Abstract
An RNA-dependent RNA polymerase activity has been detected in purified preparations of influenza virus. In contrast to the replicase activity induced in influenza-infected cells, the virion-associated enzyme has an absolute requirement for Mn++. Most of the RNA synthesized in vitro is complementary to virion RNA.
A number of animal viruses contain polymerase activities within their virions. Vaccinia virus contains a DNA-dependent RNA polymerase (1), reovirus and vesicular stomatitis virus contain RNA-dependent RNA polymerases (2, 3), and a number of oncogenic RNA viruses contain RNA-dependent DNA polymerases (4-6).
The lack of infectivity of influenza-virus RNA (7) and the demonstration that purified influenza virions contain essentially the same protein components as those that are induced in cells infected with influenza virus (8), suggest that influenza virions might also contain RNA polymerase activity. Two of us recently reported a failure to detect such an activity in influenza virus preparations by means of various solubilization and assay techniques (7). This failure was due to the fact that the influenza virion-associated RNA polymerase has an unusually stringent requirement for a divalent metal ion: a description of this manganese-activated RNA-dependent RNA polymerase activity is presented.
The NWS and PR-8 strains of influenza virus were grown either in embryonated eggs or in cell culture (7). Standard RNA polymerase assays were performed in 50 mM Tris-chloride, 6 mM P-mercaptoethanol, 25 mM (NH4)2 SO4, 0.23 mM (each) ATP, GTP, and CTP, 2 mM MnCl2, 0.15% Nonidet P40 detergent (Shell Chemical Co.), and either 10.5 pM (1100 cpm/pmol) (low U assay) or 78 pM (140 cpm/pmol) (high U assay) [ NWS influenza virus, grown in 10-day embryonated eggs and purified as previously described (7), was assayed for RNA polymerase activity. The activity detected in such assays t To whom reprint requests should be sent.
( Table 1 ) is insensitive to all of the inhibitors of DNA-dependent RNA polymerase tested, and is also insensitive to treatment with DNase. On the other hand, the preparations were devoid of activity in the presence of RNase, or in the absence of detergent or of one of the nucleoside triphosphates.
The metal-ion requirement of this polymerase is shown in Fig. 1 . In contrast to the enzymatic activity induced in influenza virus-infected cells (11), the virion-associated polymerase is inactive in the presence of any concentration of magnesium tested, but is markedly stimulated by the addition of 2-5 mM manganese. As shown in Fig. 2 , the enzyme is also stimulated about three-fold (at 2 mM Mn++) by 30 mM ammonium sulfate. NaCl, at equivalent ionic strengths in the absence of (NH4)2SO4, gave little stimulation of the activity. The stimulations observed in the presence of Mn++ and (NH4)2S04 are qualitatively similar to those reported for polymerase II from mammalian tissues (10) but, in contrast to that activity, the influenza polymerase is insensitive to aamanitin. Figs. 3 and 4 demonstrate that the incorporation of labeled nucleotide was linear over the time periods tested and that the assay was responsive to different concentrations of virus.

A number of animal viruses contain polymerase activities within their virions. Vaccinia virus contains a DNA-dependent RNA polymerase (1), reovirus and vesicular stomatitis virus contain RNA-dependent RNA polymerases (2, 3) , and a number of oncogenic RNA viruses contain RNA-dependent DNA polymerases (4) (5) (6) .
The lack of infectivity of influenza-virus RNA (7) and the demonstration that purified influenza virions contain essentially the same protein components as those that are induced in cells infected with influenza virus (8) , suggest that influenza virions might also contain RNA polymerase activity. Two of us recently reported a failure to detect such an activity in influenza virus preparations by means of various solubilization and assay techniques (7) . This failure was due to the fact that the influenza virion-associated RNA polymerase has an unusually stringent requirement for a divalent metal ion: a description of this manganese-activated RNA-dependent RNA polymerase activity is presented.
MATERIALS AND METHODS
The NWS and PR-8 strains of influenza virus were grown either in embryonated eggs or in cell culture (7) . Standard RNA polymerase assays were performed in 50 mM Tris-chloride, 6 mM P-mercaptoethanol, 25 mM (NH4)2 SO4, 0.23 mM (each) ATP, GTP, and CTP, 2 mM MnCl2, 0.15% Nonidet P40 detergent (Shell Chemical Co.), and either 10.5 pM (1100 cpm/pmol) (low U assay) or 78 pM (140 cpm/pmol) (high U assay) [ 'H]UTP. Reaction mixtures were incubated at 370C for various lengths of time, as indicated in the text, and spotted directly onto Whatman DE-81 filter-paper circles. The filters were processed and counted according to Blatti et al. (9) .
RESULTS
Properties of the influenza virus RNA polymerase NWS influenza virus, grown in 10-day embryonated eggs and purified as previously described (7), was assayed for RNA polymerase activity. The activity detected in such assays t To whom reprint requests should be sent.
( Table 1 ) is insensitive to all of the inhibitors of DNA-dependent RNA polymerase tested, and is also insensitive to treatment with DNase. On the other hand, the preparations were devoid of activity in the presence of RNase, or in the absence of detergent or of one of the nucleoside triphosphates.
The metal-ion requirement of this polymerase is shown in Fig. 1 . In contrast to the enzymatic activity induced in influenza virus-infected cells (11) , the virion-associated polymerase is inactive in the presence of any concentration of magnesium tested, but is markedly stimulated by the addition of 2-5 mM manganese. As shown in Fig. 2 , the enzyme is also stimulated about three-fold (at 2 mM Mn++) by 30 mM ammonium sulfate. NaCl, at equivalent ionic strengths in the absence of (NH4)2SO4, gave little stimulation of the activity. The stimulations observed in the presence of Mn++ and (NH4)2S04 are qualitatively similar to those reported for polymerase II from mammalian tissues (10) but, in contrast to that activity, the influenza polymerase is insensitive to aamanitin. Figs. 3 and 4 demonstrate that the incorporation of labeled nucleotide was linear over the time periods tested and that the assay was responsive to different concentrations of virus.
Localization of the RNA polymerase activity within influenza virions
The RNA-dependent RNA polymerase activity of influenza virus preparations was coincident with the hemagglutination Mixtures were made that contained 10,000 cpm of the reaction product and unlabeled PR-8 influenza virus RNA, whole-egg RNA, or no added RNA. They were made up to 0.1% sodium dodecyl sulfate, in a final volume of 0.21 ml. Where indicated in the table, samples were boiled, 0.05 ml of hot 0.15 M NaCl-0.015 M Na3 citrate was added, and annealing was allowed to occur for 120 min as the temperature was gradually reduced from 70 to 500C. The samples were chilled and divided into two equal portions, one of which was treated with 60 jug/ml of RNase A for 30 smithdylan@example.net.
analyses of virus preparations that have been previously purified by gradients of the same type.
Partial characterization of the product RNA The in vitro RNA product of the reaction was analyzed by annealing to RNA under various conditions, as shown in Table 2 . The data indicated that the primary product of the reaction is complementary to influenza virion RNA. The relatively high proportion of product that is resistant to ribonuclease after self-annealing is presumably due to the fact that the total amount of RNA synthesized in the reaction was small compared to the potential template in the reaction mixture.
DISCUSSION
The dependence on the presence of detergent for the enzymatic activity, the cosedimentation of the polymerizing activity with hemagglutination activity in rate-zonal and isopycnic centrifugation, and the fact that the RNA product of the reaction anneals with purified influenza virus RNA all indicate that the RNA polymerase activity we have measured is located within influenza virus particles.
Localization of an RNA-dependent RNA polymerase activity that synthesizes a product largely complementary to virion RNA within the influenza virion explains the lack of infectivity of influenza RNA and predicts that minus strands should be translated in influenza virus-infected cells. The fact that the virion-associated activity is dependent on the presence of manganese, while the activity induced in infected cells is active in the presence of magnesium (11) , provides direct evidence that there are at least two ribonucleotide-polymerizing enzymes involved in the infection process. Whether these activities are completely independent, or share common subunits or other factors, must await solubilization and purification of the molecular entities involved. These studies were performed in the laboratory of John J. Holland, to whom we are very grateful for the use of his laboratory, for continuing advice and support, and for the original suggestion that the influenza virion would contain the activity reported in this communication.
This investigation was supported by grant number CA-10802 from the National Cancer Institute. E. P. is a postdoctoral fellow of the National Institute of Child Health and Human Development (no. 42118-02) and S B. is a postdoctoral fellow of the National Institute of Arthritis and Metabolic Diseases (no. 39509-02).

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