73,
VIROLOGY
327-338
(1976)
RNA Synthesis
by Ribonucleoprotein-Pclymerase from Influenza Virus OLGA
Department
of Virology,
The Public Health
Complexes
Isolated
M. ROCHOVANSKY Research Institute New York 10016
Accepted April
of The City of New York, Inc., New York,
28,1976
Highly active ribonucleoprotein-polymerase complexes isolated from the AJWSN strain of influenza virus contained RNA, the nucleoprotein, and the two minor P proteins. Purified complexes retained the same RNA to polymerase activity ratio present in whole virus and polymerase specific activity (picomoles of GMP incorporated into RNA per hour per milligram of protein) was increased fivefold. In vitro cRNA synthesis by complexes and unfractionated virus showed very close similarity, with the exception of the divalent cation requirements. Mn2+ was required for polymerase activity of unfractionated virus while optimum synthesis by complexes was obtained with either Mn2+ or M$+. Studies on the time dependence of cRNA formation by complexes showed very little synthesis beyond 2 hr. Addition of inorganic pyrophosphatase and guanylyl(3’ + 5’)-guanosine increased both the rate and extent of RNA synthesis severalfold and resulted in the formation of longer transcripts. INTRODUCTION
Under optimum conditions, transcription of the WSN genome was incomplete with only 28% of the virion RNA copied (Bishop et al., 1971a, b). Many questions concerning the polymerase and the reaction it catalyzes still remain unanswered. Very little is known about the polypeptide identity of the polymerase, the template specificity of the enzyme, or its mechanism of action. Moreover, the reasons for the incomplete transcription catalyzed by the WSN polymerase in vitro are not understood. Studies on the inhibitory effects of the drugs, actinomycin D and a-amanitin, on early RNA synthesis in the replicative cycle of influenza virus (Mahy et al., 1972; Bean and Simpson, 1973), suggest that host cell factors may be required for synthesis of genome transcripts. In vitro studies with isolated polymerase-template complexes provide a simple and direct approach to detailed investigations of the transcriptive process, its components, and the possible involvement of cellular factors. The present paper de-
Influenza virus was shown by Chow and Simpson (1971) as well as by Penhoet et al. (1971) and Skehel (1971) to contain an RNA-dependent RNA polymerase that in vitro catalyzes the synthesis of RNA complementary (cRNA) to virion RNA (vRNA). Two lines of evidence strongly suggest an in viuo function of the polymerase in the synthesis of influenza mRNA. Viral-specific RNA on polyribosomes of infected cells was found to be very largely or completely cRNA (Pans, 1972, 1975; Etkind and Krug, 1974); and recently, Etkind and Krug (1975) demonstrated that only cRNA was active in directing the synthesis of complete influenza proteins in wheat germ cell-free extracts. The requirements of the in vitro reaction catalyzed by the polymerase of the A,J WSN strain of influenza virus have been studied in some detail. Conditions of ionic strength, temperature, and the divalent cations necessary for optimum polymerase activity of detergent-disrupted WSN virus were investigated by Bishop et al. (1971). 327 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
328
OLGA
M. ROCHOVANSKY
scribes the purification and characterization of highly active ribonucleoprotein-polymerase complexes from the A,,/WSN strain of influenza virus. This strain was chosen for investigation because in vitro cRNA synthesis by the virus is well characterized (Bishop et al., 1971a, b). In addition, WSN virus, because of its rather low polymerase activity and inability to synthesize complete genome transcripts in vitro (Bishop et al ., 1971b, 19721,provides a good system for investigating the effects of cellular constituents on both the rate and extent of transcription. A comparison of the requirements and characteristics of cRNA synthesis by complexes and unfractionated virus particles and the effects of various compounds on transcription are reported. MATERIALS
AND
METHODS
Virus growth and purification. The &I WSN strain of influenza virus was used throughout this study. Virus was grown in chick embryo fibroblasts (CEF) as described by Simpson and Hirst (1961) using a multiplicity of approximately lo-” plaque-forming units (PFU) per cell. Radioactive virus was prepared by adding the desired isotope to the medium one-half hour after infection as follows: [3H]amino acid mixture, 4 @i/ml, or 13Hluridine, 5 &i/ml. Virus released into the medium by infected CEF cells after a 40-hr incubation at 37” was purified according to the method of Pons and Hirst (19681, except that virus was not treated with ribonuclease. Preparation of polymerase-template complexes. Step 1. Mixtures for disrupting
virus particles contained the following components in a final volume of 1 ml: 0.38 ml of a lysolecithin solution (10 mg per ml in HzO>, 0.06 ml of HzO, 0.08 ml of 70% glycerol, and 0.40 ml of a solution containing Tris-HCl, pH 8.1, 280 n-&f; ATP, CTP, UTP, and GTP, all at a concentration of 1.4 m&f; KCl, 400 n&f; dithiothreitol, 3.3 n-&f; MgC&, 50 m&f, and MnC&, 5 r&f. Finally, 0.08 ml of a virus suspension in STE (0.05 M Tris-HCl, 0.1 M NaCl, 10e7M EDTA, pH 7.2) containing 0.8 mg of virus
protein was added. After incubation at 30” for 10 min, the samples were chilled and an additional 0.08 ml of 70% glycerol was added. Sterile solutions were used in all steps of the purification procedure. Step 2. The samples were immediately layered onto chilled discontinuous glycerol gradients formed in siliconized tubes. The glycerol solutions contained 50 mM TrisHCl, pH 7.8, and 0.15 M NaCl. The glycerol composition (w/w) of the gradients was1,25mlof33%, 0.25mlof40%,0.50ml of 50%, and 1.0 ml of 70% glycerol. The gradients were centrifuged at 5” in a Spinco model L2-65B in a SW 56 rotor for 4 hr at 340,000g. Four or six such gradients were usually run, and after centrifugation the individual fractions were collected from the top and similar fractions from the gradients were combined. The gradients were usually clear except for a faint band about halfway into the 70% glycerol layer. This fraction probably contained partially disrupted virus particles (see below). A faint flocculant band infrequently observed on top of the 50% layer was removed by aspiration and discarded. Purified polymerase-template complexes were present in the 50% glycerol layer. Storage of this fraction at -20” for at least 8 months did not result in any detectable loss of polymerase activity. RNA polymerase assay. The reaction conditions were a modification of the method described by Bishop et al. (1971a) for the assay of the WSN polymerase. Activities of glycerol gradient fractions were determined using samples no larger than 0.05 ml. Assay mixtures in a final volume of 0.25 ml contained 16 pmol of Tris-HCl, pH 8.1, 8.3 pmol of (NH&SO,, 1 pmol of dithiotreitol, 0.5 pmol of MnCl,, 0.4 pmol of ATP, 0.2 pmol of CTP and UTP, and 0.05 pmol of 13H1GTP.The counts per minute per picomole of the labeled nucleotide are given for each experiment. When the activity of unfractionated WSN virus was assayed, 10 ~1 of a lysolecithin solution containing 10 mglml dissolved in H,O were added to each assay tube to disrupt virus particles. Approximately 50 to 100 pg of viral protein were used in assays of WSN virus.
RNA
SYNTHESIS
BY INFLUENZA
Assay mixtures were incubated at 31” and the reactions terminated by the addition of 0.2 ml of a mixture containing 2.5 mg/ml of yeast RNA, 2.5 mg/ml of bovine serum albumin, and 5 pmol/ml of sodium pyrophosphate, immediately followed by the addition of 1 ml of 10% trichloroacetic acid. After 30 min at 4”, the precipitates were collected on Whatman GF/A filters and washed first with 50 ml of cold 6% trichloroacetic acid containing 0.05 M sodium pyrophosphate, and finally with 20 ml of cold 1% trichloroacetic acid. The remaining acid on the filters was neutralized by the addition of 0.5 ml of 0.1 M NaOH to the counting vials. Radioactivity was determined after addition of 5 ml of Bray’s solution in a Nuclear-Chicago Mark II counter. Estimation of protein. Protein was determined by the microbiuret method of Munkres and Richards (1965).
RNP+P
329
first precipitated by the addition of an equal volume of 20% trichloroacetic acid. After 3 hr at 4”, the precipitated protein was centrifuged, washed twice with cold acetone, and prepared for electrophoresis as described. Estimation of the size of RNA transcripts. Reaction mixtures were prepared
and incubated as described in the legend to the appropriate figure. Reactions were terminated by the addition of an equal volume of SLA (0.5% recrystallized sodium dodecyl sulfate, 0.14 M LiCl, 0.01 M sodium acetate, pH 4.9). RNA was extracted and precipitated as previously described (Pons and Hirst, 1968). After a second precipitation, the RNA was dissolved in 0.3 ml of 0.01 x SSC (0.15 M NaCl, 0.015 M sodium citrate, pH 7.2). Samples of 0.2 ml were diluted to 1 ml with 0.01 x SSC and the mixtures heated for 5 min at 85”. After rapid cooling, the mixtures were layered Sedimentation analysis of polymeraseonto 35-ml glycerol gradients (5-30% w/w) template complexes. Velocity sedimentain STEU (0.1 M NaCI, 1O-3M EDTA, 0.05 tion studies on [3H]uridine-labeled com- M Tris-HCl, pH 7.2, 1 M urea). Gradients plexes were carried out in preformed 34-ml were centrifuged at 5” in a Spinco model linear glycerol gradients (20-70% w/w) L2-65B for 17 hr at 110,000 g in a SW 27 containing 0.05 M Tris-HCl, pH 7.4, and rotor. Fractions of 1 ml, collected from the 0.05 M NaCl by a modification of the bottom of the tubes, were divided into 2 method of Pons et al. (1969). 0.4-ml samples. One sample was precipiIn order to detect polymerase activity in tated by addition of 0.1 ml of serum albufractions of a linear gradient carried out min (10 mg per ml) and 1 ml of 20% trichlowith unlabeled complexes, the peak frac- roacetic acid. To the other sample was tions (identified by comparison with a gra- added an equal volume of RNAse (2 pg of dient of radioactive complexes run in par- Tl and 20 /Ig of A per ml). After 20 min at allel) were first concentrated by the follow- 37”, the samples were precipitated as ing procedure: the fractions were com- above. The precipitated RNA was collected bined, diluted to 36 ml with 0.05 M Tris- on Whatman GF/A filters and washed HCl, pH 7.8, and centrifuged for 17 hr in a with 20 ml of 1% trichloroacetic acid. RaSpinco model L2-65B for 17 hr at 60,000 g dioactivity was determined as described in a SW 27 rotor. Polymerase activity was above. Enzymes and chemicals. Lysolecithin detected in the bottom 1 ml of the centrifuge tube by the assay procedure described was in part a kind gift from Dr. Eric Cole, concentration of and the rest was purchased from the above. A similar [3H]uridine-labeled peak fractions gave a Grand Island Biological Company. Nurecovery of 80% of the radioactivity in the cleotide triphosphates were obtained from the Sigma Chemical Company; guanosine, same position. Polyacrylamide gel electrophoresis. The GpG, spermidine, and spermine from Calpolypeptides present in the fractions ob- biochem; and poly(A) from Miles Laboratained from discontinuous glycerol gra- tories, Inc. Radioactive isotopes were obdients were determined by the electropho- tained from New England Nuclear and resis method described by Schulze (1970). from Amersham/Searle. Inorganic pyroThe proteins in the gradient fractions were phosphatase, purified from yeast by the
330
OLGA
M. ROCHOVANSKY
method of Petrack and Ratner (1958), was kindly provided by Dr. Sarah Ratner. RESULTS
The procedure used by Bishop et al. (1972) for isolation of polymerase-template complexes from the WS strain of influenza virus yielded purified preparations containing only RNA, the nucleoprotein (NP), and the P proteins. However, approximately 90% of the initial polymerase activity present in the virus was lost by this method (Hefti et al., 1975). In order to study the enzymatic properties of purified polymerase-template complexes of the WSN strain under more favorable conditions, a procedure for the separation of highly active complexes was developed. Characterization of Polymerase-Template Complexes Isolation of complexes. Several different detergents effective in disrupting influenza virus were examined for their effect on polymerase activity in order to establish optimum conditions for step 1 of the isolation procedure. Virus treated with lysolecithin gave an activity at least 15% higher than did virus treated with nonidet P-40, Triton, or sodium deoxycholate. Lysolecithin was therefore chosen for disruption of the virus. The second step in the isolation procedure, separation of complexes on discontinuous glycerol gradients, is a modification of the sucrose gradient procedure developed by Compans and Caliguiri (1973) for isolation of similar complexes from influenza virus-infected cells. Both sucrose and glycerol were found to inhibit virion polymerase activity. A comparison of the effects of the two materials at concentrations necessary for the isolation of complexes showed that inhibiton by sucrose was about 20% greater. Three different preparations of virus, labeled with (a) [3H]uridine, (b) 13H]amino acids, or (c) unlabeled, were disrupted and each preparation was then fractionated as described under Materials and Methods. The distribution of radioactivity and polymerase activity in the five gradient fractions is shown in Table 1. Comparisons of
fractionation profiles were possible since numerous experiments carried out with isotopically labeled and unlabeled preparations of virus gave virtually identical results. Substantial polymerase activity was detected only in gradient fractions 4 and 5. Fraction 4 contained 65% of the total polymerase units present in unfractionated virus, 64% of the RNA, and 13% of the protein. Only 29% of the total enzyme activity was present in fraction 5 with 30% of the RNA and 18% of the protein. The active fractions of the gradients (3, 4, and 5) contained a total of 1736 enzyme units or 95% of the activity in unfractionated virus. These recovery values were highly reproducible . The fractionation resulted in a substantial purification of the virion polymerase activity. The specific activity of 2975 obtained for material in gradient fraction 4 was 5-fold higher than that of unfractionated virus (Table 2). The activity of fraction 5 represented an increase of only 1.5fold. Addition of lysolecithin to assays of TABLE 1 ISOLATION OF POLYMERASE-TEMPLATE COMPLEXES FROM A,,IWSN VIRUS" Sample
[:‘HlRNA total cpm
1:‘HlProtein total cpm
Polymerase activity total units”
WSN Fr 1 Fr 2 Fr 3 Fr 4 Fr 5
1,067,000 43,690 3,710 14,200 677,510 318,410
350,000 146,930 81,290 6,670 44,540 62,610
1,824 0 0 26 1,190 520
(1 Fractionation on discontinuous glycerol gradients of F’Hluridineand 1”Hlamino acid-labeled and unlabeled preparations of WSN virus disrupted by lysolecithin. In each case, WSN virus containing 3.2 mg of protein was used. Glycerol concentrations (w/w) in the gradient fractions were: Fr 1 (sample layer) 15%; Fr 2,33%; Fr 3,40%; Fr 4,50%; Fr 5,70%. Values for fractions 1-5 are to be compared with the starting samples of unfractionated virus given in line 1. ’ Total units are expressed as picomoles of F’HIGMP incoporated per hour per total fraction volume at 31”. The [3HlGTP used for assay contained 40 cpm/pmol. Assay values were corrected for a zero time blank of 50 cpm. Value for unfractionated virus in line 1 represents the total units of polymerase activity per 3.2 mg of viral protein.
RNA TABLE
SYNTHESIS
2
BY INFLUENZA
RNP+P
331
gels by Coomassie blue, showed the P and NP proteins; no other polypeptides were Total Specific detected. The P protein on stained gels protein activity’ consisted of two very closely moving but W discrete bands (not shown). WSN 1824 3.2 570 Analysis of the sedimentation characFr 3 26 0.06 433 teristics and the protein composition (Fig. Fr 4 1190 0.40 2975 1) showed that the RNA-protein structures Fr 5 520 0.58 897 in fraction 4 consisted of RNP and the P a Data taken from Table 1 proteins. Assays showing that the fraction b Protein was determined by a microbiuret contained active polymerase (Tables 1 and method except for the Fr 3 value which was esti2) further indicated that the structures mated from the protein data given in Table 1. were ribonucleoprotein-polymerase comC Specific activity is expressed as picomoles of plexes, designated RNP+P for conveni13HlGMP incorporated per hour per milligram of ence. The recovery of 65% of the total poprotein at 31”. lymerase units arid-64% of the viral RNA in the RNP+P (Table 1) demonstrated fraction 5 did not increase the activity, that the complexes closely resembled insuggesting that this fraction contained partially disrupted particles with accessi- tact virus in the ratio of RNA to polymerase activity. Similar ratios were obtained ble RNA polymerase. for the other two less active gradient fracSedimentation characteristics of comtions (3 and 5). On this basis it was conplexes. Fraction 4 material obtained from cluded that comparisons of the requireL3Hluridine-labeled virus was further anaments and factors influencing the in vitro lyzed on linear glycerol gradients, and its activities of the isolated complexes and sedimentation position was compared to whole virus preparations were valid. that of ribonucleoprotein (RNP) obtained from nonidet P-40-treated virus by the method of Pons et al. (1969). The material in fraction 4 sedimented in one discrete peak in a position identical to that of the RNP marker (not shown). Peak fractions of a parallel gradient carried out with unlabeled fraction 4 were assayed for polymerase activity as described in Materials and Methods. Approximately 40% of the total enzyme activity originally present in fraction 4 was detected. Protein composition of complexes. The polypeptides associated with the RNA-protein structures in the [3H]amino acid-labeled fraction 4 were analyzed by polyacrylamide gel electrophoresis. Only the P 0 40 80 120 (MW 92,000) and NP (MW 65,000) polypepFraction No tides were clearly present (Fig. 1). AnalyFIG. 1. Polyacrylamide gel analysis of the proses of the other gradient fractions showed complexes. that polypeptides HA, HA,, HAB, NA, and tein composition of polymerase-template 13H]amino acid-labeled complexes obtained from a M were present in the upper fractions (1 discontinuous glycerol gradient were prepared and and 2) and all of the viral proteins except analyzed as described in Materials and Methods. polypeptide NA were found in gradient The protein profile of complexes is shown with the fraction 5 (not shown). profile obtained for intact WSN virus labeled with An examination of the polypeptides in [3H]amino acids. Symbols: (O-O), polymerase fraction 4 by a second method, staining of template complexes; (O- - -01, WSN virus. PURIFICATION OF RNA Sample Total enzyme units”
POLYMERASE ACTIVITY
332 Conditions
OLGA
for Optimum
RNP-tP
M. ROCHOVANSKY
Activity
Temperature and ionic strength requirements. The conditions for maximum polymerase activity of RNP+P complexes were similar to those reported by Bishop et al. (1971a) for unfractionated WSN virus. Optimal RNP+P activity was obtained at 31” at an ionic strength of 0.1. An enhancement of polymerase activity of about 20% occurred when ammonium sulfate at a concentration of 33 mM was substituted for either KC1 or NaCl at equivalent ionic strengths. Penhoet et al. (19’1) reported that polymerase activity of the unfractionated NWS strain of influenza virus was stimulated threefold by ammonium sulfate. Although the effect of this salt on polymerase activity of unfractionated WSN virus and RNP+P complexes was much more modest, stimulation was consistently obtained and ammonium sulfate was, therefore, routinely added to polymerase assays. Divalent cation requirements. Previous studies on the divalent cation requirements for in vitro activity of the WSN polymerase have resulted in some apparent inconsistencies. Chow and Simpson (1971) found that optimal activity was obtained with Mr?+ ions alone but subsequently Bishop et al. (1971a) reported that both Mn’+ and MgZ+ ions were required. The divalent cations required for polymer-
ase activity of unfractionated WSN virus were, therefore, investigated and compared to the requirements of RNP+P complexes. Polymerase activity of unfractionated WSN virus in the presence of increasing concentrations of either Mn’+ or Mg2+ is illustrated in Fig. 2A. Optimum activity was obtained with 2 mM Mr?’ alone. In comparison, the Mg2+-dependent activity at the optimal concentration of 10 mM was only 18% of the maximum MI?+-dependent activity. The results are in agreement with earlier findings that Mn2+ is the preferred divalent cation for the polymerase of unfractionated WSN virus (Chow and Simpson, 1971; Penhoet et al., 1971; Skehel, 1971). The response of RNP+P complexes to increasing concentrations of either Mn2+ or MgZ+ ions is shown in Fig. 2B. In contrast to the results with unfractionated virus (Fig. 2A), identical maximum activities were obtained with either 2 mM Mn2+ or 10 mM Mg2+. Addition of increasing concentrations of Mg” to assays of unfractionated WSN virus and RNP+P complexes containing 2 mM Mn2+ resulted in increasing inhibition of polymerase activity. At a concentration of 8 m&f Mg2+, polymerase activity of WSN virus and RNP+P were inhibited 24 and 38%, respectively. However, addition of 8 mM Mg2+ did increase activities about 30% in assays carried out with suboptimal con80:
r
-~--
~-
-
-1
Mn++
I-_-.-
8 A
Mnst
or Mg+‘(
mM)
B
Mn”
or Mqft
12 (mM 1
FIG. 2. Dependence of polymerase activity on divalent cations. (A) WSN virus (72 Fg of protein) and (B) RNP+P complexes (12.2 Fg of protein) were assayed for polymerase activity with increasing concentrations of MnCl, or MgCl,. Reaction mixtures containing L3HlGTP (30 cpm/pmol) were incubated for 2 hr. Symbols: (O-O), Mnz+-dependent activity; (O---O), M&+-dependent activity.
RNA
SYNTHESIS
BY
centrations of Mn*+ (1 mM). Concentration dependence of RNP +P polymerase activity. As shown in Fig. 3,
INFLUENZA
333
RNP+P
cases a nonlinear increase was observed, and by 4 hr the reactions had terminated. The possibility of ribonuclease activity in the RNP+ P preparation was investigated by adding phenol-extracted L3Hluridine-labeled virion RNA to RNP+P under assay conditions in the absence of labeled nucleotide. The mixture was incubated for 4 hr at 31” and the sedimentation position of the incubated RNA on a 5-30% (w/w) glycerol gradient was compared with untreated RNA. No change in the sedimentation characteristics was observed. Although the presence of trace amounts of nuclease activity cannot be completely ruled out, contamination seems unlikely in view of the following experiments which show that both the rate and extent of RNP+P activity were increased by the addition of specific cellular metabolites.
polymerase activity of RNP+P complexes increased linearly with concentrations from 0.01 to 0.03 ml (2.5 to 7.5 pg of protein). With concentrations larger than 0.03 ml, a marked departure from linearity was observed. Polymerase activity of unfractionated virus showed a linear response to concentrations as high as 48 pg of protein. A small departure from linearity was observed with higher concentrations of virus. The results obtained with increased concentrations of complexes were apparently due, at least in part, to an inhibitory effect of the glycerol present in RNP+P preparations. Additions of 0.01 and 0.05 ml of the 50% glycerol solution used for isolation of complexes to assays containing 0.05 ml of an RNP+P preparation caused a 10 and 45% inhibition of polymerase activity, re- Stimulation of RNP+P Polymerase Activspectively. Thus far, it has not been possiity by Cellular Constituents ble to remove the inhibitory material by Studies on the in vivo effects of the dialysis with retention of active com- drugs, actinomycin D and a-amanitin, on plexes. Time dependence of RNP+P polymeruse activity. The activity dependence of
1
RNP+P complexes and unfractionated virus on time is illustrated in Fig. 4. In both
Time,
ol/,
001
1’
003
RNPtP
005
j
al
0.07
or WSN (ml)
FIG. 3. Dependence of polymerase activity on concentration. Preparations of WSN virus and RNP+P complexes that were tested for concentration dependence contained 1.2 and 0.25 mg of protein per ml, respectively. Reaction mixtures containing the indicated volumes of virus or complexes and [3H]GTP (80 cpm/pmol) were prepared and incubated for 1 hr. Symbols: (O-O), WSN activity; (O-O), RNP+P activity.
hours
FIG. 4. Dependence of polymerase activity on time. The activities of RNP+P complexes and WSN virus were examined as a function of time. For assay of RNP+P complexes, a 1.5ml reaction mixture containing the standard concentrations of reaction components, RNP+P complexes (73 pg of protein), and 13H1GTP (40 cpm/pmol) was prepared. The reaction mixture for assay of the WSN polymerase contained 0.48 mg of viral protein. At the indicated times 0.2-ml samples were removed from the mixtures and the acid-insoluble radioactivity was determined. Symbols: (O-O), WSN activity; (O-O), RNP+P activity.
334
OLGA
M. ROCHOVANSKY
The effect of S-adenosyl-L-methionine (SAM) was examined because several viruses were recently shown to contain a methylase activity that incorporates methyl groups from SAM into mRNA synthesized by the viral polymerases. The rate of RNA synthesis by reovirus and vaccinia virus polymerases was unaffected by the methyl donor (Shatkin, 1974; Wei and Moss, 1974) but transcription of the cytoEffect of polyamines, S-adenosylmeplasmic polyhedrosis virus genome was thionine and poly(A). The polyamines, completely dependent on the presence of spermidine and spermine, have been SAM (Furuichi, 1974). Addition of SAM at found in a number of viruses including concentrations of 0.2 to 0.8 mJ4 had no influenza virus (Bachrach et al., 1974). effect on the rate of RNP+P activity; simiThe role of polyamines in viruses is not lar results were obtained with unfractionunderstood but may include regulatory as ated WSN virus. well as structural functions (Russell, Poly(A) was tested because influenza 1970). It was therefore of interest to test mRNA isolated from polyribosomes of inthe effects of spermidine and spermine on fected cells contains poly(A) segments (EtRNA synthesis by RNP+P complexes. As kind and Krug, 1974; Macnaughton et al., may be seen in Table 3, both compounds at 1975). The mechanism of poly(A) addition concentrations higher than 0.4 m&f in- to transcripts of the influenza genome is hibited RNA formation. Lower concentra- not understood nor is it known whether tions had no effect on polymerase activity. the activity is virus or cell-mediated. Polymerase activity of RNP+P complexes was inhibited by concentrations of poly(A) TABLE 3 as low as 1.0 pg (Table 3); lower concentraEFFECT OF CELLULAR CONSTITUENTS ON RNP+P tions had no effect. POLYMERASE ACTIVITY
the synthesis of early influenza virus-specific RNA (Mahy et al., 1972; Bean and Simpson, 1973) suggest that in uiuo transcription may be controlled by cellular constituents absent from in vitro reaction Specific compounds either mixtures. known to be present in virus particles or to structurally modify mRNA molecules were examined for their effect on cRNA synthesis by RNP+P complexes.
Addition
Tjcoco$s
Activity (%I
incorpora. ted” None Spermidine
(0.4 n&f) (0.8 mM) (4 mM) Spermine (0.8 n&f) Poly A(2 pg) (5 CLP) None Guanosine (0.08 nuI4) (0.15 mkf) (0.5 m&f) GpG (0.08 m&f) (0.15 m&f) (0.20 mA4) GpG (0.15 n-&f) + SAM (0.4 d) PPaseb
21.5 26.2 23.9 10:6 24.2 21.2 17.9 25.4 30.0 32.3 21.3 38.6 49.5 41.2 49.9
100 95 87 39 87 77 65 100 118 127 84 152 195 162 196
32.3
127
U Assays were carried out as described under Materials and Methods with [3H]GTP containing 80 cpm per pmol. Incubations were for 1 hr. b Assay contained 0.5 units of PPase. A unit of activity is that liberating 1 pmol of inorganic phosphate per min at pH 7.2 and 25”.
Effect of guanosine, guanylyl43 + 5’) guanosine, and inorganic pyrophosphatase . As shown in the lower part of Table 3,
the only components that were found to increase the polymerase activity of RNP+P complexes were guanosine, the dinucleoside phosphate, guanylyl-(3’ + 5’)guanosine (GpG), and inorganic pyrophosphatase (PPase). Stimulations similar to those shown were obtained when the activity of unfractionated WSN virus was tested. Addition of SAM to assays containing GpG caused no further stimulation over that given by GpG alone. McGeoch and Kitron (1975) recently reported that polymerase activities of several influenza viruses were stimulated severalfold by 0.5 mM concentrations of guanosine and GpG. The data presented here are in general agreement with the earlier results except that the stimulations of RNP+P activity were considerably smaller and the optimum concentrations of guanosine and GpG were lower. The reasons for these differences are not under-
RNA
SYNTHESIS
BY INFLUENZA
stood but may be due, at least in part, to the use of WSN virus, RNP+P complexes obtained from WSN virus, or to different assay conditions. Many enzymatically catalyzed synthetic reactions in which inorganic pyrophosphate (PPi) is formed exhibit product inhibition. Addition of PPase to such reactions removes inhibitory PPi thereby accelerating synthesis. Since PPi is formed in the synthesis of RNA, it was of interest to test the effect of PPase on in vitro activity of RNP+ P complexes. Commercial preparations of the enzyme are contaminated by variable amounts of nuclease activity. In the present study, highly purified PPase (5 x recrystallized) prepared by the method of Petrack and Ratner (1958) was dialyzed and tested for nuclease activity by incubating the enzyme with [3Hluridine-labeled virion RNA and then by examining the sedimentation characteristics of the RNA. No RNA degradation was detected. As shown in Table 3, addition of the enzyme to in vitro assays of RNP+P complexes caused a small increase in activity. In order to provide support for the suggested role of PPase, inhibition by PPi of RNP+P polymerase activity and the release of inhibition by the enzyme were examined in greater detail. As shown in Table 4, addition of increasing concentrations of PPi to assays of RNP+P complexes resulted in decreased enzyme activity. At the highest level of PPi tested, 5 nmol, activity was inhibited by 87%. In the presence of PPase and 5 nmol of PPi no inhibition was observed; assays carried out with and without addition of PPi gave identical activity values. The results suggest that the pyrophosphatase effect would be more pronounced in highly active systems in which the initial rate of RNA synthesis was faster with a concomitant accumulation of higher amounts of PPi. Prolonged synthesis of RNA in presence of PPase and GpG. The results in Table 3 indicated that the rate of the reaction catalyzed by RNP+P complexes was increased by PPase and GpG. In order to determine if the extent of RNA synthesis was also increased, the effects of PPase and GpG on the activity of RNP+P complexes as a function of time were examined. As illus-
335
RNP+P
trated in Fig. 5, addition of PPase increased activity over the entire time course with a maximum stimulation of 40% at 4 hr (curve 21, but the shape of the TABLE
4
EFFECT OF INORGANIC PYROPHOSPHATE ON RNP+P ACTIVITY” Nanomoles PPi addedb
Picomoles lJHIGMP incorporated
0 0.05 0.10 0.50 1.0 2.0 5.0 0 + PPase’ 5 + PPase’
24.1 21.0 18.2 14.1 10.9 8.4 3.2 31.3 31.6
Activity (%) 100 87 76 59 45 35 13 130 131
(1Assays were carried out as described under Materials and Methods with [SH]GTP containing 80 cpm/pmol. Incubations were for 1 hr. b Indicated concentrations of PPi were added to assays at zero time. C PPase (0.5 units) was added to the assays at zero time. ,
0
120
60 Time,
180
24
minutes
FIG. 5. Stimulation of polymerase activity of RNP+P complexes. Reaction mixtures containing the standard concentrations of reaction components, RNP+P complexes (36.5 pg of protein), 13H]GTP (80 cpm/pmol), and additional components as indicated were prepared in a final volume of 1.25 ml. At the indicated times, 0.20-ml samples were withdrawn and the acid-insoluble radioactivity was determined. Further additions were as follows: curve 1 (O-O), none; curve 2 (O-O), 2.5 units of PPase; curve 3 (A-A), 0.15 mM GpG, curve 4 (O-Cl), 2.5 units of PPase and 0.15 mM GpG.
336
OLGA M. ROCHOVANSKY
curve was similar to that obtained with complexes alone (curve 1). In both cases a rapid decline in the rate of RNA synthesis occurred after 90 min when about 75-80% of the total RNA was synthesized. Greater stimulation of RNP+P activity was obtained by addition of GpG with the maximum effect observed in the early time periods (curve 3). Comparison with the activities given by RNP+P alone (curve 1) showed that GpG stimulated RNA synthesis by 210, 106, and 77% at 30, 60, and 90 min, respectively. The rate of synthesis declined rapidly in the presence of GpG, and at 4 hr only 66% more total RNA was formed. The greatest effect on the rate and extent of RNA synthesis was observed when both PPase and GpG were added (curve 4). Comparison with the activity of complexes alone (curve 1) showed that the rate of the reaction was increased fourfold at 30 min and then steadily declined with time. RNA synthesis continued for at least 4 hr at a diminished rate and the total amount of RNA formed was increased by almost threefold. Size of product RNA. In order to determine if the prolonged synthesis of RNA in the presence of PPase and GpG resulted in the formation of transcripts of longer lengths, RNA from 4-hr reaction mixtures was isolated, melted to obtain singlestranded structures, and analyzed by centrifugation on glycerol gradients. RNAse sensitivity of the material indicated that predominantly single-stranded RNA was present. Transcripts formed in the presence of PPase were of quite small size, ranging from about 4-12 S with a mean value of 6 S (Fig. 6A). A broader distribution of sizes (6-17 S) was obtained with RNA formed in the presence of both PPase and GpG (Fig. 6B). Although about 60% of the product was small (6-8 S), the remaining RNA was of larger size, mainly 12 and 15 S. A portion of the RNA from A and B taken before centrifugation was hybridized to added unlabeled vRNA. Product RNA was at least 95% complementary to vRNA (data not shown). These data indicate that addition of PPase and GpG did result in the synthesis of longer product RNA, although only a
6 4 2 0 5
IO Froci~on
15
20
25
30
i 3! 5
Number
FIG. 6. Velocity sedimentation of RNA transcripts. (A) Reaction mixtures contained the standard concentrations of reaction components, RNP+P complexes (136 pg of protein), PPase (9 units), and [3H]UTP (60 cpmlpmol) in a finalvolume of 4.5 ml. (B) Conditions as in (A), except that the final volume of 3 ml contained RNP+P complexes (87 pg of protein), PPase (6 units), and GpG (0.15 mM). After a 4-hr incubation, RNA was extracted, treated, and centrifuged as described in Materials and Methods. Symbols: (O-O), RNA, (O-O), RNA after RNAse treatment.
very small portion of the transcripts, if any, were actually completed. If the entire lengths of the virion RNA segments were copied, completed transcripts would be expected to fall into three average size classes, 10, 18, and 22 S (Pans, personal communication). DISCUSSION
Composition and characteristics of RNP +P complexes. Procedures described
here have made possible the isolation of purified, highly active ribonucleoproteinpolymerase complexes from the WSN strain of influenza virus. Information obtained from studies on both the composition and polymerase activity of the RNP+P complexes strongly suggests that complexes retained the complete in vitro transcriptional capacity of whole virus. This conclusion is supported by the follow-
RNA
SYNTHESIS
BY INFLUENZA
ing data: Purified complexes retained the same ratio of RNA to polymerase activity found in whole virus. With the exception of divalent cation requirements, complexes showed the same characteristics of RNA synthesis as unfractionated virus. All of the eight segments of RNA which comprise the viral genome (Pons, 1976) were shown by polyacrylamide gel analysis to be present in RNP+P preparations (Rochovansky and Hirst, 1976). In addition, examination of RNP + P reaction mixtures by velocity sedimentation indicated that product RNA was associated with the large, medium, and small size classes of RNP template (Rochovansky, in preparation). In addition to RNA the complexes contained the viral nucleoprotein, NP (MW 65,000>,and the two minor P proteins (MW -92,000). A similar composition was previously reported for complexes isolated from the WS strain of influenza virus (Bishop et al., 1972) and for complexes obtained from cells infected with the WSN strain (Caliguiri and Compans, 1974). The polypeptides responsible for polymerase activity are not known and their identification must await isolation of the purified enzyme in a functionally active form. The isolation in high yield of purified complexes containing only three viral proteins may prove useful for this purpose and methods for dissociating the polymerase from complexes and for reconstitution of an active system are currently under investigation. Comparison of the properties of purified complexes and whole virus preparations revealed generally similar requirements for RNA synthesis. The only difference observed was in the divalent cations required for optimum polymerase activity. In contrast to the results with unfractionated virus showing that Mn2+ was the preferred cation, optimum activity of complexes was obtained in the presence of either Mn2+ or Mg*+. This surprising result is not understood but a possible explanation currently under investigation is that one or more of the viral proteins absent from RNP+P complexes may influence the response of the enzyme to Me+. In this regard it is interesting that the RNA-dependent RNA
RNP+P
337
polymerase present in the microsomal fraction of cells infected with influenza virus was more active in the presence of Mg2+ than Mn2+; product RNA formed by this enzyme was 93% complementary to virion RNA (Hastie and Mahy, 1973). Stimulation of polymerase activity. The stimulator-y effect of PPase on RNA synthesis by RNP+P complexes can be ascribed to removal of PPi, an inhibitory product of the reaction. Only a modest stimulation was observed when the initial rate of the reaction was low but under conditions of increased initial activity, obtained by addition of GpG, the effect of PPase was much more pronounced. Since PPase is a normal constituent of many different cells, the in vitro stimulation reported here may reflect an in vivo function of the enzyme in transcription. The effect of GpG on RNA synthesis was most pronounced in the early time periods suggesting that the dinucleoside phosphate was involved in an early step in transcription. This finding is compatible with the demonstration by McGeoch and Kitron (1975) that guanosine was incorporated at the 5’-termini of fowl plague viral cRNA synthesized in vitro. Maximum stimulation of RNA synthesis was observed in the presence of both GpG and PPase. Not only was the initial rate of the reaction increased but RNA synthesis continued for at least 4 hr with the formation of longer transcripts. Although the role of GpG in the stimulation of influenza cRNA synthesis is not presently understood, it seems likely that the dinucleoside phosphate was, like guanosine, incorporated at the 5’-termini of the RNA. Initiation of cRNA chains with suitable primers may therefore result in an increased rate of cRNA synthesis and the formation of longer transcripts. ACKNOWLEDGMENTS The author wishes to thank Dr. George K. Hirst for his many contributions to this investigation. Many helpful and stimulating discussions with Drs. M. W. Pons and A. Gregoriades are gratefully acknowledged, as is the technical assistance of Mr. Peter J. Ciraolo. This work was supported in part by Grants No. AI-04360 and No. AI-11614 of the National Institute
OLGA
338 of Allergy and Infectious Health Service.
Diseases,
M. ROCHOVANSKY
U. S. Public
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