1OOOg. The supernatant fluid was centrifuged for 90 min at 30,000 rpm in a Beckman 60 rotor, and the pelleted virus was resuspended in a buffer containing: Tris-HCl, pH 7.4, 10 m&f; NaCl, 100 mM; and EDTA, 1 n&f. This material constituted a standard virus stock. Under these conditions, the yield of VS virus was about 500-1000 plaque-forming particles per GMK-Vero or mouse L cell.

Plaque assay. The wild type, HR-C, and the different ts mutants, in 0.3 ml of attachment solution (Marcus and Sekellick, 1974) were adsorbed in 60-mm dishes of confluent mouse L cells or Vero cells for 35 min at 37”. After the adsorption period, the cells were overlaid with 0.8% agarose and 3% fetal bovine serum in MEM for L cells or with 0.8% agarose and 6% calf serum in NC1 medium (attachment solution) for Vero cells. The cell monolayers were incubated at 30 or 40”, and 68-70 hr later the plates were stained with a sterile neutral red solution (l/10,000) for 2 hr prior to counting plaques,

Cell-killing particle assay. Procedures for generating single-cell survival curves for the CK particle assay have been described in detail previously (Marcus, 1959; Marcus and Sekellick, 1974). This assay scores the capacity of virions to kill cells, i.e., to prevent colony formation from single cells, and does not depend upon the p~uction of new virions as is required for the detection of infectious virus.

Radioisotopic labeling ofproteins. Mon- olayers containing 2 X lOi mouse L cells (loo-mm Falcon dishes) were infected at a multiplicity of 100 or 500 PFP. Virus in 0.6 ml of attachment solution was adsorbed for 35 min at 37”. After the adsorption period, the cells were overlaid with 15 ml of MEM supplemented with 9% fetal bovine serum, prewarmed at 30 or 40”. After a 4.5-hr incubation at 30 or 40”, the medium was removed and the cells washed with a phosphate-bu~ered saline solution at the appropriate temperature. Immedi- ately, 3 ml of prewarmed (30 or 40°) MEM lacking amino acids and containing 16 &i of 3H-labeled amino acid mixture/ml was

added to the monolayer. After a 30-min incubation at 30 or 40”, the radioactive medium was removed rapidly, and further incorporation of radioactivity was stopped by adding 1.5 ml of Triton X-100 in RSBK buffer (Tris-HCl, pH 7.8, 10 n-&f; Mg-ace- tate, 1.5 ti; KCl, 10 n&f) cooled to 0”.

Protein extraction and analysis. The Triton X-loo-treated cells were scraped from the dishes and centrifuged for 10 min at 1OOOg. Proteins in the supernatant fluid were precipitated by adding cold acetone (80% final concentration) for 12 hr at 4”. The precipitate was pelleted by centrifuga- tion for 15 min at 2000g and washed twice with cold 80% acetone. The final pellet was dissolved in 0.2 ml of electrophoresis sam- ple buffer (Tris-HCl, pH 6.8, 0.625 M; SDS, 2.3%; glycerol, 10%; /3-mercapmetha- nol, 5%) and heated for 3 min at 100” to dissociate the proteins. Thirty microliters of this solution was analyzed by polyacrylamide slab-gel electrophoresis according to the technique of Laemmli (1970) and using the apparatus described by Studier (1973). Three concentrations of acrylamide were used per slab: 7.5, 10, and 12.5%. Electro- phoresis was carried out for 5 hr at a con- stant voltage of 60 V and an amperage of 20 mA per slab gel at the beginning of the electrophoresis.

Fluorographic conditions. Slab gels were treated with a solution of 16% PPO in DMSO, according to the technique of Bon- ner and Laskey (1974), and dried on a filter paper sheet in an Hoeffer gel-dryer apparatus The dried gel was exposed for 24 hr at -70” against a Kodak R.P. royal film. Samples were compared by scanning the fluorographic patterns on a Joyce-Loebl densitometer.

RESULTS

Revertant Titers of ts Mutants All virus stocks were assayed at 40.0” for

revertants to ensure that, in the protein synthesis inhibition studies carried out at high multiplicities, infection did not produce spurious results attributable to virus of wild-type phenotype. As reported previously, no revertants were detectable in stocks of tsG114(1) (Hunt and Wagner, 1974; Marcus and Sekellick, 1975). The plaque-forming particle (PFP) titers of ts

270 MARVALDI ET AL.

mutant stocks at permissive temperature ranged from 2 to 30 x lo9 PFP/ml, and at nonpermissive temperature the titers were 5 2 x lo4 PFP/ml. At the highest multiplicity used for the ts mutants (mDfp = 5001, revertant virus was present, in the extreme case, at mpfp = 0.02, but usually at m pfp 5 0.002. Since detection of cellular protein synthesis inhibition by wild-type virus at mpfp 5 5 was marginal at best within the 5-hr test period (data not shown), the low levels of revertant virus present in our stocks of ts mutants would not appear to affect the results of protein synthesis experiments carried out at high multiplicities.

Host Protein Synthesis In~~bit~o~ and the Cell Keening rapacity of VS Virus ts Mutants

Complementation group I. The virion- associated transcriptase of mutant tsG114 is extremely heat labile (Szilagyi and Prin- gle, 1972), to the extent that primary transcripts do not accumulate in the infected cell (in uiuo) held at nonpermissive temperature (Unger and Reichmann, 1973; Marcus and Sekellick, 1974), thus qualify- ing this mutant as phenotypically RNA-. In contrast, tsO5 and tsGl1 appear to be phenotypically RNA+, producing low but measurable amounts of primary transcripts (Printz-Ane et al., 1972; Szilagyi and Pringle, 1972; Flamand and Bishop, 1973) at nonpermissive temperature.

The capacity of these mutants to kill GMK-Vero cells at permissive and nonpermissive temperatures was compared by generating cell-killing survival curves as described previously (Marcus and Sekel- lick, 1975). Figure 1 illustrates the results from representative experiments and dem- onstrates that individual virions of mutants tsG114 and tsGl1 do not kill cells at 40” whereas those of mutant tsO5 do. Simi- lar results were obtained with mouse L cells as hosts (data not shown). Results from one other group I mutant, tsT1026, also are illustra~d in Fig. 1. Although this mutant does not kill cells at 40.0”, significant killing ensues if the temperature is lowered 1 or 2” (not illustrated), an obser- vation consistent with other characteris-

tics of tsT1026 as reported by Stanners et al. (1975).

Inhibition of cellular protein synthesis by three of these mutants following infection (mpfp = 100 or 5001 for 4.5 hr at 30 or 40” was measured as described in Materi- als and Methods. Mouse L cells were pulse-labeled with 3H-labeled amino acids for 30 min, and the proteins extracted and analyzed electrophoretically. Figures 2, 3, and 4 illustrate densitometer scans of fluorographic patterns which represent cellular and/or viral proteins synthesized (labeled) between 4.5 and 5.0 hr postinfection. At permissive temperature (Fig. 2, left column), cells infected with tsG114, tsG11, or tsO5 all produce virus-specific components (proteins M, NS, N, G, and L) identifiable on the scanning pattern by comparison with the wild-type HR marker proteins (Fig. 2, bottom left panel). A control experiment shows the fluorographic

FRACTION

OF

SURVIVING

CELLS

(-=30-l

(cv-0=40”)

0,2345012345Ol2345

CELL KiLLiNG PARTICLE ~LTlPL!ClTY

FIG. 1. Survival curves of GMK-Vera cells exposed to low multiplicities of ts mutants representing the five complementation groups and wild-type VS virus (Ind.). Experimental details have been described (Marcus and Sekellick, 1975).

CELL KILLING BY VSV 271

16 GROUP n /

I

022

‘i L+-.-L

MIGRATION (CM.1

FIG. 2. Gel electrophoresis of proteins from mouse L cells infected with ts mutants representing the five complementation groups and wild-type VS virus (Ind.1. The scanning profiles of fluorographic patterns obtained as described in Materials and Methods represent protein synthesis during a 30- min period 4.5 hr postinfection at melp = 100 and incubation at permissive temperature, 30”.

profile of cellular proteins from mock-infected cells (Fig. 2, top left panel),

At nonpermissive temperature (Figs. 3 and 4, left columns) el~trophores~s-fluo-

rographic profiles from cells infected with tsG114 or tsGl1 revealed a protein pattern identical to that of uninfected control cells (top left panel in each figure), both at m,,fp = 100 (Fig. 3) or mpfp = 500 (Fig. 4). Analy- sis profiles from cells infected with tsO5 exhibited a different pattern, revealing a marked inhibition of cellular protein syn-

MIGRATION f CM. f

Fxc. 3. Gel electrophoresis: scanning profiles of ~uorographic patterns obtained as described in the legend of Fig. 2, except that infected cells were incubated at nonpermissive temperature, 40”. Scans were made from the fluorographic patterns shown in Fig. 5b.

MARVAL~I ET AL.

1

MIGRATION (CM.1

FIG. 4. Gel electrophoresis: scanning profiles of fluorographic patterns obtained as described in the legend of Fig. 2, except that cells were infected at m Pf,, = 500 and incubation was carried out at nonpermissive temperature -40”. Scans were made from the fluorographic patterns shown in Fig. 5a.

thesis and possibly some synthesis of viral proteins, especially N and NS [these represent translation products of the two genes most proximate to the single initiation site at the 3’-end of the genome RNA (Ball and White, 1976; Abraham and Ba-

nerjee, 1976)l. Clearly, infection with mutants tsG114 or tsGl1 does not culminate in the turnoff of cellular protein synthesis at nonpermissive temperature, whereas infection with ts05 does.

Complementation groups II and IV. Three mutants, tsWl0 and tsG41 of group IV, and tsG22 of group II, were tested for their effect on both cell killing and cellular protein synthesis at 30 and 40”. All three mutants possess a temperature-stable virion transcriptase and are capable of synthesizing primary transcripts at 40” in ho, but apparently are defective in some subsequent replicative function (Wong, et al., 1972; Unger and Reichman, 1973; Mar- cus and Sekellick, 1977). At 40” mutants tsG41 and tsWl0 produce large and small amounts of primary transcripts, respee- tively, in the presence or absence of cycloheximide (Marcus and Sekellick, 1977).

The cell-killing survival curves generated by these three mutants demonstrate that tsWlO(IV) produces a lethal dose of the putative cell-killing factor (Marcus and Sekellick, 1975) under the restrictive temperature (Fig. 1). In sharp contrast, mutant tsG41(IV), also of complementation group IV, does not kill cells at 40”, nor does mutant tsG22(II) (Fig. 1). The cell- killing characteristics of two other group IV mutants also are illustrated in Fig. 1. Mutants tsOlOO(IV) and tsOlll(IV) are like tsWlO(IV) in their capacity to kill cells at 40”.

Inhibition of cellular protein synthesis was determined for mutants tsWlO(IV), G41(IV) and tsG22(11). Representative fluoro~aphic scan patterns for protein synthesis between 4.5 and 5.0 hr after infection are illustrated in Figs. 2, 3, and 4 (right columns). Virus-specific protein profiles typical of wild-type controls were obtained at permissive temperature for these three mutants (Fig. 21, except that L protein was always obtained in low amounts.

At 40” the fluorographic profiles of proteins synthesized in cells infected with tsG4lfIV) or tsG22(11) were virtually identical to protein patterns from uninfected cells, indicating that neither of these mutants turned off cellular protein synthesis, whether at m,,, = 100 or 500 (Figs. 3 and 4). In sharp contrast, and under the same


conditions, the protein synthesis profile obtained from cells infected with mutant tsWl0~~~ at 40” shows the presence of specific viral components and a marked reduction in the accumulation (synthesis) of cellular proteins (Figs. 3 and 4).

Clearly, mutants of group II and IV which inhibit cellular protein synthesis at 40” also kill cells at that temperature.

Complementatio~ groups III and V. Two mutants, tsG3l(III) and t&45(V) were tested both for their capacity to inhibit cellular protein synthesis and to kill cells. In addition, tsOllO(V), ts044(V), and tsG32(IIl) were tested for cell-killing activity. At restrictive temperatures these mutants all exhibit an active transcriptase and replicase activity in ho, quali~ing them as RNA+ ts mutants (Lafay, 1974; Szilagyi and F’ringle, 1972). Cells were infected, processed, and tested for the lethal ef%ect of the virus or inhibition of protein synthesis as described above.

The cell-killing survival curves generated by these mutants at 30 and 40” are presented in Fig. 1. They demonstrate that both of the group III mutants tested kill cells at the restrictive temperature, albeit tsG32(111) with somewhat less efficiency than tsG31(IlI). The same may be said of cell killing by two group V mutants, tsOll0 and ts044. Figure 1 reveals that only about 1 of every 2 virions of tsG32(111) or t&44(V) that kill cells at 30”, are lethal at 40”. In the case of ~045(V) there is a marked decrease in the slope of the survival curve generated at 40” relative to that at 30”. Determination of the slope from data obtained at very high multiplicities indicates that an average of about 40 cell-killing particles must interact with a cell held at 40” to achieve the level of killing obtained by m&v = 1 at 30”.

As for all the mutants tested at permissive temperature typical virus specific profiles were obtained for tsG31(III) and tsO45(V) (Fig. 2). At nonpermissive temperature, the fluorographic scans revested a significant inhibition of cellular protein synthesis, and demonstrated production of viral specific proteins. For these two mutants in particular and at both multiplicities tested (m,,,, = 100 and 500), synthesis of two cellular proteins which migrate be-

tween viral proteins L and G appears quite resistant to inhibition.

Comparison of Cell-Killing Particle Actiu- ity and Cellular Protein Synthesis In- ~i~it~on by ts mutants of Vesicu~ur Stoma&is Virus

A summary of our data for cellular protein synthesis inhibition by ts mutants representing all five complementation groups of VS virus (Ind.) ts mutants are presented in Fig. 5. They are typical of those used to generate the ~uoro~aphic scans of Figs. 2, 3, and 4. A comparison of cell-killing particle activity and host cell protein synthesis by these mutants is presented in Table 1 and reveals that they may fall into two classes within a complementation group. One class consists of, in addition to wild-type virus, mutants tsom), tsG31(111), tsWlO(IV), and tsOllO(V); all are capable of host cell protein synthesis inhibition and eelI-hilling particle activity at 40” and may be termed ttisi+.cklj+ . Members of the other class do not inhibit cellular protein synthesis significantly (even at mpfp = 500), nor do they kill cells at 40” (tested at PYZ 4 5). butts tsGl14(1), tsGll(I), tsG22(II), and tsG41(IV) constitute this class, and may be termed tSl,si-.rPu .

DISCUSSION

These data extend the observations from earlier studies in this series (Marcus and Sekellick, 1974, 1975, 1976) and demonstrate that cell killing and the inhibition of cellular protein synthesis by vesicular stomatitis (VS) virus both require the same gene functions and that not all viral proteins need by fully functional. In every instance tested, with the reservation for ~045(V) as discussed be10w,~ there was an exact correlation between the inhibition of cell protein synthesis, measured at mrtfn = 100-500, and cell-killing particle activity, scored by the action of single virions.

4 Studies of eelI killing by ~045(V~ at high multiplicity reveal a special requirement for large numbers of virions to kill cells at 40” (40 CK particles at 40” = 1 CR particle at 30”) and will be the subject of a subsequent communication. In this context, the use of preformed G (group V) protein to rescue tsO45(V) was convincingly demonstrated by the experiments of Deutsch (1975).

274 MARVALDI ET AL.

FIG. 5. Fluorographic patterns of proteins synthesized in mouse L cells infected with ts mutants and wild-type VS virus (Ind.) and processed as described in Materials and Methods. The pattern represents proteins synthesized at 40” in cells infected at mpfp = 100 (b) or mPrp = 500 (a). Protein patterns from uninfected cells (“cell”) and viral marker proteins L, G, NS, N, and M are also illustrated.


TABLE 1

COMPARISON OF CELL-KILLING PARTICLE ACTIVITY AND PROTEIN SYNTHESIS INHIBITION BY ts MUTANTS OF VESICULAR STOMATITIS VIRUS

Complementation group and ckp, ts mutant designation Activity at 40” psi phenotypes

Cell k;tfl;g par- Protein synthesis inhibition

Wild typeckp*.W*’ W+ (HR-Cj(Ind.1 + + p.P-m- G114 - -

Gil - -

T1026 - n.t.* pp+.LN* 05 +c + ~pP-,P*i- G22 - - ~~p’kP+m+ G31 + +

G33 + n.t. IVCkP-,P*,- G41 - - ~v”k?J’m’+ WlO + +

0100 + n.t. 0111 + n.t.

vc”P+.Ps” 0110 + + 044 + n.t. 045 -c +

u The ckp+ andpsi+ phenotypes represent expression of cell-killing particle activity and protein synthesis inhibition (or the lack thereof, ckp-, psi?) tested at nonpermissive temperature, 40.0”. The virions of all ts mutants and W+ virus contain transcriptase molecules which function at 40”, albeit at varying efficiencies, and hence may be termed tP , excepting tsG114(1) which is t+-.

bn.t. = not tested. c See discussion regarding this apparent anomaly.

The failure of a ts mutant to kill cells or inhibit cellular protein synthesis at the restrictive temperature, as is the case with mutants G114(1), Gil(I), T1026(1), G22(11), G41(IV), and 045(V), reveals a requirement for that protein in the formation and expression of these viral functions. The data summarized in Table 1 demonstrate that minimally functional proteins of four complementation groups, e.g., I, II, IV, and V, appear to be necessary for the expression of cell killing and protein synthesis inhibition. However, the requirement for proteins L(1) and G(V) appears to differ in kind from that of proteins NW) and N(IV): Whereas proteins N and NS must be newly synthesized, preformed and appropriately configured, L and G proteins in the virion appear to suffice.4 These conclusions are based on the following observations: Ultraviolet -radiation data revealed an apparent target size for the expression of cell-killing particle activity by VS virus of one-fifth of the genome (Marcus and Sekellick, 1975). Those data were sufficiently accurate and reproducible for us to conclude that the

72% of the viral genome coding for proteins L and G need not be intact for the virus to express cell killing. This conclusion was strengthened further by the assignment of the L and G protein genes to the most distal point from the mandatory single initiation site at the 3’-terminus of the VS virus genome (Ball and White, 1976; Abra- ham and Banerjee, 1976). These gene posi- tions would result in an apparent target size for the cistrons of the L and G proteins equivalent to that of the entire genome and would, were new L and G proteins required for cell killing, produce the same rate of inactivation for both plaque-forming and cell-killing particle activities, a result incompatible with the observations reported earlier (Marcus and Sekellick, 1975). Consequently, we interpret these data to reflect the need for preformed but not newly synthesized, virion-associated L protein to transcribe the messenger RNAs coding for proteins N and NS, as well as a need for a critical number of minimally functional G protein molecules.4 The failure of tsG114(1), a group I mutant with a heat-labile virion-associated transcrip-

276 MARVALDI ET AL.

tase, to kill cells and inhibit protein synthesis at 40” is compatible with this expla- nation. However, the ts’.C’+~P~-ri~~~Js~~ phenotype of mutants Gil(1) and T1026(1) provide further evidence that primary transcription per se does not suffice to bring about the expression of cell killing (Mar- cus and Sekellick, 1974, 1975) or protein synthesis inhibition (Table 1) and that some additional function is required of the virion-derived L protein. At the restrictive temperature, molecules of L protein of mutants Gil(1) and T1026(1) with their ts’” character do not possess this function, whereas those of mutant 05(I) with its t&v- .d;~,-.,e~ + phenotype do.

Functional matrix protein M, representing the gene product of complementation group III does not appear to be required for the expression of protein synthesis inhibition or cell killing by VS virus.;’

The lack of protein synthesis inhibition and cell killing by some mutants of complementation groups II and IV at nonpermissive temperature indicates that the polypeptides of these two viral genes must

ts-Mutants: Gll4(ll Gil(l) G22Cll) G4l(IV)

VS virus

data from uv-survival curves for cell killing and the pattern of cell killing and protein synthesis inhibition by certain mutants of complementation groups I, II, IV, and V at nonpermissive temperature by simply considering their location on the VS virus genome relative to the single initiation site for transcription at the 3’- terminus. We conclude that transcription of genes N and NS by virion-associated transcriptase (L protein) and their translation into minimally functional proteins are required for the expression of cell killing and protein synthesis inhibition by VS virus.

Our results also allow for a subdivision of mutants into those that kill cells and inhibit cellular protein synthesis at 40”, termed ts’i”+,~lsi+ and those which do not, t#“‘-,~‘~‘+ . These important findings permit a definition of ts mutants in terms of these two activities, chp (cell-hilling particles) and psi (protein synthesis inhibitor) and allow for an ordering of mutants with respect to function, even within a complementation group.”

05(l) W+( Ind.) G3lCIIll WlO(lV) OllO(V)

protein synthesis , , RNA synthesis .,,,,.. 1

Phenotypes:

(tstra-,psi-,ckp-,pfp-)

be minimally functional to bring about these cellular responses. In this context, we note that the two genes coding for proteins NS and N (complementation groups II and IV, respectively) not only constitute one-fifth of the length of the genome, but they are the two genes most proximate to the single initiation site at its 3’-end and therefore constitute the only sequence of two genes that would present a target size proportional to their combined molecular weights, about 21% (Ball and White, 1976; Abraham and Banerjee, 1976).

As noted above, we can reconcile the ‘The requirement for minimally functional M

protein will be established when a group III mutant is found to display a uP”-.“*’ phenotype.

Thus, while properly configured wild-type N protein functions at 40” to support (i) cellular protein synthesis inhibition, (ii) cell killing, and (iii) viral replication, the N protein synthesized by tsWlO(IV) will only support functions (i) and (ii), whereas that synthesized by mutant tsG41(IV) will support neither. Presumably, the confor-

ii We have included the notation td““. to desig- nate the presence or absence, respectively, of in uiuo primary transcription at the restrictive temperature. Furthermore, by an operational definition, all mutants should be designated td’” because of their failure to form plaques (replicate) at 40”. Mutant taOll0 (IV), tested at mpfp = 100 and 40”, inhibits cellular protein synthesis (data not shown). Mutant ts045(V) was omitted for reasons considered in the discussion, see footnote 4.

CELL KILLING BY VW 277

mation of tsWlO(IV) N protein synthesized and/or held at 40” suffices to provide the minimal function required to express protein synthesis inhibition and cell-killing particle activity but not viral replication.

Although our results point out the need for primary transcription in the expression of cell killing and protein synthesis inhibition, they also establish that it is not a sufficient event. For example, under nonpermissive conditions mutant tsG41(IV) does not kill cells or inhibit cellular protein synthesis despite the accumulation of large quantities of virion-derived transcripts of normal size distribution (Unger and Reichmann, 1973; Marcus and Sekel- lick, 1977). Whether these transcripts function to produce viral proteins is ob- scured by the high (normal) levels of cellular protein synthesized upon infection with this virus at 40” and remains to be determined. Other results indicate that the quality rather than the quantity of the viral transcripts synthesized is the important determinant in cell killing and protein synthesis inhibition by VS virus. Mu- tant tsWlO(IV) synthesizes significantly fewer primary transcripts at 40” than does tsG41(IV), a tspsi-,ck”- mutant (Marcus and Sekellick, 1977), but inhibition of cellular protein synthesis and cell killing by tsWlO(IV) is just as efficient as with wild- type virus. The t@+ character of tsWlO(IV) allows us to detect viral proteins against an extremely low back- ground of cellular proteins and conclude that the transcripts of this mutant are translated at 40” into the five viral proteins characteristic of VS virus (Ind.) (Wagner et al., 1969).

Although cellular protein synthesis inhibition and cell killing by VS virus appear to be controlled by the same genes, the relationship between these two viral- induced activities is not obvious. Presum- ably, a virion capable of inhibiting cellular protein synthesis over an extended period would ultimately prevent cell division and be scored as a cell-killing particle. In this case, insight into the mechanism of viral- induced inhibition of protein synthesis would provide the raison d’e^tre for cell killing. On the other hand, inhibition of cellular protein synthesis per se may not

be responsible for the rapid onset of the cytopathology characteristic of VS virus infection and the ultimate demise of the cell. After all, cellular protein synthesis inhibition mediated by cycloheximide does not mimic in any temporal sense the cytopathic effects of viruses (Bablanian, 1970; Marcus, unpublished observations). In fact, GMK-Vero cells held for 20-24 hr in cycloheximide (50 pug/ml) show no loss in plating efficiency upon washout of the drug (Marcus and Sekellick, 1975). Con- ceivably, cell killing by VS virus requires one or more reactions subsequent to or possibly independent of protein synthesis inhibition.

Following completion of our experimental work, studies by McAllister and Wag- ner (1976) appeared reporting on the inhibition of cellular protein synthesis in mouse L cells by ts mutants of VS virus representing three of the five complementation groups. One mutant, tsG114(1), was common to their studies and ours, and gave identical results, displaying a ts”“‘. character at the restrictive temperature, as did another ts”““- mutant, G13(1). Their result with mutant tsG44(IV) was similar to ours with tsWlO(IV) both revealing a t&“‘& phenotype, whereas the t#“‘+ character of mutant 052(11) indicates that the group II protein, like that of proteins I, IV, and V, can exist in at least two configura- tions with respect to the expression of protein synthesis inhibition and viral replication. Whether the same may be said for M protein (group III) must await the testing of more mutants.

The results summarized in Table 1, and those of McAllister and Wagner (1976) with mutants tsG114(1) and tsG13(1), clearly establish primary transcription as a requisite event for protein synthesis inhibition by VS virus [as was the case for the expression of cell-killing particle activity (Marcus and Sekellick 1974,1975)]. Our results also demonstrate the need for minimally functional proteins N and NS to express the t&+” J’~~‘ phenotype, accounting for the specific one-fifth of the viral genome apparently required as a template for the transcription of genes N and NS (Marcus and Sekellick, 1975).

The intriguing observations of Dubovi

278 MARVALDI ET AL.

and Youngner (1976a) regarding the inhibition of pseudorabies virus by single particles of complete VS virus warrant com- ment: Conceivably, virtually all physical particles in the VS virus stocks prepared by Dubovi and Youngner (1976a) possessed functional transcriptase and genomes sufficiently intact to be capable of producing the newly synthesized N and NS proteins required for the expression of cell killing and protein synthesis inhibition. Hence, these particles would qualify as the nonin- fectious (defective) cell-killing particles described by Marcus and Sekellick (1974) as an excess of infectious particles (plaque- forming particles) in many stock prepara- tions of VS virus. We also note that the capacity of VS virus ts mutants to inhibit the replication of pseudorabies (psr phenotype) at nonpermissive temperature as reported by Dubovi and Youngner (1976a, Table 3) correlates exactly with the ckp andpsi phenotypes for the ts mutants that both they and we have tested. Presumably then, the same genes determine the expression of these attributes.

Since the intracellular and structural proteins of VS virus appear to be identical (Wagner, 1975), the addition to the cell of large enough numbers of viral polypeptides through infection at superhigh multiplicities might provide sufficient amounts of these proteins to inhibit cellular protein synthesis and/or kill cells, even in the absence of primary transcription as a source of newly synthesized proteins N and NS, and neglecting the possible involvement of certain viral RNA species (Marcus and Sekellick, 1974, 1975). Recent observations by others and studies now in progress on cell killing carried out at very high multiplicities indicate that sufficiently large numbers of preformed viral components (protein?) may function to inhibit (i) cellular protein synthesis (Wertz and Young- ner, 1972; Dubovi and Youngner, 1976b; Baxt and Bablanian, 1976), (ii) the replication of pseudorabies virus (Dubovi and Youngner 1976a), or (iii) cell replication (Marcus and Sekellick, unpublished observations). However, there is reason to be cautions regarding the interpretation of such results. At superhigh multiplicities it is difficult to distinguish between the ac-

tion in concert of otherwise inactive virions from the action of a single virion among a vast excess of inactive particles. Although studies are in progress to resolve this question we can report that single-cell survival curves generated by high multiplicity infection with “nonlethal” ts+“‘- mutants reveals single-hit kinetics, sug- gesting that cell killing may derive from a rate-limiting reactant produced by the action of a single virion rather than the combined efforts of many particles (Marcus and Sekellick, unpublished observations). These observations also indicate that cell killing is a more sensitive measure of gene action than are present methods for detect- ing the inhibition of cellular protein synthesis.

The relatively high resistance to inactivation by uv radiation of the cell-killing (Marcus and Sekellick, 1975) and protein synthesis-inhibiting potential of VS virus (in preparation) further complicates experiments in which this agent is used to draw conclusions regarding the need for newly synthesized viral proteins, especially when high multiplicities are used. For example, there is a suggestion from the polyacrylamide-gel electropherograms of cells infected with a superhigh multiplicity Cm = 3000-4000 particles, i.e., mpfp = 200 X 15-20 physical particles) of uv-irradiated virus that a sufficient number of copies of genes N and NS survives the radiation to produce detectable amounts of protein (Baxt and Bablanian, 1976, Fig. 4), possibly enough to account for protein synthesis inhibition. Such a result would be consistent with the high resistance of these two genes to inactivation by uv radiation (Ball and White, 1976; Abraham and Banerjee, 1976) and is in accord with our data on protein synthesis inhibition by uv-irradiated virus (in preparation).

If it is definitely established that preformed viral proteins added to cells in sufficiently high concentrations can, in the true absence of transcription and translation, inhibit cellular protein synthesis, as Dubovi and Youngner (1976b) and Baxt and Bablanian (1976) propose, our results clearly define which input viral components are required as minimally functional, namely, proteins L, N and NS. If


the sole function of the L protein was to produce mRNAs coding for N and NS (for subsequent translation), then its need would predictably disappear at multiplicities high enough to supply these two proteins at concentrations above. the threshold for the production of a putative protein synthesis inhibition (psi) factor. The lack of cellular protein synthesis inhibition at 40" by ts”“‘- mutants tsG114(1) at mpfp = 100 or 500 and tsG13(1) at n 2 100 (McAl- lister and Wagner, 1976) suggests this view may be too simplistic. Either about lo7 molecules of N and 5 x lo” molecules of NS protein per cell do not slice to produce detectable amounts ofpsi factor, or protein L serves a function beyond that of merely supplying threshold numbers of N and NS transcripts. Several investigators have im- plicated the L protein in a replicative, as well as transcriptive, function (Wertz and Levine, 1973; Perlman and Huang, 1973; Combard, et al., 19741, and it may be that the former role is required for the expression of protein synthesis inhibition (and cell killing).

We have established that minimally functional viral proteins L, N and NS (and possibly G4,“> are specifically required for the expression of cellular protein synthesis inhibition and cell killing by VS virus. As a working hypothesis we propose that a single virion of VS virus can kill a cell (and inhibit pseudorabies virus replication and possibly inhibit cellular protein and nucleic acid synthesis) through the formation and action of a putative cell-killing factor if minimally functional N and NS proteins are newly synthesized in the presence of threshold amounts of properly configured molecules of preformed virion-associated L, and possibly G**” proteins, The extent to which newly synthesized N and NS proteins can be dispensed with and re- placed by preformed molecules is presently under investigation, as are studies to determine the nature of the putative ckp and psi factor(s) produced by the concerted action of these viral proteins.

Note added in proof. Colonno, Lazzarini, Keene, and Banerjee (Proc. Nat. Acad. Sci., in press, 1977) have reported that a long defective interfering particle of VS virus (DI-LT) is active in transcribing the leader sequence of genome RNA and genes N,

NS, M and G, but lacks most of the gene for L protein. We have been able to establish that a single DI-LT particle suffices to kill a cell (Marcus, Sekellick, Johnson, and Lazzarini, manuscript submitted). These results prove that newly synthesized L mRNA or protein are not required for cell killing by vesicular s&mat&is virus, and provide further evidence for our hypothesis that cell killing requires functional vi&n-associated L protein.

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