JOURNAL OP VIROLOGY, OCt. 1972, p. 875-879 Vol. 10, No. 4 Copyright © 1972 American Society for Microbiology Printed In U.S.A. Ribonucleic Acid Synthesis in Cells Infected with Herpes Simplex Virus VI. Polyadenylic Acid Sequences in Viral Messenger Ribonucleic Acid STEVEN L. BACHENHEIMER AND BERNARD ROIZMAN Department of Microbiology, The University of Chicago, Chicago, Illinois 60637 Received for publication 28 June 1972
Evidence is presented by use of radiolabeling and pancreatic and Ti ribonuclease digestion that some of the ribonucleic acid specified by herpes simplex virus con- tains polyadenylic acid sequences. The polyadenylic sequences are not transcribed from viral DNA.
The occurrence of polyriboadenylic acid than 99% of the 3H-adenosine was contained in a (poly A) sequences in messenger ribonucleic acid molecule labile in alkali, i.e., in RNA. (mRNA) and heterogeneous nuclear ribonucleic The size of poly A isolated from polyribosomes acid (HnRNA) of a variety of eukaryotic cell of infected cells was determined by co-electro- systems has been well established (3, 4, 5, 11, 12, phoresis on a polyacrylamide gel with uninfected 16). Poly A has also been detected in the niRNA cell 4S RNA (Fig. 1). Both poly A and 4S of adenovirus (14) and vaccinia virus (9, 16), in RNA marker were denatured in 1.1 M formal- the genomic ribonucleic acid (RNA) of tumor dehyde prior to electrophoresis so that their viruses (6, 7, 10), as well as in the RNA of polio- migration and subsequent molecular-weight de- (1) and sindbis (8) viruses. Significant amounts termination would be independent of molecular of poly A were not detected in the genomic conformation (2). The molecular weight of the RNA species of Newcastle disease, influenza, or largest poly A sequence found, calculated from vesicular stomatitis viruses (6). In the instances the relationship of relative distance migrated to where poly A sequences were detected, they were log molecular weight, was 54,500, correspond- characterized as single-stranded ribonucleotide ing to about 160 nucleotides. The heterogeneity sequences resistant to pancreatic and Ti ribo- with respect to the size distribution of poly A nuclease. The isolated poly A sequences char- sequences has been noted by others (1, 10, 13) acteristically migrated in polyacrylamide gels as if in a variety of cell systems, both uninfected and they were 150 to 200 nucleotides in length. In this infected with virus. The heterogeneity may be a report we wish to extend these observations to reflection ofrandomness in the process of poly herpes simplex virus. A attachment to mRNA, or the consequence The evidence that RNA specified by herpes of removal of adenylic acid residues after poly simplex virus contains poly A sequences is based A is in the cytoplasm, or a combination. on two series of experiments. The first series of The objective of the second series of experi- experiments was based on previous work (15, 17, ments was to determine in a more rigorous man- 18) indicating that, by 4 hr after infection, the ner whether poly A sequences were contained in polyribosomes of infected cells were exclusively viral RNA. In these experiments 8P-labeled synthesizing viral proteins, and that most, if not RNA was hybridized to viral DNA immobilized all, cytoplasmic RNA specified by the virus was on filters. The hybridization was carried out in associated with polyribosomes. In the present five times concentrated standard saline citrate and experiments, RNA labeled with 3H-adenosine and 50% formamide at 43 C to insure a minimum of 14C-uridine was isolated from polyribosomes of breakage of RNA and to test for the covalent infected cells and digested with pancreatic and Ti attachment of poly A to virus-specified RNA. ribonuclease. As shown in Table 1, the residual Preliminary experiments designed to test the ribonuclease-resistant RNA contains 20% of the effects of various hybridization procedures on adenosine but only 1% of the uridine of poly- the integrity of the poly A-mRNA linkage have ribosomal RNA. To establish that the residual shown that, in general, the lower the temperature 3H-adenosine was in RNA, polyribosomal RNA at which the hybridization is carried out, the was digested with ribonuclease and then hydro- greater the stability ofthe RNA. At the conditions lyzed with 0.6 N KOH. As shown in Table 1, more selected for the hybridization tests, 77% of the 875 876 NOTES J. VIROL.
TABLE 1. Presence of polyadenylic acid sequenices 14 - u in polyribosomal RNA of herpes virus- 3H-Aid inifected cellsa 1000 r250 3H-Adenosine 14C-Uridine Polyribosomal RNA Counts/ Percent Counts/ Per- 800 200 min min cent
Expt 1 600 150 Input 10,200 100 3,100 100 After ribonuclease 2,037 20 33 1 Expt 2 400 100 Input 51,000 100 15, 500 100 I\ Wbe,^~~ >k10 After ribonuclease 107 0.2 34 0.2 \z + KOH 200 50 a 2 X 108 A monolayer of HEp-2 cells, infected Oa* oao,o'd o\ \ with 10 plaque-forming units of herpes simplex virus subtype 1 (HSV-1) per cell was labeled nI nm from 3 to 4.5 hr postinfection with 10 ,Ci of 10 20 30 40 + 3H-adenosine per ml in 25 ml of Eagle minimal fraction no. essential medium supplemented with 1% dialyzed calf serum. Cells were washed in phosphate- FIG. 1. Acrylamide gel electropherogram ofpoly A buffered saline and disrupted by resuspension in isolated from polyribosomes of infected cells. Inifected- RSB [0.01 M NaCl, 0.01 M tris(hydroxymethyl)- cell polyribosomes, labeled with 3H-adenzosine, were aminomethane (Tris), pH 7.5, 0.0015 M MgCl2J prepared, and poly A was isolated as described in the containing 0.5% Nonidet P-40 at 4 C for 15 min. legend to Table 1. After digestion with pancreatic Nuclei were removed by centrifugation at low and Ti ribonuclease, sodium dodecyl sulfate was speed. The cytoplasmic extract was layered over a added to a final concentration of 0.5%, and 100 ,g 40% (w/w) sucrose-RSB cushion, and poly- of yeast RNA was added as carrier. The solubilized ribosomes were collected as a pellet by centrifuga- digest was phenol-extracted, anid a Tris blffer, pH 9.0, tion at 102,000 X g for 2.5 hr at 5 C in a T65 rotor. wash of the interface and pheniol phase was combined The polyribosome pellet was resuspended in with the original aqueous phase an1d chloroform-ex- TSM (0.15 M NaCl, 0.01 M Tris, pH 7.5, 0.001 M tracted. Uninfected cell RNA, labeled with 14C-uridine MgCl2) containing 0.5% sodium dodecyl sulfate, and soluble in 2 M LiCl was added to the combinied and was extracted with phenol. The interphase and aqueous phases, precipitated with 7.5%7 trichloroacetic phenol phase were washed with Tris buffer (0.1 M acid, anid washed with 80% ethanol. The precipitate Tris, pH 9.0, 0.01 M KCI, 0.002 M MgCl2), to insure was resuspended in 50 jAliters of buffer containing that RNA containing poly A sequences was not 0.09 M Na2HPO4, 0.01 M NaH2PO4, pH 7.6, 1.1 m specifically lost during the extraction. The aqueous formaldehyde, heated to 65 C for 15 min, quick- phases were combined and extracted with chloro- cooled, and applied to a 10-cm, 10% acrylamide gel form-2% isoamyl alcohol, and RNA was pre- made up in 0.018 M Na2HPO4, 0.002 M NaH2PO4, cipitated with two volumes of absolute ethanol 1.1 sm formaldehyde. After electrophoresis for 3.25 hr and stored at -20 C. The precipitate was col- at 4 ma, the gel was fractioniated into 2-mm slices. lected by centrifugation and resuspended in The slices were swelled in 0.6 ml of soluenie (Packard TSM buffer. In experiment 1, a sample of the Instrument Co., Inc.) at 60 C for 2 hr and theni as- RNA was digested with deoxyribonuclease I sayed for radioactivity in 10 ml of a toluene-base (50 jg/ml) for 30 min at 37 C. Ethylenediamine- scintillant. tetraacetic acid (EDTA) was then added to a final concentration of 0.01 M, and enough 10 X SSC (1.5 M NaCl, 0.15 M sodium citrate) was added RNA retained its ability to bind to membrane to give a final concentration of 0.3 M NaCl. Pan- filters (Millipore Corp.). After 20 hr of hybridiza- creatic ribonuclease (2,ug/ml) and Ti ribonuclease tion, the RNA was eluted from the filter and (5 units/ml) were added and allowed to digest analyzed for its ability to bind to Millipore filters the RNA for 60 min at 37 C. The digest was pre- in the presence of 0.5 M KCI (11). As shown in cipitated with an equal volume of 15c%o trichloro- Table 2, of 50,000 counts/min of cytoplasmic acetic acid, collected on a membrane filter, dried, viral RNA tested, 41% bound to the filter, and counted. In experiment 2, a sample of RNA whereas 59%O passed through the filter. These was digested as described above, and then hy- drolyzed for 18 hr at 37 C in 0.6 N KOH. The data indicate that poly A is covalently linked to hydrolysate was then precipitated, potassium viral RNA. perchlorate was spun out at low speed, and any Consistent with previous reports (13, 14), the residual material in the supernatant was collected experiment described above and other experi- on a filter for counting. ments suggest that, in herpesvirus-infected cells VOL. 10, 1972 NOTES 877
TABLE 2. Binding of viral RNA to cellulose 3H-adenosine, the appearance of label in poly nitrate filtersa A-rich RNA binding to filters lags behind the appearance of the label in total nuclear RNA Total RNA RNA bound Viral RNA tested (counts/ to filters tof (Fig. 2). In agreement with these observations, min) (counts/min) the proportion of total 3H-adenosine in the poly A moiety of membrane-bound polyribosomal Cytoplasmic ...... 44,650 18,400 41 RNA declines with time after the addition of Nuclear...... 45,400 5,500 12 label (Fig. 3). As a final test for the post-transcriptional a A monolayer of 2 X 108 HEp-2 cells was in- an fected with 10 plaque-forming units of HSV-1 addition of poly A to viral-specified RNA, per cell. At 3 hr postinfection, the monolayer experiment was designed to demonstrate that was washed with Eagle minimal essential medium poly A is not a transcriptional product of viral supplemented with 1% dialyzed calf serum and DNA. The rationale was that, if poly A were containing no phosphate salts and then was covalently linked to virus-specific RNA but not labeled for 2 hr with 32p (2 mCi/ml) in 25 ml of transcribed from viral DNA templates, it would the same medium. Cells were disrupted as de- be selectively released from hybrids by digestion scribed before, and nuclei were lysed by addition with pancreatic and Ti ribonuclease at high of deoxycholate to a final concentration of 0.5% ionic strength. DNA-RNA hybridization was and deoxyribonuclease I (50 ,g/ml). Total cyto- plasmic and nuclear RNA were phenol-extracted as described in the legend to Table 1. In addition, 2500 500 RNA preparations were digested with deoxy- ribonuclease I and then repurified. Cellulose ni- trate filters (Millipore Corp., HAWP, 25 mm 2000 400j diameter) containing 5 ,ug of HSV-1 DNA were prepared by heat denaturing a solution of DNA in 2 X SSC (1 X standard saline citrate consisted 1500 .300 -0 of 0.15 M NaCl, 0.015 M sodium citrate) at 115 C = for 10 min, followed by quick cooling in an ice z c._-0 bath, diluting and adjusting the salt concentration zE0a a 1000 200
the virion RNA of poliovirus and eastern equine encepha- 11. Lee, S. Y., J. Mendecki, and G. Brawerman. 1971. A poly- litis virus. Science 176:526-528. nucleotide segment rich in adenylic acid in the rapidly 2. Boedtker, H. 1971. Conformation independent molecular labeled polyribosomal RNA component of mouse sar- weight determination of RNA by gel electrophoresis. coma 180 ascites cells. Proc. Nat. Acad. Sci. U.S.A. 68: Biochim. Biophys. Acta 240:448-453. 1331-1335. 3. Burr, H., and J. B. Lingrel. 1971. Poly A sequences at the 12. Means, A. R., J. P. Comstock, G. C. Rosenfeld, and B. W. 3' termini of rabbit globulin mRNAs. Nature N. Biol. O'Malley. 1972. Ovalbumin messenger RNA of chick 233:41-43. oviduct: partial characterization, estrogen dependence, 4. Darnell, J. E., R. Wall, and R. J. Tushinski. 1971. An adenylic and translation in vitro. Proc. Nat. Acad. Sci. U.S,A. 69: acid rich sequence in messenger RNA of HeLa cells and 1146-1150. its possible relationship to reiterated sites in DNA. Proc. 13. Mendecki, J., S. Y. Lee, and G. Brawerman. 1972. Char- Nat. Acad. Sci. U.S.A. 68:1321-1325. acteristics of the polyadenylic acid segment associated with 5. Edmonds, M., M. H. Vaughan, Jr., and H. Nakazato. 1971. messenger ribonucleic acid in mouse sarcoma 180 ascites Polyadenylic acid sequences in the heterogeneous nuclear cells. Biochemistry 11:792-798. RNA and rapidly-labeled polyribosomal RNA of HeLa 14. Phillipson, L., R. Wall, G. Glickman, and J. E. DarnelL cells: possible evidence for a precursor relationship. Proc. 1971. Addition of polyadenylate sequences to virus-specific Nat. Acad. Sci. U.S.A. 68:1336-1340. RNA during adenovirus replication. Proc. Nat. Acad. 6. Gillespie, D., S. Marshall, and R. C. Gallo. 1972. RNA of Sci. U.S.A. 68:2806-2809. RNA tumor viruses contains poly A. Nature N. Biol. 15. Roizman, B., S. Bachenheimer, E. K. Wagner, and T. Savage. 236:227-231. 1970. Synthesis and transport of RNA in herpesvirus- 7. Green, M., and M. Cartas. 1972. The genome of RNA infected mammalian cells. Cold Spring Harbor Symp. tumor viruses contains polyadenylic acid sequences. Proc. Quant. Biol. 35:753-771. Nat. Acad. Sci. U.S.A. 69:791-794. 16. Sheldon, R., Jurak, C., and Kates, J. 1972. Detection of 8. Johnston, R. E., and H. R. Bose. 1972. An adenylate-rich polyadenylic acid sequences in viral and eukaryotic RNA. segment in the virion RNA of Sindbis virus. Biochem. Proc. Nat. Acad. Sci. U.S.A. 69:417-421. Biophys. Res. Commun. 46:712-718. 17. Spear, P. G., and B. Roizman. 1968. The proteins specified 9. Kates, J. 1970. Transcription of the vaccinia virus genome by herpes simplex virus. I. Time of synthesis, transfer and the occurrence of polyriboadenylic acid sequences in into nuclei, and properties of proteins made in productively messenger RNA. Cold Spring Harbor Symp. Quant. Biol. infected cells. Virology 36:545-555. 35:743-752i 10. Lai, M. M. C., and P. H. Duesberg. 1972. Adenylic acid-rich 18. Sydiskis, R. J., and B. Roizman. 1966. Polysomes and pro- sequences in RNAs of rous sarcoma and rauscher mouse tein synthesis in cells infected with a DNA virus. Science leukaemia virus. Nature (London) 235:383-386. 153:76-78.