of the transcripts is involved in this selective inhibition. In this communication, we demonstrate that poly(riboadenylic

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 83, pp , March 1986 Biochemistry Poly(riboadenylic acid) preferentially inhibits in vitro translation of cellular mrnas compared with vaccinia virus mrnas: Possible role in vaccinia virus cytopathology (in vitro transladon/reticulocyte lysate/wheat germ' extract/protein synthesis) ROSTOM BABLANIAN* AND AMIYA K. BANERJEEt *Department of Microbiology and Immunology, State University of New York Downstate Medical Center, Brooklyn, NY 11203; and troche Institute of Molecular Biology, Roche Research Center, Nutley, NJ Communicated by Howard L. Bachrach, October 25, 1985 ABSTRACT Vaccinia virus-induced inhibition of host protein synthesis seems to be mediated by viral transcripts based on their differential inhibition of cellular mrna translation in a rabbit reticulocyte lysate system. In this study, we demonstrated that the removal of poly(riboadenylic acid) [poly(a)] from the in vitro viral transcripts abolished this inhibition in the same cell-free system. This observation led us to the finding that <1,IM poly(a) completely inhibited HeLa cell mrna translation in the reticulocyte Iysate, whereas only 50% inhibition of vaccinia virus mrna translation was observed at the same concentration. Similar results were also obtained in a wheat germ protein-synthesizing system. This inhibitory effect of poly(a) was totally abrogated by the addition of polydeoxythymidylate. This selective inhibition was highly specific for poly(a) since other homopolymers, including poly(g), poly(c), and poly(da), were not capable of causing such an inhibition. Poly(U), however, had a moderate selective inhibitory effect. Among the several mrnas tested, the translation of L-cell, encephalomyocarditis virus, and reovirus RNAs was also sensitive to poly(a). However, vesicular stomatitis virus mrna translation was strikingly more resistant. These results suggest that poly(a), which is also synthesized by the virion-associated poly(a) polymerase, may be involved in vaccinia virus-mediated host cell shutoff. Virus-induced inhibition of host cell macromolecular synthesis has been an area of active research in the study of virus cytopathology. The strategy employed by DNA- and RNAcontaining animal viruses to inhibit host cell RNA and protein synthesis seems to vary with each type of virus (1). In spite of extensive studies in this area, the virus-specific agent(s) responsible for selective inhibit'ion of host cell macromolecular synthesis has not been clearly established. We have used vaccinia virus, a DNA-containing cytoplasmic virus, as a model to study virus-cell interactions that lead to inhibition of host cell protein synthesis or shutoff. Previous work in our laboratory and others (2-9) has demonstrated that vaccinia virus-induced RNAs may play a role in this inhibition. Using vaccinia virus transcripts synthesized in vitro by the virionassociated DNA-dependent RNA polymerase, we have shown that these transcripts are capable of inhibiting host cell mrna translation in an in vitro protein-synthesizing system. In contrast, the translation of vaccinia virus mrna remains unaffected (10). Recently, we have demonstrated that this discriminatory property resides exclusively in the small ( nucleotide) nontranslating poly(a)-containing in vitro transcripts, whereas the larger transcripts were not selective inhibitors (11). In an effort to fuither characterize this inhibitory viral RNA, we found that the poly(a) moiety The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact of the transcripts is involved in this selective inhibition. In this communication, we demonstrate that poly(riboadenylic acid) can, by itself, markedly inhibit host cell mrna translation with minimal effect on vaccinia virus mrna translation. MATERIALS AND METHODS Cells and Viruses. HeLa cells were grown in suspension in Eagle's spinner medium and supplemented with 5% fetal bovine serum. The cells were infected with plaque-purified WR strain of vaccinia virus and purified according to the method of Joklik (12). Cell-Free Protein-Synthesizing Systems. Reticulocyte lysates were prepared according to the method of Schreier and Staehelin (13) and treated with micrococcal nuclease (14). The 25-,ul reaction mixtures [56% (vol/vol) lysate] described previously (10) were programmed with various mrnas as described below. Incubation was carried out for 60 min at 320C. Wheat germ extract prepared according to Both et al. (15) was a generous gift of Donald Nuss. Preparation of mrna from Mammalian Cells. HeLa or L-cell spinner cultures (1 x 109) were treated with cycloheximide (300,ug/ml) and incubated at 370C for 4 hr. Total cytoplasmic RNA was extracted according to the method of Krystosek et al. (16). Cytoplasmic RNA from vaccinia virus-infected cells (300 particles per cell) was similarly treated and extracted as above. Poly(A)-containing mrnas from infected and uninfected cultures were collected by oligo(dt)-cellulose chromatography (Collaborative Research, Waltham, MA, type 3). Encephalomyocarditis (EMC) virus RNA was purified according to Kerr and Martin (17). Vesicular stomatitis virus (VSV) mrna was transcribed in vitro according to the method of Testa et al. (18). Reovirus mrna was a generous gift of Aaron J. Shatkin. Preparation of Vaccinia Virus in Vitro Transcripts. The viral cores containing the transcription mixture (10) were incubated for 1 hr at 370C and the reaction was stopped by the addition of lithium d6decyl sulfate (0.1%) in 0.5 M lithium acetate. After centrifugation for 15 min (39,000 x g) the supernatant was added to oligo(dt)-cellulose equilibrated in 0.5 M lithium acetate. The poly(a)-containing RNA was selected by using the batch absorption method (19). Elution of the poly(a)+ RNA was carried out with H20. The RNA was precipitated with 0.2 M lithium acetate and 2.5 vol of ethanol at -200C. Ribopolymers and Deoxyribopolymers. Poly(A), poly(c), poly(u), poly(g), and poly(da) were obtained from Miles Laboratories. The polymers were dissolved in sterile H20 and the concentrations were calculated from their extinction Abbreviations: VSV, vesicular stomatitis virus; EMC, encephalomyocarditis.

2 Biochemistry: Bablanian and Banedee coefficients. Oligo(dT)12_18 used in these experiments was obtained from Sigma. RESULTS Effect of Removal of Poly(A) from Vaccinia Virus Small Transcripts on mrna Function. In an effort to characterize further the small poly(a)-containing viral transcripts involved in the selective inhibition of host mrna translation, we undertook a series of experiments to delineate what structural changes would bring about a reversal of this inhibition. To demonstrate if poly(a) plays a role in this inhibition, we enzymatically removed the poly(a) moiety of the small transcripts by digestion with RNase H in the presence of oligo(dt). The remaining fraction was assayed for inhibitory activity in the reticulocyte lysate translation system programmed by HeLa cell and vaccinia virus mrna. The result of this experiment is shown in Fig. 1. Based on hot trichloroacetic acid-insoluble radioactivity, the small vaccinia virus transcripts were found to significantly inhibit (80%) HeLa cell polypeptide synthesis. This degree of inhibition was also evident when densitometric tracing was carried out on HeLa cell polypeptides (compare lanes 3 and 2). The small transcripts, by themselves, had no translational activity (lane 8). Upon the removal of the poly(a) moiety, the * Proc. NatL. Acad. Sci. USA 83 (1986) 1291 in vitro transcribed small RNAs were markedly reduced (<10%o) in their ability to inhibit HeLa cell polypeptide synthesis (compare lanes 4 and 2). The small transcripts, however, had a lower inhibitory effect (<40%) on vaccinia virus-directed protein synthesis (compare lanes 6 and 5), as demonstrated previously (11), and this inhibitory effect was essentially abrogated (<10%) when the poly(a) moiety of the small transcripts was enzymatically removed (compare lanes 7 and 5). These results strongly suggested that poly(a) may play a direct role in this selective inhibitory process. We, therefore, used synthetic poly(a) to study its effect on this phenomenon. Effect of Poly(A) on HeLa Cell and Vaccinia Virus mrna Function. As shown in Fig. 2, poly(a) (average chain length of 170), at-a concentration of 1 pg/25-,al reaction mixture (0.8 gm), completely inhibited HeLa cell polypeptide synthesis (compare lanes 3 and 2). In contrast, poly(a) inhibited vaccinia virus polypeptide synthesis only by <20% (compare lanes 8 and 7). This result clearly indicated that poly(a) was selectively inhibiting HeLa cell polypeptide synthesis, a situation that mimicked the effect of vaccinia virus infection on HeLa cell polypeptide synthesis (Fig. 1). To demonstrate that poly(a) was directly involved in this inhibitory activity, we added oligo(dt) to the reaction mixture to study whether the inhibitory effect of poly(a) would be abrogated due to the formation of a duplex. It can be seen that, at the lower concentration of oligo(dt) (1 ug/25-,ul reaction mixture) used, the inhibitory effect of poly(a) on HeLa cell polypeptide synthesis was diminished by 78% (compare lanes 4 and 2 in Fig. 2). At the higher concentration of oligo(dt) (2,g/25-,ul reaction mixture), the HeLa cell polypeptide synthesis was 120% of control (compare lanes 5 and 2), suggesting that oligo(dt) may complex not only with the exogenously added poly(a) but also with poly(a) that may be found with HeLa cell RNA. This contention was further supported by w il -20 FIG. 1. Effect of removal of poly(a) from vaccinia virus transcripts. Vaccinia virus in vitro transcripts labeled with [a-32p]cmp were electrophoresed on 1.5% agarose containing 6 M urea in 25 mm sodium citrate at ph 3.0 (11). Following autoradiography the RNA band was divided into five size classes. The eluted RNA from the smallest size class fraction possessing the selective inhibitory activity was treated with RNase H and oligo(dt) as described (20). Reticulocyte lysates, treated with micrococcal nuclease, were programmed with either HeLa cell (lane 2) or vaccinia virus mrnas (lane 5). The treated in vitro transcripts were added together with the HeLa cell mrnas (lane 4) or vaccinia virus mrnas (lane 7). Lane 3 depicts the effect of untreated small in vitro transcripts on HeLa cell mrna function, and lane 6 shows the effect of these small transcripts on vaccinia virus mrna translation. Lane 1 had no additions, and lanes 8 and 9 received untreated and treated small in vitro transcripts, respectively. Total cytoplasmic RNA from either uninfected (15,g/25 ul) or infected (10 pyg/25 Al) cells was used to program the cell-free system. The amount of in vitro transcripts used for the untreated and treated was 0.1 pg/25 Wu. The lysates were incubated for 1 hr at 32 C and the 35S-labeled products (20-,Ul samples) were subjected to 5-15% NaDodSO4/polyacrylamide gel electrophoresis and autoradiography (10). Molecular weights are shown as Mr x Bf "low - 67 FIG. 2. Effect of poly(a) on HeLa cell and vaccinia virus mrna translation. Reticulocyte lysates, treated with micrococcal nuclease, were programmed with total cytoplasmic RNA obtained from HeLa cells (15 big; lanes 2-6) or from infected HeLa cells (10,ug; lanes 7-11). Lanes 3 and 8 received poly(a) at a concentration of 1 tig/25-al reaction mixture (0.8,uM). Lanes 4 and 9 received oligo(dt) (1 ±g/25,l) in addition to poly(a) (1 ug/25 ul), and lanes 5 and 10 received oligo(dt) (2,ug/25,ul) in addition to poly(a) (1 u&g/25 Al). Lanes 6 and 11 received only oligo(dt) (1,ug/25 /ul). Lanes 1 and 12 contained no exogenous RNA and poly(a) (1,ug/25 /l), respectively. The products of the reaction were treated as in Fig. 1. Molecular weights are shown as M, X 10-3.

3 1292 Biochemistry: Bablanian and Banerjee ~~~~~~ C Poly(A),,g/25-,ul reaction mixture FIG. 3. Dose-response of the inhibition of protein synthesis by poly(a). Reticulocyte lysates, treated with micrococcal nuclease, were programmed with either total HeLa cell RNA (15 ug/25 u4) or vaccinia virus-infected RNA (10,ug/25,ul) in the presence of various concentrations of poly(a). After 1 hr of incubation at 32 C, the [I"S]methionine-labeled products (5,ul) were acid-precipitated and assayed for radioactivity in a scintillation counter. The concentration of poly(a) was plotted against the percent inhibition of the untreated controls. o, Vaccinia virus-infected HeLa cell RNA; e, HeLa cell RNA. the fact that when oligo(dt) was added without poly(a), the HeLa cell polypeptide synthesis was 133% of control (compare lanes 6 and 2). Similarly, the addition of oligo(dt) not only abolished the slight inhibitory effect of poly(a) in the reticulocyte lysate system programmed with vaccinia virus RNA but also stimulated 2-fold viral polypeptide synthesis (compare lanes 9 and 10 with lane 7). As shown above for Proc. Natl. Acad. Sci. USA 83 (1986) HeLa cell polypeptide synthesis (lane 6), addition of oligo(dt) without poly(a) stimulated viral polypeptide synthesis by >2-fold (compare lanes 11 and 7). We conclude from the above results that poly(a) plays a direct role in this selective inhibitory phenomenon. Fig. 3 shows the concentration curves of poly(a) required to inhibit HeLa cell and vaccinia virus mrna translation in the reticulocyte lysate system. It is important to note that the discriminatory inhibition becomes more striking at the higher concentrations used (0.25-1,ug per reaction mixture). At lower concentrations, however, this selective inhibition is less pronounced. These results suggest that the cellular mrnas are more dependent on some translational factor(s) that is affected by poly(a). Effect of Homopolymers on HeLa Cell and Vaccinia Virus mrna Translation. To determine whether this selective inhibitory property was specific for poly(a), we studied the effect of additional homopolymers on mrna translation. As shown in Fig. 4, poly(c), poly(g), and poly(da) at 1 pug per reaction mixture had virtually no effect on HeLa cell mrna translation (lanes 3, 4, and 12, respectively). Similarly, vaccinia virus mrna translation was unaltered by the same homopolymers (lanes 7, 8, and 14, respectively). These results strongly suggest that the selective inhibition is mediated specifically by poly(riboadenylic acid). It is noteworthy that poly(u) also inhibited HeLa cell mrna translation by 57% (lane 5) and vaccinia virus mrna translation by 24% (lane 9) (discussed below). Effect of Poly(A) on Translation of HeLa Cell and Vaccinia Virus mrna in Wheat Germ Extracts. As shown in Fig. 5, in wheat germ extracts, poly(a) completely inhibited HeLa cell mrna translation (lane 3), whereas vaccinia virus mrna translation was inhibited by about 50% (lane 6). As observed previously, the inhibitory effect of poly(a) was totally abrogated by the addition of oligo(dt) (lanes 4 and 7). These results are identical to those observed in the reticulocyte in vitro translation system, indicating that the observed selective inhibition by poly(a) is a faithful phenomenon. Effect of Poly(A) on Translation of Various mrnas in the Reticulocyte Cell-Free System. In view of the above results, it was of interest to determine the effect of poly(a) on trans _ FIG. 4. Effect of various homopolymers on HeLa cell and vaccinia virus protein synthesis in the reticulocyte cell-free system. Lysates treated with micrococcal nuclease were programmed with HeLa cell RNA (15 Zg; lanes 2-5 and 11-12) or with infected HeLa cell RNA (10,ug; lanes 6-9 and 13-14). Lanes 3 and 7 received poly(c); lanes 4 and 8 received poly(g); lanes 5 and 9 received poly(u); and lanes 12 and 14 received poly(da). The concentration of the homopolymers was 1,ug/25,ul. Lanes 1 and 10 had no additions. Lanes were from a separate experiment. The products of the reactions were treated as in Fig. 1. Molecular weights are shown as Mr x

4 Biochemistry: Bablanian. and Banedee Proc. Natl. Acad. Sci. USA 83 (1986) _- I.Q., low a'i t.. t. -45 * \ w _ _ - FIG. 5. Effect of poly(a) on the translation of HeLa and vaccinia virus mrnas in the wheat germ system. Wheat germ extracts (1:5) were programmed with oligo(dt)-selected HeLa cell RNA (0.5 gg/25,l; lanes 2-4) or with infected HeLa cell RNA (0.25,ug/25 bl; lanes 5-7). Lanes 3 and 6 received poly(a) (1 Ag/25 Al). In addition to poly(a) (1 Ag/25 1.l), lanes 4 and 7 also received oligo(dt) (2 Ag/25 1.l). The control (lane 1) contained no added RNA but translated some endogenous mrnas. The products of the reaction were treated as in Fig. 1. Molecular weights are shown as M, x lo-3. lation of other viral and host cell mrnas. It can be seen in Fig. 6 that, like HeLa cell mrna, L-cell mrna function was completely inhibited by poly(a) (lane 7). Similarly, reovirus and EMC viral mrnas were not capable of translation in the presence of poly(a) (lanes 11 and 13). On the other hand, we have routinely observed that VSV mrna translation was resistant to the effect of poly(a) (lane 9) albeit not equal to the degree of resistance shown by vaccinia virus mrna (lane 5). It is interesting to note that the inhibition of VSV polypeptide synthesis by poly(a) was predominantly directed toward the synthesis of the preterminated polypeptides (compare lanes 8 and 9). However, the degree of inhibition ofthe VSV (N and NS) polypeptides (see top band in lane 9) was inhibited by 50% and M polypeptide (bottom band in lane 9) was inhibited by 70%. Thus, VSV mrna translation appears to be resistant to the poly(a) effect, similar to that seen for vaccinia virus mrna. DISCUSSION It has been suggested by several investigators (3-5) that vaccinia virus poly(a)-containing small transcripts may play a role in the inhibition of host protein synthesis in infected cells. Using an in vitro protein-synthesizing system, Coppola and Bablanian (10) demonstrated that in vitro synthesized vaccinia virus transcripts selectively inhibited host mrna function. The precise nature of the inhibitory molecule(s) in the total in vitro transcripts was not determined, except that they were found primarily in the small nontranslating poly(a)-containing fraction of the transcript (11). Moreover, it was demonstrated that this selective inhibition was not the result of a vast excess ofbiologically competent viral mrnas from infected cells (11). To further study this selective inhibition we removed poly(a) from these small transcripts. This resulted in a dramatic abrogation of the selective inhibitory effect (Fig. 1). To determine whether this effect FIG. 6. Effect of poly(a) on various cellular and viral mrna translations. Reticulocyte lysates treated with micrococcal nuclease were programmed with oligo(dt)-selected HeLa cell RNA (0.5,g/25,l; lanes 2 and 3) or with vaccinia virus-infected cell RNA (0.25 Ag/25,ul; lanes 4 and 5). Lanes 6 and 7 were programmed with L-cell RNA (0.5,g/24 jul), lanes 8 and 9 with VSV mrna (0.3 ug/25,l), lanes 10 and 11 with reovirus RNA (0.25,g/25,l), and lanes 12 and 13 with EMC RNA (0.5 ug/25,l). In addition to various mrnas, lanes 3, 5, 7, 9, 11, and 13 also received 1 gg/25,ul of poly(a). Lane 1 is the control with no added RNA. The products of the reactions were treated as in Fig. 1. Molecular weights are shown as Mr X was related to free poly(a) or poly(a)-containing small transcripts (3-5), we used synthetic poly(a) in our in vitro translation system. In the present work, we demonstrate that poly(a) at a concentration of <1,uM completely inhibited HeLa cell mrna translation with only 50% inhibition of vaccinia virus mrna translation (Figs. 2 and 3), a phenomenon that may be involved in the virus-induced shutoff. This inhibitory effect was directly associated with poly(a) since addition of oligo(dt) completely reversed this effect (Fig. 2). Probably, heteroduplex formation between poly(a) and oligo(dt) resulted in abolition of its inhibitory activity. This is also supported by the fact that oligo(dt) by itself stimulated the translation of HeLa cell and vaccinia virus mrnas (Fig. 2), possibly due to hybrid formation with the endogenous free poly(a) likely to be present in both of the mrna preparations. The selective inhibitory effect was highly specific for poly(riboadenylic acid) since poly(da) was totally ineffective in the translation system (Fig. 4). Furthermore, poly(g) and poly(c) were also not capable of inhibiting translation of either host or viral mrna. Interestingly, poly(u) had an effect similar to that of poly(a), although at a much reduced level (Fig. 4). This observed inhibition may be due to complex formation with endogenous free poly(a) resulting in a doublestranded RNA, which is known to be a potent inhibitor of translation in the reticulocyte cell-free system (21, 22). This is also borne out by the fact that poly(u), in contrast to oligo(dt), failed to reverse the poly(a) inhibitory effect in the reticulocyte system (data not shown). However, in the wheat germ extract, which is insensitive to the effect of doublestranded RNA (23, 24), the selective inhibitory effect of poly(a) was abolished by the addition of oligo(dt) (Fig. 5) and also by the addition of poly(u) (data not shown). Unlike vaccinia virus mrnas, the translation of several viral

5 1294 Biochemistry: Bablanian and Banejee mrnas, such as reovirus and EMC virus, was found to be sensitive to poly(a) (Fig. 6). It is interesting to note that VSV mrna was moderately resistant (about 30%) to the effect of poly(a). Whether poly(a) also is involved in VSV-mediated shutoff remains to be determined. The mechanism by which poly(a) selectively inhibits host mrna translation remains unknown. A possible role of poly(a) in protein synthesis has been proposed in which free poly(a) may compete with the poly(a) tract on mrna (25). In addition, these authors proposed that purified poly(a) may sequester some component in the lysate that would have otherwise interacted with mrna poly(a) tracts. In our experiments, however, the selective inhibitory effect of free poly(a) was evident with either poly(a)+ or total mrna. Poly(A) has also been shown to block polypeptide chain initiation (26). It is possible that poly(a) may interact with a factor(s) present in the translation machinery that is much more essential for initiation of the host mrna translation than the viral mrna. Recently, involvement of small RNAs in virus-induced shutoff has been shown or implicated in several virus-cell systems, such as adenovirus (27, 28), influenza virus (29), and VSV (30, 31). It remains to be determined whether the mechanisms of poly(a)-induced inhibition of protein synthesis are mediated by the regulation ofphosphorylation of initiation factor(s) ofprotein synthesis. Finally, the above studies gave us an insight into the possible link between poly(a) and vaccinia virus infection. It is known that vaccinia virus contains a poly(a) polymerase that not only adds poly(a) tracts to the transcript but also synthesizes free poly(a) in vitro (4, 32). Thus, it is tempting to speculate that in the infected cells, poly(a) synthesized by the input virions may be directly involved in the inhibition of host mrna translation, whereas vaccinia virus mrnas are resistant to the effect of poly(a), leading to selective inhibition. It will be important to determine the levels of poly(a) synthesized in the cell during infection and also the amount of poly(a) required to inhibit host mrna translation in vivo. Further studies along this line will be needed to study the role of poly(a) in vaccinia virus cytopathology. We thank Santo Scribani for expert technical assistance. This work was supported by Grant DMB from the National Science Foundation. 1. Bablanian, R. (1984) in Comprehensive Virology, eds. Fraenkel-Conrat, H. & Wagner, R. R. (Plenum, New York), Vol. 19, pp Bablanian, R. (1975) Prog. Med. Virol. 19, Rosemond-Hornbeak, H. & Moss, B. (1975) J. Virol, 16, Proc. Natl. Acad. Sci. USA 83 (1986) 4. Gershowitz, A. & Moss, B. (1979) J. Virol. 31, Paoletti, E., Lipinskas, B. R. & Panicali, D. (1980) J. Virol. 33, Bablanian, R., Esteban, M., Baxt, B. & Sonnabend, J. A. (1978) J. Gen. Virol. 39, Schrom, M. & Bablanian, R. (1979) J. Gen. Virol. 44, Schrom, M. & Bablanian, R. (1979) Virology 99, Bablanian, R., Coppola, G., Scribani, S. & Esteban, M. (1981) Virology 112, Coppola, G. & Bablanian, R. (1983) Proc. Nati. Acad. Sci. USA 80, Bablanian, R., Coppola, G., Masters, P. S. & Banejee, A. K. Virology, in press. 12. Joklik, W. K. (1962) Biochim. Biophys. Acta 61, Schreier, M. H. & Staehelin, T. (1973) J. Mol. Biol. 73, Pelham, H. R. B. & Jackson, R. J. (1976) Eur. J. Biochem. 67, Both, G. W., Lavi, S. & Shatkin, A. J. (1975) Cell 4, Krystosek, A., Cowthon, M. L. & Kabat, D. (1975) J. Biol. Chem. 250, Kerr, I. M. & Martin, E. M. (1972) J. Virol. 9, Testa, D., Chanda, P. K. & Baneiee, A. K. (1980) Cell 21, Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). 20. De, B. P. & Baneree, A. K. (1985) Biochem. Biophys. Res. Commun. 126, Hunter, T., Hunt, T., Jackson, R. J. & Robertson, H. D. (1975) J. Biol. Chem. 250, Farrell, P. J., Balkow, K., Hunt, T., Jackson, R. J. & Trachsel, H. (1977) Cell 11, Grill, L. J., Sun, J. D. & Kandel, J. (1976) Biochem. Biophys. Res. Commun. 73, Reijnders, L., Albers, A. M. J., Van Kammen, A. & Bern, A. J. M. (1975) Biochim. Biophys. Acta 390, Jacobson, A. & Favreau, M. (1983) Nucleic Acids Res. 11, Lodish, H. F. & Nathan, D. G. (1972) J. Biol. Chem. 247, Thimmappaya, B., Weinberger, C., Schneider, R. J. & Shenk, T. (1982) Cell 31, Schneider, R. J., Weinberger, C. & Shenk, T. (1984) Cell 37, Katze, M. G., Chen, Y. T. & Krug, R. M. (1984) Cell 37, Dunigan, D. & Lucas-Lenard, J. (1983) J. Virol. 45, Dratewka-Kos, E., Kiss, I., Lucas-Lenard, J., Mehta, H. B., Woodley, C. L. & Wahba, A. J. (1984) Biochemistry 23, Kates, J. & Beeson, J. (1970) J. Mol. Biol. 50,