Reprinted fro111 the PROCEEDINGS Oli THE Na.rroh-.a. ACADhllY 01` SCIENCES Vol. 50, No. 5, pp. 905911. Ncvember, 1963. AN RNA "REPLICASE" INDUCED BY AND SELECl'IVE FOR A VIRAL RNA: ISOLATION AND PROPE'RTIES* BY I. HARUNA, K. xOZU,t Y. OHTAKA, AND s. SPIEGELMAN DEPARTMENT OF MICROBIOLOGY, UNIVERSITY OF ILLINOIS, URBANA Communicated by M. M. Rhoades, August 26, 1963 The existence of the RNA viruses raises obvious questions concerning information transfer in organisms with an RNA genome. Doi and Spiegelman' have shown that neither before nor after infection can sequcnces be detected in the DNA of the host cell which are complementary to the viral RXh. These results suggest that RNA viruses do not employ DNA as an informed intermediary in any synthetic step required for components unique to the formation of virus particles. On this ground and others one is led to predict2, a mechanism of RNA replication involving an RNA-dependent RNA polymerase, which we will hereafter refer to as a "repli- case." (We have elsewhere pointed out2 that the repetitive use of such phrases as "DKA-dependent RNA polymerase," "RNA-dependent RKA polymerase," etc., can be avoided by introducing three simple terms. As noted, the RNA-dependent RYA polymerase which functions to turn out RKA replicas is termed a "replicase." The corresponding enzyme which synthesizes DKA4 duplicates can be called a "duplicase." Finally, the transcribing enzyme which employs DNA as a template to synthesize complementary RNA can be referred to as a "transcriptase." These terms have the advantage of brevity, precision, and a useful alliterative allusion.) Available evidence makes it unlikely that an enzyme using an RKA template functions in uninfected cells. All recognized cellular RKA components, including the "message" fra~tion,~~~ ribo~omal,~~ and amino acid transfer RXA9* have been shown to be complementary to some sequences in homologous DNA. Consequently, the pathway of their formation can be adequately explained by the DNA-dependent RNA synthesizing mechanism. This conclusion is further strengthened by the observations that actinomycin D inhibits" normal cellular RNA synthesis without interfering with the appearance of viral RNA.l2 The following two predictions are generated by the considerations just sum- marized: (1) the infecting RNA strand of the virus must serve as a message and be conserved during its translation into protein; (2) an enzyme should be obtainable from cells infected with RNA viruses which is uniquely dependent on RNA to exhibit RNA polymerizing activity. The first prediction was ~onfirmed'~ through the use of double labeling (NI5 and P3'9 and the demonstration that the two isotopes could be recovered in the same RNA strands at the end of a complete lytic cycle. It is the purpose of the present paper to offer evidence which confirms the expectation that a new type of polymerase is induced by an RNA virus. The experiments to be described were performed with the RSA bacteriophage MS42 used in the earlier investigations noted,', l3 and is similar to the f2 of Loeb and Zinder.14 A search for a unique RKA-dependent polymerase is complicated by the presence of a variety of enzymes15 which can incorporate ribonucleotides either terminally or subterminally into pre-existent RNA chains. In addition, there are others (e.g., 905 906 BIOCHEMISTRY: HARUNA ET AL. PROC. N. A. S. VOL. 50, 1963 BIOCHEMISTRY: HARUNA E2' AL. 907 RNA phosphorylase, l6 DNA-dependent RNA polymera~e,'~ polyadenylate syn- thetase`*) which can effect extensive synthesis of polynucleotide chains. Many of these sources of confusion can be avoided by suitable adjustment of the assay conditions and supplementary tests for a requirement of all four triphosphates, etc. The most serious difficulty is introduced by the DSA transcriptase, since it appears that, when isolated, it can employ certain types of RNA as substitutes for DNA as templates for polyribonucleotide 2o Under these circum- stances, the use of actinomycin D or DNAase does not ensure against observing its activity. The only certain way to avoid interference with the DNA-dependent enzyme is to eliminate it from the fraction of interest. It is obvious that a claim for a new type of RNA polymerase must be accompanied by evidence for RNA dependence and a demonstration that the enzyme is distinguishable in one or more of its properties from previously known enzymes with which it can be confused. Several recent reports offer suggestive evidence of polyribonucleotide synthesis activity induced by RNA viruses of animalz1 and bacterial hosts.22 However, in no case was an enzyme isolated in a sufficiently purified state to permit a definitive demonstration of RNA dependence. Under these circumstances, investigation of template specificity was obviously impossible. It is the purpose of the present paper to show that an RNA-dependent polymerase can be isolated from E. coli cells infected with an RNA bacteriophage (MS42). Further, the enzyme shows a selective preference for its own RXA as a template. Materials and Methods.-1. Bacteria and virus: The bacterial virus, MS42, was provided by Dr. Alvin J. Clark; it was grown and assayed according to the procedures of Loeb and Zinder.14 Preparation of virus stocks and purified viral RNA followed the methods of Doi and Spiegel~nan.'~ 2. Preparafion of znfected cells: The medium used contained the following in gm/liter: Bacto- tryptone, 10; NaCI, 10; Difco yeast extract, 5; glucose, 1; to each liter was added 1.3 ml of 2 M CaC1,. The procedure was as follows: (1) one liter of a log phase culture (0.D.6e0 of 0.150) is inoculated with phage at multiplicity of 0.1-0.2 and allowed to go into complete lysis and then used as a source of virus inoculation for larger batches. Thirty-five liter quantities of cells are grown up in cyclone pumps to an O.D.,, of 0.3. They are then infected with a multiplicity of 10 and allowed to aerate for several min. The aeration is interrupted 10 min for absorption, re- instituted, and the infection allowed to proceed 3040 min. The process is stopped by chilling with crushed ice, the cells are harvested, and then stored frozen at -20°C. Such cells retain their ability to yield active enzyme for periods exceeding 4 months. Uninferted cells are prepared by the same procedure and stored in the same manner. C14-labeled riboside triphosphates were all purchased from Schwarz BioResearch, Inc. They were used at the following specific activities: ATP-C14, 2 X 6 cpm/uM; GTP-C14, 0.72 x 106 cpm/pM; UTP-C14, 1.8 X lo6 cpm/pM; CTP-C14, 1.7 X 106 cmp/uM. P3,-labeled nucleotide was prepared by a modification of Tendsz3 procedure suggested to us by A. M. Michelson. The method is illustrated by the synthesis of UMP3,: a mixture of 100 pM of inorganic phosphate and 200 pM of isopropylidine is dissolved in 3 ml of dry pyridine, and the solution evaporated to dryness. More pyridine is added, and the procedure is repeated several times. The residue is finally dissolved in 0.1 ml of dimethylformamide and 1 ml of pyridine, to which is added 200 pM of p-cyanoethanol and then 1,000 pM of dicyclohexylcarbodiimide, and the mixture is left at 37°C overnight under anhydrous conditions. Pyridine is removed under reduced pressure, and to the residue is added 2 ml of 2 X NaOH; the mixture is kept at 100°C for 30 min. To remove the protecting isopropylidene residue, the mixture is adjusted to pH 1.0 with 2 A' HCl, kept at 100°C for 45 min, and then neutralized to pH 8.0. (If applied to purine nucleosides, the acidic hydrolysis should be at pH 2.0 at 100°C for 30 min.) Usually 80-90% conversion of in- organic phosphate into nucleoside-5'-phosphate is achieved. Conversion to the triphosphate is accomplished by the baker's yeast kinase as described by WeimZ4 The UTP32 was employed at 2 X IO6 cmp/wM. 3. Labeled substrates: 4. Reagents: Unlabeled rihoside triphosphates were from Pabst Laboratories, Milwaukee, Wisconsin. DNAase was 2 X recrystallized from Worthington Biochemical Company, Freehold, New Jersey. It was further purified on DEAE columns to remove contaminating ribon~clease.2~ Phosphoenolpyruvate (PEP) and the corresponding kinase (PEP-kinase) were from C. F. Boehringer and Soehne, Mannheim, Germany. Lysozyme was purchased from Armour and Company, Kankakee, Illinois. Poly A, Poly U. and Poly C were obtained from the Miles Chem- ical Company, Clifton, Yew Jersey. Poly G was a gift from Dr. J. Fresco, and turnip yellow mosaic virus (TYMV) RNA was kindly provided by Dr. It. Haselkorn. Tobacco mosaic virus RNA was isolated and purified from infected plants supplied by Dr. L. M. Black. 5. Assay sf enzyme acfivity by incorporation of radioadive nucleotides: The standard reaction of 0.25 nil contained the following in pM: Tris-HCI pH 7.5, 21; MgCl,, 1.4; MnCI,, 1.0; KC1, 3.75; mercaptoethanol, 0.65; spermine, 2.5; phosphoenolpyruvate, 1.0; ( NH4),S04, 70; CTP, ATP, GTP, and UTP, 0.5 each. In addition, it contained pyruvate kinase, 5 pg, DNAase, 2.5 pg, and, where indicated, 10 pg of the polynucleotide being tested as template. Enzyme was assayed at levels of 50-300 pg protein per sample. DNAase was always omitted in assaying for DNA- dependent polymerase act>ivity. Incubations were carried out at 35°C for 10 min and terminated by placing the reaction mixture in an ice bath and by the addition of 0.15 ml of neutralized satu- rated pyrophosphate, 0.15 ml of neutralized orthophosphate, and 0.1 ml of 80% trichloracetic acid (TCA). The precipitate was washed onto a millipore filter and washed five times wit,h 10 ml of cold 10% TCA containing 0.9yo of Na pyrophosphate. The millipore membrane was then dried and counted in a liquid scintillation counter, as described pre~iously.~ The pyrophosphate was included in the wash, since we found in agreement with Kammen et that its presence lowered zero time backgrounds to acceptable levels (40-70 cpm per sample containing input counts of 1 X lo6 cmp). 6. Preparation of enzyme: A suitable aliquot of the frozen infected cells is removed and put through the following procedure which is specified for 2 liters of in- fected cells at 1 X lo9 cells per ml. (a) The frozen cells are suspended in "standard buffer" (0.01 M Tris, pH 7.2; 0.005 M MgC12; 0.0005 M mercaptoethanol) and allowed to thaw. To this, 5 pg/ml of DNAase and 1 mg/ml lysozyme are added, and the mixture is frozen and thawed twice, as described by Hayashi and S~iegelman.~ The resulting mixture is allowed to incubate for 10 min at 20°C after increasing the level of DNAase to 10 pglml. (b) The extract is centrifuged at 15,000 g for 20 min, and the supernate and pellet are separated. The pellet is frozen with dry-ice acetone and ground in prechilled ( - 15OC) mortar for 10 min. It is then resuspended in 5 ml of "standard buffer" and again centrifuged at 15,000 g for 20 min. The two supernates are combined and possess an O.D.,,, of about 300. (c) The crude supernate is made up of 25 ml with "standard buffer" and adjusted to 0.01 211 EDTA and incubated at 0°C for 5-15 min. The appearance of a light, white precipitate indicates the onset of ribosomal destruction. The extract is then centrifuged at 15,000 g for 20 min and the precipitate discarded. (d) The extract is now subjected to a protamine fractionation which is designed to remove the DNA-dependent RNA polymerase, while leaving the RNA-dependent enzyme in t,he super- natant. The amount of protamine required to remove virtually all of the DNA-dependent activity varies from 11-13 mg per ml and must be titrated by assay for DNA-dependent activity. After appearance of the precipitate, the extract is centrifuged at 10,000 g for 10 min. (e) The super- nate of the protamine fraction is kept, and to 40 ml is added 23.6 ml of a saturated ammonium sulfate (pH adjusted to 7). After 10 min the material is centrifuged at 10,000 g for 10 min. (f) The activity is found in the supernate, and to it is added a further 12 ml of saturated ammonium sulfate followed by centrifugation at 10,000 g for 10 min. (9) The supernate is discarded; the precipitate is dissolved in 6 nil of "standard buffer" and dialyzed against 1 liter of the same buffer for 2 hr. (h) The dialysate is then put on a DEAE cellulose column (1.2 x 7 cm) and washed with 40-100 ml of the "standard buffer" made 0.1 M with respect to sodium chloride. This effectively removes remaining protamine sulfate and contaminating nucleases. Following the 0.1 M wash, the enzyme is eluted with 20 ml of the "standard buffer" made 0.3 M with respect to sodium chloride. (i) To 20 ml of the eluted enzyme is added 20 ml of a saturated ammonium sulfate, and the enzyme is centrifuged down at 10,000 g for 10 min. (j) The precipitate is dissolved in 4 mi of the "standar I buffer." The usual preparation obtained from the above procedure contains 7.4 mg protein per ml PROC. N. A. s. 008 BIOCHEMISTRY: HARUNA ET AL. VOL. 50, 1963 BIOCHEMISTRY: HARUNA ET AL. 909 with an 0.D.280/0.D.260 ratio of 1.2. The enzyme activity is unstable unless the ionic strength is restored. The addition of (NH4)2SO4 to 10% of saturation was found to prevent detectable decay of activity for several weeks providing the enzyme is stored in a 0°C ice bath. Results.-Reasons for including certain components in our ('standard assay mixture" (see Methods, 8 5) may be specifically noted. The riboside triphosphate generating system (PEP and PEP-kinase) was routinely included to avoid con- tributions by RNA-phosphorylase to the observed incorporation. DNAase was added to eliminate DNA-dependent synthesis. Spermine had previously been found2' to be an effective neutralizer of ribonuclease commonly found in crude extracts. From our first examinations of extracts from infected cells, evidence was obtained for DNAase-resistant incorporating activity associated with the pellet fraction which was obtained at 100,000 g in 2 hr (100G120P). This feature is illustrated in the data of Table 1 in which the pellet contains a fair amount of DNA-dependent activity of which 16 per cent continues in the presence of DNAase. If the pellet fraction is treated with EDTA as in Methods, 5 6(c) and the supernatant then frac- tionated with protamine as in Methods, 0 6(d), activities exhibited are as shown in Table 2. Prior to the protamine fractionation there is considerable DKA-dependent activity. The protamine step effectively removes this, the ratio of DNA- to RNA- dependent activity changing from 23.3 to 0.09. It was found with experience that separation nf the extract into the 100G120P pellet and supernatant was unnccessary, and the procedure described in Methods 0 6 was adopted for routine purposes. Table 3 shows some of the general character- istics of the enzyme fraction thus obtained. It has a clear requirement for viral RKA, and synthesis is only observed when all four triphosphates are present. The omission of any one of them completely abolished incorporation of UMPY2. It should also be noted (from the last line of Table 3) that this preparation does not contain dctectable amounts of the polyadenylate synthetase. TABLE 1 ~~OCATION OF EXZYME ACTIVITY IN INFECTED CELL EXTRACTS NT incorporated in Fraction Template mpM/10 min/mg protein Pellet MS42-RNA 0.34 0 0.28 CT-DNA* 1.94 0 <0.02 Supernatant MS42-RNA