BIOCHIMIE, 1982, 64, 969-973.

Murine retrovirus genome directs the synthesis of gag protein precursor early after infection.

Jo~lle TOBALY *, Luc d 'AURIOL *, Wen K. YANG **, Jorge PERIES * and Rodica EMANOIL-RAVICOVITCH * o.

(Re9ue le 16-6-1982, accept~e le 8-9-1982).

* Ddpartement d'Oncologie Expdrimentale, U. 107 INSERM, Institut de Recherches sur les Maladies du Sang, H~pital Saint-Louis, 75475 Paris Cddex 10, France.

** Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 USA.

R ~ s u m ~ .

Le RNA g~nomique 35S des r~trovirus de type C est prdsent au niveau des polysomes 4 heures aprOs infection dans des lign~es cellulai- res non diff&enci~es et diff&enci~es d&iv~es du t&atocarcinome de la souris.

Une prot~ine sp~cifique virale de 65.000 dal- tons est alors produite dans les celhdes infect~es.

Ces r~sultats montrent que le g~nome viral ]onctionne comme RNA messager pour la syn- those du pr~curseur des protOines gag Pr65 ?l une ~tape prdcoce du cycle infectieux des r~trovirus ~cotropiques murins.

Mots-el~s : r~trovirus murins / RNA messager / prot~ine gag.

S u m m a r y .

Incoming type C retroviral genomic 35S RNA is present in polysomes of undifferentiated and differentiated murine teratocarcinoma cell lines at 4 hours after infection.

A t the same time a 65,000 daltons viral specific protein is produced by the infected cells.

These data present evidence that incoming viral RNA serves as messenger for the synthesis of gag protein precursor Pr65 early in the infec- tious cycle of ecotropic murine retrovirus.

Key-words: murine retrovirus / messenger RNA / gag protein.

I n t r o d u c t i o n .

It is well established now that the retroviral genomic RNA serves as template for viral DNA synthesis by the reverse transcriptase in the cyto- plasm of exogenously infected cells [1]. Recent observations strongly suggest that the genomic viral RNA may also serve in the early steps after virus infection as messenger for the synthesis of virus specific proteins [2, 3, 4]. In the case of avian tumor viruses, it was shown that the pre- cursor to the internal structural proteins, Pr76, is already detectable at about 3 hours after infec- tion even in the presence of actinomycin or cyto-

<> Please send requests to Dr. R. EmanoH-Ravicovitch.

sine arabinoside [2] ; however for murine retro- viruses no such early translational products have as yet been determined.

We previously reported that in undifferentiated teratocarcinoma ceils (PCC4), the replication cycle is blocked before transcription of proviral DNA [5]. Therefore, the absence of newly trans- cribed viral RNA makes PCC4 cells a clean sys- tem to study the fate of input genome RNA.

Comparative studies were performed with dif- ferentiated teratocarcinoma cells (PCD1) permis- sive to viral multiplication [5, 6].

9 7 0 I . Tobaly and coll.

I n this repor t , we p r e sen t e v i d e n c e tha t the in- c o m i n g g e n o m i c R N A serves as m R N A for the synthes is of the v i ra l gag p ro t e in p r e c u r s o r (Pr65 g~g) ear ly in the in fec t ious cycle of m u r i n e l e u k e m i a re t rov i ruses .

T h e poss ib le ro le of ea r ly P r 6 5 g"g in the r e t ro - v i ra l r e p l i c a t i o n cycle wil l be d iscussed.

rotor for 2.5 hours. The pellet was rinsed with 1.5 ml of solution A that was added to the << non polysomal frac- tion >> obtained by coUecting the layer above the sucrose cushion after ultracentrifugation.

The polyribosomal pellet was treated with 50 Ixl of 0.2 M EDTA and 100 ~1 of 1 per cent SDS for 5 minutes to dissociate polyribosomes. 850 ~1 of NTE buffer were added, adjusted to 0.5 M NaC1 before loading onto oligo (dT)-cellulose column. The non polysomal fraction was extracted twice with phenol-chloroform/isoamyl alcohol and the aqueous phase was precipitated with 2.5 volumes of ethanol.

Materials and Methods.

Cells and viruses.

The undifferentiated cell line PCC4 and myoblast diffe- rentiated cell line PCD1, both originally derived from transplantable teratocarcincma of mouse strain 129, were used. Characterization of these two lines has been repor- ted [6]. The source of Gross strain N-tropic type C retro- viruses has been previously described [7].

Cells were grown in Dulbecco's modified Eagle's mini- mum essential medium supplemented with 10 per cent fetal calf serum, 100 jxg/ml streptomycin, and 100 Ixg/ml penicillin.

Twenty four hours after polybrene treatment (2 ~g/ml) teratocarcinoma cells were infected with Gross strain N-tropic virus at a multiplicity of infection in the range of 2 to 3 as determined by XC plaque assay [8]. The virus titers determined as plaque-forming units per ml of cellular supernatant were 5 × 105 for PCD1 and less then 101 for PCC4 cells.

RNA extraction and isolation of poly (A) containing RNA.

The cytoplasmic R N A was extracted in NTE (100mM NaCI, 10 mM Tris HCI pH 7.3, 1 mM EDTA) after treat- ment with 1 per cent SDS and deproteinized by phenol- chloroform-isoamyl alcohol [9]. Viral RNA was extracted from concentrated culture supern.atants, as described by Callahan et al. [9].

Cytoplasmic RNA was fractionated into polyadenylated poly (A +) and non poIyadenyIated poly (A-) R N A by two passages over an oligodeoxythymidylate oligo (dT)-cell~- lose column, as described by Aviv et Leder [10].

Preparation of polyribosomes.

Total polyribosomes were isolated as described [4] by centrifugation of cytoplasmic extracts through a 6 ml 1.5 M sucrose cushion in solution A (10 mM Tris HC1

p H 7.6, 100 mM KCI, 5 mM MgCle). The polyribosomes were sedimented by centrifugation at 45 K in an Ti 50

Electrophoresis and molecular hybridization.

Cytoplasmic RNAs were treated with glyoxal [11], frac- tionated by electrophoresis i.n 1 per cent agarose gel and transferred to diazobenzyloxymethyl (DBM)-paper [12]. The paper was then reacted with 32p labelled eDNA from genomic RNA in a molecular hybridization mixture con- taining dextran sulfate [13]. Hybridization of cDNA to RNA was detected by autoradiography at - - 8 0 ° C with the use of an X-ray film and intensifier screens.

32p cDNA was prepared by using 70S RNA of Gross strain N-tropic virus as template, essentially according to the procedure of Taylor et al. [14].

Preparation o[ cytoplasmic extracts.

At indicated times after infection, cells on 90 mm Petri dishes were starved for 40 rain in medium lacking methio- nine, followed by a 20 minutes labelling period with 500 ,txCi of [35S] methionine (specific activity greater than 1 000 Ci/mmol) in 2.5 ml of methionine free minimum essential medium (MEM), supplemented with 10 per cent heat inactivated fetal calf serum. After being washed twice with phosphate buffered saline, the cells were lysed in 3 volumes of immune buffer (0.5 per cent DOC, 1 per cent Triton X 100, 0.1 per cent SDS, 0.25 M NaC1, 1 mM EDTA, 0.05 M Tris HCI pH 7.5). The lysates were clarified by centrifugation at 10,000 rpm for 15 mi- nutes.

lmmunoprecipitation and protein analysis.

Approximately 30 × 106 trichloroacetic acid precipi- table counts of the cytoplasmic extracts were used to per- form each immune reaction in a 0.5 ml final volume of immune buffer. The antiserum used was, goat anti Raus- cber Leukemia virus pr65 (precursor protein) Batch 76S-454 obtained from the National Cancer Institute. The pr65gag antiserum (2 to 4 ~l) was added and the mixtures were incubated for 2 hours at 4°C. The antigen-antibody complexes were precipitated by addition of 100 ~tl of a 10 per cer~t formalin in.activated Staphylococcus aureus suspension.

Protein sample were analyzed in SDS polyacrylamide slab gels by the method of Laemmli [15] with a 8-16 per cent separating and a 5 per cent stacking gel and deve- lopped by fluorography according to Bonner and Laskey [16].

BIOCHIMIE, 1982, 64, n ° 10.

Messenger function oJ retroviral genome. 971

Resu l t s .

I. - Detection o] virus specific R N A in the cyto- plasm of teratocarcinoma cells early (4 hours) after infection with N-tropic MuLV.

Viral R N A was analyzed as a function of time after infection of teratocarcinoma cells with eco- tropic murine retrovirus.

II. - Detection of genomic viral R N A in poly- ribosomes at 4 hours after infection.

To further characterize the input viral R N A detected at 4 hours post infection, polyribosomes were isolated from the cells. Poly (A+) R N A isolated from this material by chromatography on oligo dT cellulose and total R N A from the non polysomal fraction (see Methods) were analyzed by molecular hybridization.

FIG. 1. - - Virus spe- cific RNA in teratocar- cinoma cell lines at dif- ferent times after hzJec- tion.

In this and subsequent figures ceils were infec- ted with Gross strain N tropic virus at a multi- plicity of infectien ef 2.

Polyadenylated RNA was isolated from the cytoplasm, treated with glyoxal, fractionated by electrophoresis in 1 per cent agarose gel, trans- ferred to DBM paper and hybridized with MuLV[a2P] cDNA. Au- toradiography was car- tied out at --80°C for 24 hours.

Small arrows indicate position of 28S and 18S ribosomal RNAs, as judged by staining with acridine orange.

In PCC4 cells (figure 1A) a 35S viral R N A was detectable 4 hours after virus inoculation and disappeared after longer periods of time such as 8, 12 and 24 hours post infection. This R N A is the input viral genome since no integration and therefore no neotranscription could have occurred, according to our previous kinetic analysis of pro- viral DNA formation [5].

In PCD1 cells (figure 1B) viral R N A is present 4 hours after infection as well as at longer times (8, 12 and 24 hours). The 35S viral R N A detec- ted at 4 hours is most likely input viral R N A since at this early time, integration could not have occurred [17]. However at later times, the viral R N A detected is a newly synthesized product of the replicat ion process. In addition at 24 hours post infection, 24S viral R N A is detectable. Such a species has been described, resulting from spli- cing of newly transcribed viral R N A [18].

BIOCHIMIE, 1982, 64, n ° 10.

The autoradiograms in figure 2 shows that a 35S viral specific R N A is detected on polyribo- somes in PCC4 and PCD1 cell lines (lanes G and H). In contrast no viral specific R N A was detec- ted in the non polyribosomal fraction of both cell lines (lanes E and F). No virus specific R N A was present in either polyribosomes or << non polyri- bosomal fraction >> after mock infection (lanes A to D).

These results showed clearly that most viral R N A binds to polyribosomes at 4 hours post infection.

III . - Early synthesis of gag protein precursor Pr65, 4 hours post infection.

The observation that the incoming viral genome is bound to polyribosomes at 4 hours post infec- tion (p.i.) suggests that the 35S R N A may serve

972 J. Tobaly and coll.

as messenger for the synthesis of gag proteins . In o rder to invest igate this possibi l i ty, cells were pu l se - labe led for 20 minutes with [zsS] methio- nine at 4 hours post infection. The lysates were immunoprec ip i t a t ed with an ant i serum against M u L V Pr65 g"g and analyzed by SDS-po lyac ry l - amide gel e lectrophoresis .

As shown in f igure 3, a 65 ,000 M W polypep- t ide which is immunolog ica l ly re la ted to M u L V Pr65 gag is synthet ized in PCC4 and PCD1 cells in response to infection. This p ro te in is ident ical in size to Pr65 gag synthet ized f rom newly trans- c r ibed viral R N A in PCD1 cells at 48 hours pos t infect ion (da ta not shown).

FIG. 2. - - Size o[ virus specific RNA associated with polyribosomes early after infection o] teratocarcinorna cell lines.

Lanes A, B, E, F : RNA from the <~ non polysomal frac- tion ~> as described under Methods.

Lanes C, D, G, H: Polyribosome-associated poly (A ÷) RNA.

Autoradiography was carried out at - - 8 0 ° C for 24 hours.

Abbreviations: C4, PCC4 cells; D1, PCDI cells; M, mock infected cells; I, infected cells.

3. - - Synthesis o] in teratocarcinoma

;s early after infec-

utoradiogram shows per cent polyacryl- gel of anti-Pr65g ag ; precipitates from of uninfected (A or infected (B and

~. Cells were pulse- for 20 minutes with .~thionine (200 I~Ci/

Lard t4C molecular markers were used : 3rylase b (92K) bo- urn albumine (69K), nine (46K) and car- ~nhydrase (30K). exposure time was

BIOCHIMIE~ 1982, 64, n ° 10,

Messenger function of retroviral genome. 973

D i s c u s s i o n .

In this study we have shown that 35S viral polyadenylated R N A is detectable at 4 hours after infection of both differentiated and undif- ferentiated teratocarcinoma cell lines with an ecotropic MuLV.

The fact that no new viral R N A is synthetized in PCC4 cells after infection [5] and that see- mingly no proviral integration occurs 4 hours post infection [17] indicates that the 35S viral R N A present at 4 hours post infection can only be the input viral RNA, originated from the infec- ting virions. At this time most of the genomic viral R N A is found associated to polyribosomes.

With the use of a highly sensitive method [13] we detected no viral R N A smaller than the 35S species, in either polyribosomes or cytoplasmic extracts, contrary wise to what was found by Shurtz et al. [4]. These authors described two major size classes of 38S and 23S viral RNAs present in polyribosomes early after infection of N I H / 3 T 3 cells with Moloney leukemia virus.

To further study the infecting virion R N A mes- senger function, we examined whether it is trans- lated to yield gag precursor proteins. Our results indicate that Pr65 gag is synthetized in both cell lines 4 hours post infection. This finding is con- sistent with in vitro translation experiments [19] and demonstrates for the first time for a mamma- lian type C retrovirus that the input viral R N A functions as a messenger for the early synthesis of a gag polypeptide precursor.

The current view of retrovirus replication pos- tulates a role for structural proteins only during particle formation. The synthesis of gag protein precursor early after infection raises the question of its role in the infectious cycle of retroviruses.

A recent report demonstrated that the first 4 hours after virus infection constituted the most sensitive period for cycloheximide inhibition of covalently closed supercoiled D N A forma- tion [20].

These results indicate that an early synthesis of viral or cellular protein(s) is required for viral replication. Based on these and our findings, we

suggest that the pr65 g"~ translated from the in- coming viral R N A early after infection could be involved in the viral supercoiled DNA formation.

A c k n o w l e d g e m e n t s .

We thank Mrs Frida Hojman and Mr. Filippo Cava- lieri for valuable discussions o[ these data and critical reading o/ the manuscript, and M. C. PwuJ and M. Pas- quet ]or typing this paper.

This work was supported by DGRST grant 81 L 0728 and DGRST grant A 650 1926.

REFERENCES.

1. Varmus, H. E., Heasley, S., Kung, H. J., Oppermann, H., Smith, V. C., Bishop, J. M. & Shank, P. R. (1978) J. Mol. Biol., 120, 55-82.

2. Gallis, B. M, Eisenman, R. N. & Diggelman, H. (1976) Virology, 74, 302-313.

3. Salzberg, S., Robin, M. S. & Green, M. (1977) Viro- logy, 76, 341-351.

4. Shurtz, R., Dolev, S., Aboud, M. & Salzberg, S. (1979) J. Virol., 31, 668-676.

5. d'Auriol, L., Yang, W. K., Tobaly, J., Cavalieri, F., Peries, J. & Emanoil-Ravicovitch, R. (1981) J. Gen. Virol., 55, 117-122.

6. Jacob, F. (1977) Proceeding o/ the Royal Society, Series B, 201, 1144, 249-270.

7. Yang, W. K., Tennant, R. W., Rascati, R. J., Ollen, J. A., Shutter, B., Kiggans, J. O., Myer, F. E. & Brown, A. (1978) J. Virol., 27, 288-299.

8. Rowe, W. P., Pugh, W. E. ~ Hartley, J. W. (1970) Virology, 4'2, 341-351.

9. Callahan, R., Benveniste, R. E., Lieber, M. M. & Todaro, G. J. (1974) J. Virol., 14, 1394-1403.

10. Aviv, H. & Leder, H. (1972) Proc. Nat. Acad. Sci. U.S.A., 69, 1408-1412.

11. McMaster, G. K. & Carmichael, C. G. (1977) Proc. Nat. Acad. Sci. U.S.A., 74, 4835-4838.

12. Alwine, J. C., Kemp, D. J. & Starck, G. R. (1977) Proc. Nat. Acad. Sci. U.S.A., 74, 5350-5354

13. Wahl, G. M., Stern, M. & Stark, G. R. (1979) l~roc. Nat. ,4cad. Sci. U.S.A., 76, 3683-3687.

14. Taylor, J. M., Illmensee, R. & Summers, J. (1976) Biochem. Biophys. Acta, 442, 324-330.

15. Laemmli, U. K. (1970) Nature, 227, 680-685. 16. Bonner, W. M. & Laskey, R. A. (1974). Eur. J. Bio-

chem., 46, 83-88 . 17. Gianni, A. M., Smotkin, D. K. & Weinberg, R. A.

(1975) Proc. Nat. Acad. Sci. U.S.A., 72, 447-451. 18. Fan, H. & Verma, I. M. (1978) J. Virol., 26, 468-478. 19. Murphy, E. C. & Arlinghaus, R. B. (1978) Virology,

86, 329-343. 20. Yang, W. K., Yang, D. M. & Kiggans, J. O. (1980)

J. Virol., 36, 181-188.

BIOCHIM1E, 1982, 64, n ° 10.