Mutant retrovirus particles package vesicular stomatitis virus mRNA during mixed infection

V1K0L0CY

109, 183-187

(1981)

Mutant

Imperial

Cancer

Retrovirus Particles Package Vesicular Virus mRNA during Mixed Infection

EMANUEL

YAKOBSON’

Research

Laboratories,

Fund

Accepted

AND Lincoln’s October

We have investigated whether retroviruses can package unrelated mRNA into virions by superinfecting virus-producing cells with a mutant vesicular stomatitis virus (VSV) which produces abundant VSV mRNA without shutting off retrovirus synthesis (1). NIH/3T3 cells producing Moloney strain murine leukemia virus (MoLV) particles were superinfected by the tsG31 mutant of vesicular stomatitis virus (VSV) in the presence of actinomycin D at nonpermissive temperature. Under these conditions synthesis of MoLV genomic RNA is inhibited while production of uninfectious “empty” MoLV virions continues at a considerable rate for several hours. VSV tsG31 RNA synthesis occurs at nonpermissive temperature at a level similar to that of cell RNA synthesis in the absence of actinomycin D. We could not detect any VSV RNA in MoLV “empty” virions formed after actinomycin D treatment, indicating a high specificity of RNA packaging into MoLV particles. A quail cell line (1SQ) producing Rous Sarcoma virus similarly releases “empty” virions following actinomycin D treatment and VSV tsG31 superinfection. Another quail cell line (SEBlQlb) carries a mutant of RSV which is naturally deficient in packaging genomic RNA (2). By contrast to the actinomycin D experiments with wild type retroviruses, superinfection of SEBlQlb cells with VSV tsG31 results in

ROBIN Inn

Fields,

Stomatitis

A. WEISS? London

WCZA

SPX,

England

6. 1980

uptake of VSV RNA into retrovirus particles. However, it appears that mRNA for only two of the five VSV proteins (NS and M) are packaged into SE2lQlb virions. Retroviruses may package very small amounts of cellular mRNA. Ikawa et al. (3) estimated that Friend leukemia virus, released from a mouse cell line induced to make hemoglobin, packaged about one globin mRNA molecule per thousand 60 S viral RNA molecules. We decided to investigate whether any significant packaging of mRNA occurs under conditions when virion RNA of a retrovirus is not available. Levin et al. (4) and Paskind et al. (5) showed that the intracellular pool of genomic retrovirus RNA could be eliminated by treatment of infected cells for 2-3 hr with a low concentration of actinomycin D. Under these conditions the production of retrovirus proteins nevertheless continues because of the relative stability of viral mRNA in polyribosomes, resulting in the formation of so-called “empty” virions for several hours following actinomycin D treatment. These C-type particles are devoid of genomic RNA, but are otherwise similar to normal particles in their physicochemical properties (size, buoyant density); they contain all the virion proteins (6), have normal levels of reverse transcriptase (7), and the usual complement of host tRNA (8). No RNA of higher molecular weight was detected in “actinomycin D” particles (8 ), but this could be due to the unavailability of newly synthesized host mRNA which is inhibited by actinomycin D. The synthesis of viral RNA following infection by VSV is not sensitive to actinomycin D. By superinfecting MoLV-

’ Present address: Department of Biology, University of California, San Diego, La Jolla, Calif. 92093. L Present address: Institute of Cancer Research, Chester Beatty Research Institute, Fulham Road, London SW3 6JB, England. To whom reprint requests should be addressed. 183

0042-6822/81/0301X3-05$02.0010 Copyright Ail rights

1; 19x1 by Aradem1c Press. of reprc~luct~on in any form

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184

SHORT TABLE

RNA

SYNTHESIS

Experimental Untreated Actinomycin Actinomycin infected Actinomycin infected

IN NIHi3T3

conditions

cells at 37” D treated D treated, at 32” D treated, at 39.5”

1 MoLV-INFECTED

CELLS

[“HIUridine incorporated (cpm/9-cm plate)

3 at 37 tsG31

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x 10”

2 x 10” 5 x 10”

tsG31 3 x 10”

Note. Subconfluent monolayer cultures of NIHi3T3 MoLV-infected cells were infected with VSV tsG31 at a multiplicity of infection of 5 PFUicell and incubated at the appropriate temperature in medium containing 0.2 pgiml of actinomycin D for 5 hr. The cells were then labeled for 5 hr with [“Hluridine (50 &i/ml, 20 Wmmol) and the TCA-insoluble radioactivity was determined in cytoplasmic lysates obtained by treatment with 0.5% NP40.

producing cells with a VSV temperaturesensitive mutant (tsG31) that synthesizes RNA at nonpermissive temperature (9) but does not produce virions (lo), we have found that even under these conditions of abundant newly synthesized mRNA, no packaging of VSV RNA could be detected. In contrast, tsVSV superinfection of quail cells producing mutant avian sarcoma particles, SE21Qlb (2), results in packaging of mRNA for two of the five VSV proteins. Mouse NIH/3T3 cells, MoLV, and MoLVinfected NIH/3T3 cells were kindly provided by N. Teich. MoLV was assayed by XC test and reverse transcriptase as previously described (11). The VSV mutant tsG31 was originally provided by C. Pringle and was plaque purified as previously described (1). SE21Qlb cells (2) were kindly provided by M. Linial. The data presented in Table 1 show that infection of NIH/3T3 MoLV-infected cells by VSV tsG31 resulted in actinomycin D-insensitive, VSV-specific RNA synthesis at levels comparable with overall cellular RNA synthesis in the absence of the drug. This result was expected, because tsG31 is a “late” RNA+ ts mutant (9). Figure 1 (A-G) presents an analysis of MoLV particle production under various

conditions. Following 2 hr actinomycin D (0.2 pg/ml) treatment no significant reduction of particles labeled with [3”S]methionine occurred (Figs. 1B and C). The reverse transcriptase activity of these particles was also only twofold lower than that of control cultures not treated with actinomycin D whereas the infectivity assayed by XC test was 20- to loo-fold lower (results not shown). Actinomycin D treatment apparently inhibited the synthesis and packaging of MoLV virion RNA into MoLV particles (Figs. 1E and F), in agreement with earlier studies (4-S). When MoLV-producing cells were infected with tsG31 mutants of VSV at 39.5” in the presence of actinomycin D there was a reduction of MoLV production (Figs. 1B and D), although a significant proportion of particles (>25%) was still produced. However, incorporation of actinomycin D-insensitive VSV-specific RNA into MoLV particles was not detectable under these conditions (Fig. 1G). These results show that MoLV particles devoid of genomic RNA do not assemble mRNA or genomic RNA of VSV which is abundant in the cytoplasm. Very small amounts of VSV RNA may be packaged int.o the C-type particles, as found for globin mRNA in Friend leukemia virus, but the absence of newly synthesized genomic MoLV RNA due to actinomycin D treatment does not permit the ready assembly of alternative RNA species. Analysis of SEZlQlb quail cells superinfected by VSV tsG31 mutant at nonpermissive temperature (Fig. 1, H-J) indicates that in this system an efficient packaging of actinomycin D-resistant VSV RNA occurs. Parallel experiments using 16Q quail cells (12) which produce Bryan strain RSV particles did not show VSV mRNA packaging and no virus particles were detected in normal quail cells infected with VSV tsG31 at nonpermissive temperature (results not shown). A greater yield of virus particles with higher reverse transcriptase activity was obtained from VSV tsG31 infected SEBlQlb cells by omitting actinomycin D treatment. The RNA extracted from SE2lQlb particles was assayed for template activity in a

SHORT

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FIG. 1. Formation of MoLV in NIHi3T3 mouse and SEZlQlb quail cells superinfected by the tsG31 mutant of VSV at nonpermissive temperature. Subconfluent monolayer cultures of NIHi3T3 MoLV-producing cells were infected with VSV tsG31 at a m.o.i. of 5, incubated at 39.5”, treated with actinomycin D (0.2 fig/ml) 2 hr after VSV infection, and labeled with either [“Slmethionine (100 &i/ml, 800 Ciimmol) or [“Hluridine (125 &i/ml, 20 Ciimmol) from 4 to 8 hr after infection. After lowspeed centrifugation virus particles were concentrated from culture fluids by centrifugation at 100,000 9 for 2 hr and banded in sucrose density gradients (20-45%, w/v, in PBS) for 4 hr at 120,000 g. Aliquots of gradient fractions were assayed for radioactivity. Culture fluids from one g-cm plate when labeled with [%]methionine (A-D) and eight g-cm plates when labeled with [:‘H]uridine (E-G) were used to determine MoLV particle production. (A) Uninfected cells; (B) and (E), MoLV-infected cells; (C) and (F), MoLV-infected, actinomycin D-treated cells; (D) and (G), MoLV-infected, actinomycin D-treated cells superinfected with tsG31 VSV. Monolayer cultures of SE21Qlb cells were infected and labeled with [“Hluridine as for (E-G), except culture fluids of two 90-mm plates were used to determine virus particle production. (H) SEZlQlb uninfected cells; (I) actinomycin D-treated SEZlQlb cells; and (J), actinomycin D-treated SEZlQlb cells superinfected with VSV tsG31.

RNA-dependent reticulocyte messenger lysate. Figure 2A (lane b) shows that virion RNA from retrovirus particles of SE2lQlb cells acts as an efficient template for many proteins. The pattern of proteins synthesized by RNA species packaged into SEBlQlb virions is similar to that reported by Gallis et al. (13), who showed by immunological and peptide mapping studies that the majority of proteins synthesized appear to be quail cell proteins. The protein pattern of virion RNA from retrovirus particles of SEZlQlb cells superinfected with VSV tsG31 (lane g) appears simpler because VSV infection inhibits

some of the host protein synthesis. Immunoprecipitations with anti-VSV multivalent antibody reveals two VSV-specific proteins among the products of the cell-free protein syntheses (Fig. 2A, lane e). Figure 2B presents evidence that the two VSV-specific proteins translated from RNA extracted from virus particles (lane c) corn&rate with VSV NS and M proteins translated in vitro from mRNA extracted from VSV-infected cells. Virions of VSV tsG31 are not produced at the nonpermissive temperature (9) and the negativestrand genomic RNA of VSV would not be translated into identifiable proteins. We

SHORT

A.a

b

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B.

a

b

c

COMMUNICATIONS

d

FIG;. 2. (A) Template activity of RNA in retrovirus particles from SEBlQlb cells superinfected with VSV tsG31. SE21Qlb cells were infected with VSV tsG31 as in Fig. 1. but in the absence of actinomycin D. Virion RNA from purified retrovirus particles was extracted as previously described (16) and 0.c5 wg of RNA (1 mgiml) was added to a messenger RNA-dependent reticulocytr lysate, containing 15 &i ]:“S]mrthioninc per 20 ~1 of reaction mixture ( t 7 ). The protein products from translation reactions were fractionated by electrophoresis in a 10% SDS-polyacrylamide slab gel as described previously (16 ) and the fluorograph is shown. The letters on the right-hand side indicate the positions of respective VSV proteins from purified virions in this gel. Lanes: (a) no RNA added; (b) 0.5 pg of virion RNA from SEZlQlb particles of uninfected cells; (c) the same as (b) but the material aas precipitated by 20 ~1 of 10% suspension of Sfnpllylococcws nurr~cs (18); ((1) the same as (b) but protein products were reacted with anti-VSV multivalent sheep serum (1:5000 final dilution (10) followed by precipitation with 20 ~1 S. n~~rcw; (e), (f ), (g), similar to (d), (c). (b), respectively, but 0.5 pg RNA added were from SEZlQlb retrovirus virions of cell superinfected with VSV tsG31. The reaction with antibody and vvashes of.?. o,(rc~(.s precipitate were carried out in the presence of .5 mgiml of bovine serum albumin except for the last t\vo washes to reduce nonspecific adsorption. Equal aliquots of protein synthesis reaction mixtures were analyzed in all lanes (a-g). (B) Synthesis of VSV proteins directed by retrovirus virion DNA of SEZlQlb cells superinfected with VSV fs31 and by VSV mRNA from Vero cells in messenger RNA-dependent reticulocyte lysates. Cytoplasmic

conclude, therefore, that the mRNA for the NS and M proteins is packaged into the retrovirus particles released from SEZlQlb cells. VSV fsG31 overproduces all five mRNA species (for L, G, N, NS, and M proteins) in equimolar quantities (14 ). The messenger RNA for L protein is usually poorly translated in cell-free systems (15) and \ve were unable to detect the protein product of I, mRNA when mRNA from VSV-infected cells was used as a template (Fig. 2B, (a) and (d)). Thus our results indicate that only two VSV mRNA species could be detected in retrovirus virions of SE21Qlb cells. Linial and associates (2, 13) concluded that SE2lQlb retrovirus particles, which fail to assemble their own retroviruses genomic RNA, package cellular mRNA efficiently. Our experiments support this conclusion and moreover indicate that packaging is not random. We do not knoiv why mRNA species are selectively assembled into retrovirus virions. One explanation for such nonrandom packaging could be the local concentration of different species of mRNA at the site of retrovirion assembly; alternatively, the primary structure of the mRNA may be important for assembly or stability in retrovirus particles. In summary, our VSV superinfection experiments indicate that RNA incorporapoly A-containing RNA (1 mg/ml, 0.5 pg per reaction mixture) was extracted as described previously (15 ) from VSV-infected Vero cells 6 hr after infection with 10 PFU of VSV and Ivas translated as described for Pig. 2A. Virion RN.4 from purified retrovirus particles of SEBlQlb cells superinfected with VSV tsG31 as described in Fig. 1, but in the absence of actinomycin D. was translated as described in Fig. 2A. Concentration of RNA was 1 mg/ml and 0.5 ~g was added to 20 ~1 reaction mixture. Protein products of translation reactions were fractionated by electrophoresis in a 10% SDS-polyacrylamide slab gel and the fluorograph is sho\vn. Lanes: (a) and ((I), cytoplasmic poly A RNA from WV-infected Vero cells; (b) virion RNA from purified retrovirus particles of SESlQlb-uninfected cells; (c) virion RNA from purified rctrovirus particles of SE2lQlb cells superinfected with VSV tsG31 at nonpermissive temperature. Protein products of trdnslation of reaction (b) and (c) were immunoprecipitated with anti-VSV multivalent sheep serum as in Fig. 2A ((1. e).

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COMMUNICATIONS

tion into retrovirus particles is a highly specific process. Analysis of the aberrant assembly of RNA into the RSV mutant, SE21Qlb, demonstrates the occurrence of selective packaging into retrovirions of only some of the mRNA species present in the cell. ACKNOWLEDGMENTS IYe are most grateful to Clive Dickson for help in performing cell-free protein synthesis and to Maureen Harrison for preparing SE21Qlh cell cultures. E;.U. ackno\~ltd~es the receipt of an EMBO fellowship. REFERENCES I. WEISS, R. A., BOF:TTIGER, D., and MURPHY, H. M.. Vlirology 76, 808-825 (1977). 2. LINIAL, h!I., MEDEIROS, E.. and HAYWARD, W. S., Cell 15, 1371-1381 (1978). .I. IKAIVA. I’., Ross, J., and LEDER, P., Proc. Nat. Acrrd. Sci. C/‘SA 71, 1154-1158 (1974). 4. LE:VIN, J. G., GRIMLEY, P. M., RAMSEUR, J. M., and BERE~ESKY. I. K.. ,I. Viral. 14, 152-161 (1974). .i. PASKIND, M. P.. WEINBERG, R. A., and BALTIMOREZ. D., Virology 67, 242-248 (1975).

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6. LEVIN, J. G., and ROSENAK, M. J., Proc. Nat. Acad. Sci. USA 73, 1154-1158 (1976). GER~IN, B. I., and LEVIN, J. G., b. Viral. 24. 478-488 (1977). 8. LEVIN. J. G., and SIUDMAN, J. G., J. Viral. 29, 328-335 (1979). PRINGLE, C. R., and DUIXAN, I. B., J. Viral. 8, 56-61 (1971). 10. SCHNITZER, T. J., and LODISH, H. F., d. Viral. 29, 443-447 (1979). 11. TEXH. N., WEISS. R. A., MARTIN, G. R.. and LOIVY, D. Ft., Cel( 12, 973-982 (1977). 13. MURPHY, H. M.. Virology 77, 705-721 (1977). IS. GALLIS, B., LINIAL, M., and EISEXMAX, R.. Virology 91, 146-161 (1979). 16. CLINTON, G. M., LITTLE, S. P., HAGEN, F. S., and HUANG, A. S., CrZl 15, 1455-1462 (1978). 15. MORRISON. T.. STAMPFER, M., BALTIMORE, D.. and LODISH, H. I’., J. Viral. 13, (52-72 (1974). 16. YAKOBSOX, E., PRIVES, C., HARTMAN, .J. R.. WINOCOUR. E., and RF,VEI.. M., Ce// 12, 73-81 (1977). 17. DAHL, H.-H. M., and DICKSON, C., .I. Viral. 29, 1131-1141 (1979). IX.