Proc. Natl. Acad. Sci. USA Vol. 75, No 10, pp. 4868-4872, October 1978 Biochemistry 04-(5'-Uridylyl)tyrosine is the bond between the genome-linked protein and the RNA of poliovirus* (iodination/acid and alkali hydrolysis/enzymatic degradation/paper electrophoresis and chromatography) PAUL G. ROTHBERG, TIMOTHY J. R. HARRISt, AKIO NOMOTOt, AND ECKARD WIMMER Department of Microbiology, School of Basic Health Sciences, State University of New York at Stony Brook, Stony Brook, Long Island, New York 11794 Communicated by Seymour S. Cohen, August 3, 1978 ABSTRACT Virion RNA of poliovirus type 1 has been an- MATERIALS AND METHODS alyzed for the linkage between genome-protein VPg and the polyribonucleotide chain. Hydrolysis of the linkage with acid Poliovirus was grown and labeled with phosphorus-32 in HeLa or alkali and enzymatic degradation lead to the conclusion that cell suspension cultures as previously described (9). VPg was the bond is neither a phosphodiester such as nucleotidyl-(P- labeled with [3H]tyrosine as follows: 2.5 X 109 HeLa cells were O-serine (or threonine) nor a phosphoramidate such as washed twice with Earle's saline, suspended in 200 ml of Earle's nucleotidyl4(P-N)amino acid. VPg-RNA can be iodinated by the saline containing 1 mg of actinomycin D, and infected with 50 Bolton and Hunter reagent liodinated 3(4-hydroxyphenyl)pro- plaque-forming units of PV1 per cell. After 25 min at, room pionic acid N-hydroxysuccinimide ester] but not by the chlo- temperature, 200 ml of medium containing 168 ml of Earle's ramine-T or lactoperoxidase procedures, an observation saline, 4 ml of an antibiotic solution (10,000 units of penicillin suggesting that VPg does not contain accessible tyrosine. per ml, 10,000 ,ug of streptomycin per ml; GIBCO), 4 ml of However, VPg can be labeled with [3H]tyrosine in vivo. Hy- vitamins (1OOX; GIBCO MEM vitamin solution), 20 ml of di- drolysis of VPg-32PJpUp with 5.6 M HCI at 1100 yielded 32p- alyzed calf serum, 4 ml of 1OX amino acids (lacking cysteine labeled O&(3'-phosphio-5'uridylyl)tyrosine that could be cleaved and tyrosine) and 1 mg of cysteine were added to the infected with micrococcal nuclease to 04432P]phosphotyrosine and culture, which was then incubated at 370. One-half hour after uridine 3'-[MPlphosphate. These data establish that VPg is linked infection, 10 mCi of [3H]tyrosine (52.1 Ci/mmol, New England to the poliovirus genome by a bond between the 04 of tyrosine Nuclear) in 1 ml of 50% ethanol was added; Cells were har- and the 5'-P atom of the terminal uridylic acid residue. The 5' vested and virus and virion RNA were purified as previously end of polio genome RNA can now be described as VPg(Tyr- described (9). Poliovirus was labeled with 50 ,Ci of [14C]lysine O~pU-U-A--A-A-C-A-G ... (New England Nuclear, 250 Ci/mol) and purified essentially as described for [3H]tyrosine except that labeling was in ly- Picornaviruses may be unique among animal RNA viruses sine-free F-14 medium (GIBCO) and started 2.5 hr after in- because their single-stranded genome is covalently linked to fection. Virus was recovered from sucrose gradient fractions a small protein, called VPg (1-7). The protein is virus-coded by acetone precipitation (2 vol of acetone, -20°, overnight). (2, 7) and linked to the 5'-terminal uridylate of the RNA (4, 7, VPg-pUp labeled with 32p or with [3H]tyrosine was isolated 8). Polio VPg is hydrophobic (1, 2) and basic (net charge of +7 by digesting purified virion RNA with RNase T2 and separating at pH 7.5, ref. 4) and has a molecular weight of 4000-6000 as the nucleotidyl-protein from mononucleotides by ion-exchange determined by polyacrylamide gel electrophoresis (2, 5, 7). chromatography on DEAE-cellulose at pH 5 (2, 4). [tyrosyl- Biochemical studies have shown that VPg is linked not only 3H]VPg-pUp was always purified together with VPg-[32P]pUp to virion RNA, but also to negative strand (genome comple- to facilitate detection of the protein by monitoring for Cerenkov ment) RNA (4) and to nascent strands of the replicative inter- radiation. VPg-pUp was digested with Pronase (Sigma protease the basis of these we and type VI) to yield (aa) -pUp as described (2, 4), or with 1 ml of mediate (3, 4). On findings, (2, 4) 5.6 M HCl (freshly distilled) in a vacuum-sealed hydrolysis tube others (3) have suggested that VPg may be involved in genome (Pierce) for 2 hr at 1100. After hydrolysis the HC1 was evapo- replication, possibly in the initiation of RNA synthesis (4). rated under reduced pressure and the residue was dissolved in Viral mRNA, on the other hand, may be the product of water and lyophilized. Digestion products of VPg-pUp were "processing" of newly made plus strand RNA because its 5' further degraded with highly purified snake venom 3'-exo- terminus is pUp (refs. 9 and 10 and literature cited therein). We nuclease (generous gift of M. Laskowski, Sr.), as previously have speculated that cleavage of the bond between protein and described (2, 4); with 1.0 unit of bacterial alkaline phosphatase RNA may have a regulatory function; it is possible that only (Worthington Biochemical Co.) in 10 mM Tris-HCl, pH 7.5/1 protein-linked RNA is encapsidated, whereas RNA without the mM EDTA, for 1 hr at 370; or with 1.0 unit of micrococcal protein serves as mRNA or as a transcription template (2, 4, nuclease (Staphylocus aureus, Foggi strain, grade VI; Sigma 9). Chemical Co.) in 50 mM Tris-HCI, pH 8.5/10 mM CaCl2 for For an analysis of the function of VPg, the bond linking the 1 hr at 370. protein to the RNA is of interest. Here we report that VPg of Abbreviations: VPg, the genome-linked viral protein of poliovirus; PV1, poliovirus type 1 (PV1) is bound to the RNA via a phospho- poliovirus type 1; NaDodSO4, sodium dodecyl sulfate; Tyr-P,- diester bond between the phenolic hydroxyl group of tyrosine 04-phosphotyrosine; for further information on the nomenclature of and the 5' phosphoric residue of uridylic acid. The 5'-terminal phosphorus-containing compounds, see (1977) Proc. Nati. Acad. Sci. can now be described as USA 74,2222-2230. structure of the poliovirus genome * This is paper 5 in the series "The genome-linked protein of picor- VPg(Tyr-O)-pU-U-A-A-A-A-C-A-G ... naviruses." Paper 4 is ref. 7. t On leave from the Animal Virus Research Institute, Pirbright, The publication costs of this article were defrayed in part by page Woking, Surrey, United Kingdom. charge payment. This article must therefore be hereby marked "ad- * Present address: Department of Public Health, School of Pharma- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate ceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, this fact. Tokyo, Japan. 4868 Downloaded by guest on September 25, 2021 Biochemistry: Rothberg et al. Proc. Nati. Acad. Sci. USA 75 (1978) 4869 04-Phosphotyrosine was synthesized by heating 4.1 ml of acetate as described (2, 4); thin-layer chromatography was on 85% (wt/vol) phosphoric acid, 4.2 g of phosphoric acid anhy- cellulose plates (Polygram CEL 300, Macherey, Nagel and Co.) dride, and 1.4 g of L-tyrosine to 1000 for 72 hr (11). The mixture or on EM silica gel F-254 plates (Brinkmann Instruments) in was then diluted to 65 ml with crushed ice and Tyr-P was iso- the following solvent systems: (A) saturated ammonium sul- lated by a modified procedure of Mitchell and Lunan (12) as fate/i M sodium acetate/isopropanol (40:9:1, vol/vol/vol; ref. follows: The solution was percolated through a Dowex 50 col- 20); (B) first dimension: isobutyric acid/0.5 M ammonia (5:3, umn (3.1 X 28.5 cm, hydrogen form) and the column was vol/vol), second dimension: isopropanol/concentrated HCl/ washed with water. Fractions eluting from the column con- water (70:15:15, vol/vol/vol; ref. 21); (C) isopropanol/cor- tained two peaks as monitored by absorbance at 280 nm: peak centrated ammonia/water (7:2:1, vol/vol/vol; ref. 22). Tyr-P I (beginning at the void volume of the column) that contained was detected on cellulose plates by spraying them with 0.3% Tyr and Pi, and peak II, eluting over a large number of frac- (wt/vol) ninhydrin in 1-butanol (Gelman Instrument) and tions. Those fractions of peak II with an absorbance of >0.8 heating to 1000 for 5 min. The silica gel plates were precoated were combined, concentrated to 150 ml by flash evaporation, with a fluorescent indicator making it possible to visualize mixed with 160 ml of ethanol, and stored overnight at 40. A Tyr-P by absorbance under shortwave ultraviolet light. white precipitate was recovered by filtration (800 mg) and Radioisotopes in dried gels or thin-layer plates were detected shown to be Tyr-P, which was at least 97% pure as judged by by autoradiography at -70° using Kodak SB5 film and a Du thin-layer chromatography (see below) or by release of Pi after Pont Cronex intensifying screen. treatment with bacterial alkaline phosphatase. Virion RNA was labeled with 125I by one of three methods: (i) VPg-RNA (250 jig) in 150 Ml of 0.1 M sodium phosphate, pH RESULTS 7.4/0.1% sodium dodecyl sulfate (NaDodSO4) was mixed with 2.5 of carrier-free Na'25I (100 mCi/ml, 11-17 mCi/Ag) and Elimination of Phosphoramidates or Nucleotide(P-O- with 10 AL of chloramine-T (N-chloro-p-toluenesulfonamide; serine (or Threonine) as LinkageGroups.