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The effect of polyvinylsulfate on the ribonucleoprotein of influenza virus
VIROLOGY
41, 382-384 (1970)
The Effect of Polyvinylsulfate
on the Ribonucleoprotein
of Influenza
Virus’
samples were collected and aliquots counted Polyvinylsulfate (PVS), a negative11 charged polymer, has been shown to be a in a dioxane base cocktail. The appropriate competitive inhibitor of RKase (1) and is corrections for spillover of counts into other also capable of replacing messenger RXA on channels mere made in the usual way. Polyvinylsulfate labeled with 3% was preribosomes (2). Came et al. (3) reported that PVS inhibits the replication of influenza vi- pared by the method of Bernfeld et al. (9). rus both in vivo and in vitro. Scholtissek and Chlorosulfanic acid-35S (5.3 mCi) was obtained from the Amersham/Searle CorporaRott (4) have shown that PVS and another tion (sp. act. 39 mCi/mM). Polyvinyl alcopolyanion, dextran sulfate, inhibit the RKAhol was obtained from Matheson Coleman dependent RN,4 polymerase obtained from & Bell. The yield of PVS35 was too low to fowl plaque virus-infected cells but not that obtained from the cells infected with Ken-- enable accurate determination of the specific activity of the compound. In most cases, castle disease virus. however, the virus was treated with approxiRecently, a report from this laboratory mately 20.0 pg/ml PVS3j (581,000 cpm/ml suggested that PVS completely displaced RKA from the ribonucleoprotein (RSP) of final concentration). Two preparations of influenza virus, one influenza virus (5). Electron microscopy and velocity gradient analyses showed that the labeled with 3H-amino acids, the other with 32P04 , were mixed in equal amounts (2 X morphological and sedimentation characterlo5 HA units total) and treated with nonidet istics of the intact and PVS-treated RKP were identical, indicating that PVS may P-40 (NP40), a nonionic detergent, (final concentration 1%) in standard buffer (0.1 ~11 have replaced the RNA in the RNP complex NaCl, lop3 J/ EDTA in 0.01 M Tris-HCl in such a way as to preserve the helical pH 7.2 containing 1 M urea). Velocity gradistructure. The present report demonstrates conclusively that PVS (labeled with 36S)does ent sedimentation analysis of the mixed preparation showed that RNA (labeled with indeed replace the RNA on the RKP helix. Influenza virus (WSN strain) was pre- 32P) and protein (labeled with 3H) cosedimented at 3% (Fig. la). It has been shown pared in chick fibroblast monolayers (CFAI) already that the 38s material is the RNP (6, 7). The growth and purification of virus labeled with 32P04 and 3H-amino acids were and that the HA and neuraminidase proteins sediment in a broad peak in the center of the performed as previously described (5, 8). Velocity gradient sedimentation analyses gradient’ with phospholipids at the t,op (5, 10). were carried out on 30-ml linear glycerol Treatment of a double labeled preparation gradients (lo-70 % w/w) containing 0.01 :II Tris-HCl pH 7.2, 0.3 M SaCl, 1 dl urea, with NP40, followed immediately by treatand 1O-3 111EDTA. Samples n-ere layered on ment with approximately 20 pg PVS3j altered gradients through a 5-ml layer of mineral oil the sedimentation profiles as shown in Fig. lb. PVS3” and protein (labeled with “Hand were centrifuged in a Spinco Model L2-65B using a SW-27 rotor, at 25,000 rpm amino acids) cosedimented at 38s. The for 17 hours at 20”. The bottoms of the tubes 32P-labeled RKA sedimented at 2OS, which is the sedimentation value obtained for phenolwere punctured with a needle, and l-ml SDS extracted influenza virus RNA (‘i”). 1 This investigation was supported in part by There is coincidence between 35S-labeled PVS Grant AI-04360 of the National Institute of and other proteins in the gradient, indicating Allergy and Infectious Diseases, U.S. Public that the polymer can associate with other Health Service. viral proteins. Velocity sedimentation of 382
SHORT COMMUNICATIONS
b.
383
IO FRACTION
20 NO.
FIG. 2. Polyvinylsulfate labeled with % was centrifuged through a glycerol gradient as described in Fig. 1. A.-.-A, 32P.
raphy, and the treated and untreated fractions were incubated
at 37” for 2 hours prior
to resedimentation through glycerol gradients. Figure 3a shows that in the untreated material the PVS35 and protein resedimented at 38s while small amounts of RNA and FRACTION NO. PVS35 sedimented at 18-21s and Ids, reFIG. 1. (a) Purified WSN, labeled with 3H- spectively. Figure 3b shows that after pronase treatment the digested protein was at amino acids and 32POd,was treated with l”/b nonithe top of the gradient while the PVS3j sedidet in the presence of 1 M urea. (b) Purified doubly labeled WSN treated with lo/ nonidet followed by mented at about 14s. This is the sedimenta20 @g/ml polyvinylsulfate, labeled with %, in the tion coefficient of free PVS3j. These results presence of 1 dl urea.. Both preparations were show that no particular size of PVS3” is relayered over lo-70y0 glycerol gradients and cen- quired for this reaction. They also show that trifuged for 17 hours at 20” in the SW-27 rotor the PVW had complexed with the nrot,ein of 25,000 rpm of a Spinco Model L2-65B centrifuge. the RNP. One-milliliter fractions were collected, and O.OlThe following experiment was carried out ml aliquots were counted in a liquid scintillation to determine how PVS reacts with the disascounter. Appropriated corrections for spillover of sembled RNP. 32P-3H-labeled RNP was counts were made. O--O, 3H; X-----X, 32P; treated with 10 pg/ml RNase for 10 minutes A.-.-A, 93, at 37”, and half was further treated with PVS3j alone showed that it sedimented as a 300 pg/ml unlabeled PVS. Both fractions broad peak with an average sedimentation were subjected to velocity sedimentation coefficient of about 12-14s (Fig. 2). analysis. As shown previously (5), the RSA In order to demonstrate more conclusively is found at the top of the gradient while the free protein sediments at about 4s. In t,he the existence of a PVS35-protein complex (PVWP), the appropriate fractions from presence of PVS, however, the protein sedithe 3% region of the gradient (Fig. lb) were mented in a broad band in the center of the pooled and chromatographed on Sephadex gradient (18-30s). Clearly, the formation of G-50 columns to remove the glycerol so that an orderly helical PVS-P complex requires the material could be resedimented. Pronase that the PVS replace the RNA in an intact (1 mg/ml) was added to part of the material structure. recovered from the Sephadex chromatogThe reaction of PVS with the RSI’ of in-
SHORT COMMUNICATIONS
384
RNP alone, it does appear to have some specificity for the influenza virus RNP. The RNP of SV5 is not affected by PVS (unpublished work done in collaboration with Dr. R. Compans, The Rockefeller University), nor is the RNP of vesicular stomatitis virus af-
fected by PVS (Obejeski and Simpson, personal communication). REFERENCES
CPM 0.1 ml
IO FRACTION
20 NO.
FIG. 3. 38s PVS35-P was isolated from the glycerol gradient depicted in Fig. lb. The PVSSS-P was freed of glycerol on a Sephadex G-50 column equilibrated with STE buffer containing 1 M urea. (a) An aliquot of this material was recentrifuged through another glycerol gradient as described in Fig. 1. (b) An aliquot of this material was treated with 1 mg/ml pronase for 2 hours at 37” prior to recentrifugation. (The pronase had been preincubated for 2 hours at 37” to destroy RNase activity.) O-0, 3H; X-----X, 32P; A.-.-A, ‘5s.
fluenza virus may serve as a useful tool in identification of this material in the infected cell. Although the PVS does complex lvith other viral proteins and is not specific for the
1. FELLIG, J., and WILEY, C. W., Arch. Biochem. Biophys. 85.313-316 (1959). 2. SHINOZAWA, T., YAHARA, I., and IMAHORI, K., J. Mol. Biol. 36,305-319 (1968). 3. CAME, P. E., LIEBERMAN, M., PASCALE, A., and SHIMONASKI, G., Proc. Sot. Exp. Biol. Med. 131, 443-446 (1969). 4. SCHOLTISSEK, C., and ROTT, R., J. Gen. Viral. 4, 125-137 (1969). 5. PONS, M. W., SCHULZE, I. T., and HIRST, G. K., Virology 39,250-259 (1969). 6. SIMPSON, R. W., and HIRST, G. K., Virology 15.436-451 (1961). 7. PONS, M. W., Virology 31, 523-531 (1967). 8. PONS, M. W., and HIRST, G. K., Virology 34, 386-388 (1968). 9. BERNFELD, J. S., ?JISSELBAUM, J. S., BERKELEY, B. J., and HANSON, R. W., J. Biol. Chem. 235, 2852-2859 (1966). IO. SCHULZE, I. T., PONS, M. W., and HIRST, of Large RNA G. Ii., In “The Biology Viruses” (R. D. Barry and B. W. J. Mahy, eds.), Academic Press, New York, 1970 (in press). ELLEN A. GOLDSTE& MARCEL W. PONS Department of Virology The Public Health Research Insfitute of the City of New York, Inc. New York, New York 10016 Accepted March 30, 1970 2 Submitted in partial fulfillment of the requirements for the degree of Master of Science at New York University.
41, 382-384 (1970)
The Effect of Polyvinylsulfate
on the Ribonucleoprotein
of Influenza
Virus’
samples were collected and aliquots counted Polyvinylsulfate (PVS), a negative11 charged polymer, has been shown to be a in a dioxane base cocktail. The appropriate competitive inhibitor of RKase (1) and is corrections for spillover of counts into other also capable of replacing messenger RXA on channels mere made in the usual way. Polyvinylsulfate labeled with 3% was preribosomes (2). Came et al. (3) reported that PVS inhibits the replication of influenza vi- pared by the method of Bernfeld et al. (9). rus both in vivo and in vitro. Scholtissek and Chlorosulfanic acid-35S (5.3 mCi) was obtained from the Amersham/Searle CorporaRott (4) have shown that PVS and another tion (sp. act. 39 mCi/mM). Polyvinyl alcopolyanion, dextran sulfate, inhibit the RKAhol was obtained from Matheson Coleman dependent RN,4 polymerase obtained from & Bell. The yield of PVS35 was too low to fowl plaque virus-infected cells but not that obtained from the cells infected with Ken-- enable accurate determination of the specific activity of the compound. In most cases, castle disease virus. however, the virus was treated with approxiRecently, a report from this laboratory mately 20.0 pg/ml PVS3j (581,000 cpm/ml suggested that PVS completely displaced RKA from the ribonucleoprotein (RSP) of final concentration). Two preparations of influenza virus, one influenza virus (5). Electron microscopy and velocity gradient analyses showed that the labeled with 3H-amino acids, the other with 32P04 , were mixed in equal amounts (2 X morphological and sedimentation characterlo5 HA units total) and treated with nonidet istics of the intact and PVS-treated RKP were identical, indicating that PVS may P-40 (NP40), a nonionic detergent, (final concentration 1%) in standard buffer (0.1 ~11 have replaced the RNA in the RNP complex NaCl, lop3 J/ EDTA in 0.01 M Tris-HCl in such a way as to preserve the helical pH 7.2 containing 1 M urea). Velocity gradistructure. The present report demonstrates conclusively that PVS (labeled with 36S)does ent sedimentation analysis of the mixed preparation showed that RNA (labeled with indeed replace the RNA on the RKP helix. Influenza virus (WSN strain) was pre- 32P) and protein (labeled with 3H) cosedimented at 3% (Fig. la). It has been shown pared in chick fibroblast monolayers (CFAI) already that the 38s material is the RNP (6, 7). The growth and purification of virus labeled with 32P04 and 3H-amino acids were and that the HA and neuraminidase proteins sediment in a broad peak in the center of the performed as previously described (5, 8). Velocity gradient sedimentation analyses gradient’ with phospholipids at the t,op (5, 10). were carried out on 30-ml linear glycerol Treatment of a double labeled preparation gradients (lo-70 % w/w) containing 0.01 :II Tris-HCl pH 7.2, 0.3 M SaCl, 1 dl urea, with NP40, followed immediately by treatand 1O-3 111EDTA. Samples n-ere layered on ment with approximately 20 pg PVS3j altered gradients through a 5-ml layer of mineral oil the sedimentation profiles as shown in Fig. lb. PVS3” and protein (labeled with “Hand were centrifuged in a Spinco Model L2-65B using a SW-27 rotor, at 25,000 rpm amino acids) cosedimented at 38s. The for 17 hours at 20”. The bottoms of the tubes 32P-labeled RKA sedimented at 2OS, which is the sedimentation value obtained for phenolwere punctured with a needle, and l-ml SDS extracted influenza virus RNA (‘i”). 1 This investigation was supported in part by There is coincidence between 35S-labeled PVS Grant AI-04360 of the National Institute of and other proteins in the gradient, indicating Allergy and Infectious Diseases, U.S. Public that the polymer can associate with other Health Service. viral proteins. Velocity sedimentation of 382
SHORT COMMUNICATIONS
b.
383
IO FRACTION
20 NO.
FIG. 2. Polyvinylsulfate labeled with % was centrifuged through a glycerol gradient as described in Fig. 1. A.-.-A, 32P.
raphy, and the treated and untreated fractions were incubated
at 37” for 2 hours prior
to resedimentation through glycerol gradients. Figure 3a shows that in the untreated material the PVS35 and protein resedimented at 38s while small amounts of RNA and FRACTION NO. PVS35 sedimented at 18-21s and Ids, reFIG. 1. (a) Purified WSN, labeled with 3H- spectively. Figure 3b shows that after pronase treatment the digested protein was at amino acids and 32POd,was treated with l”/b nonithe top of the gradient while the PVS3j sedidet in the presence of 1 M urea. (b) Purified doubly labeled WSN treated with lo/ nonidet followed by mented at about 14s. This is the sedimenta20 @g/ml polyvinylsulfate, labeled with %, in the tion coefficient of free PVS3j. These results presence of 1 dl urea.. Both preparations were show that no particular size of PVS3” is relayered over lo-70y0 glycerol gradients and cen- quired for this reaction. They also show that trifuged for 17 hours at 20” in the SW-27 rotor the PVW had complexed with the nrot,ein of 25,000 rpm of a Spinco Model L2-65B centrifuge. the RNP. One-milliliter fractions were collected, and O.OlThe following experiment was carried out ml aliquots were counted in a liquid scintillation to determine how PVS reacts with the disascounter. Appropriated corrections for spillover of sembled RNP. 32P-3H-labeled RNP was counts were made. O--O, 3H; X-----X, 32P; treated with 10 pg/ml RNase for 10 minutes A.-.-A, 93, at 37”, and half was further treated with PVS3j alone showed that it sedimented as a 300 pg/ml unlabeled PVS. Both fractions broad peak with an average sedimentation were subjected to velocity sedimentation coefficient of about 12-14s (Fig. 2). analysis. As shown previously (5), the RSA In order to demonstrate more conclusively is found at the top of the gradient while the free protein sediments at about 4s. In t,he the existence of a PVS35-protein complex (PVWP), the appropriate fractions from presence of PVS, however, the protein sedithe 3% region of the gradient (Fig. lb) were mented in a broad band in the center of the pooled and chromatographed on Sephadex gradient (18-30s). Clearly, the formation of G-50 columns to remove the glycerol so that an orderly helical PVS-P complex requires the material could be resedimented. Pronase that the PVS replace the RNA in an intact (1 mg/ml) was added to part of the material structure. recovered from the Sephadex chromatogThe reaction of PVS with the RSI’ of in-
SHORT COMMUNICATIONS
384
RNP alone, it does appear to have some specificity for the influenza virus RNP. The RNP of SV5 is not affected by PVS (unpublished work done in collaboration with Dr. R. Compans, The Rockefeller University), nor is the RNP of vesicular stomatitis virus af-
fected by PVS (Obejeski and Simpson, personal communication). REFERENCES
CPM 0.1 ml
IO FRACTION
20 NO.
FIG. 3. 38s PVS35-P was isolated from the glycerol gradient depicted in Fig. lb. The PVSSS-P was freed of glycerol on a Sephadex G-50 column equilibrated with STE buffer containing 1 M urea. (a) An aliquot of this material was recentrifuged through another glycerol gradient as described in Fig. 1. (b) An aliquot of this material was treated with 1 mg/ml pronase for 2 hours at 37” prior to recentrifugation. (The pronase had been preincubated for 2 hours at 37” to destroy RNase activity.) O-0, 3H; X-----X, 32P; A.-.-A, ‘5s.
fluenza virus may serve as a useful tool in identification of this material in the infected cell. Although the PVS does complex lvith other viral proteins and is not specific for the
1. FELLIG, J., and WILEY, C. W., Arch. Biochem. Biophys. 85.313-316 (1959). 2. SHINOZAWA, T., YAHARA, I., and IMAHORI, K., J. Mol. Biol. 36,305-319 (1968). 3. CAME, P. E., LIEBERMAN, M., PASCALE, A., and SHIMONASKI, G., Proc. Sot. Exp. Biol. Med. 131, 443-446 (1969). 4. SCHOLTISSEK, C., and ROTT, R., J. Gen. Viral. 4, 125-137 (1969). 5. PONS, M. W., SCHULZE, I. T., and HIRST, G. K., Virology 39,250-259 (1969). 6. SIMPSON, R. W., and HIRST, G. K., Virology 15.436-451 (1961). 7. PONS, M. W., Virology 31, 523-531 (1967). 8. PONS, M. W., and HIRST, G. K., Virology 34, 386-388 (1968). 9. BERNFELD, J. S., ?JISSELBAUM, J. S., BERKELEY, B. J., and HANSON, R. W., J. Biol. Chem. 235, 2852-2859 (1966). IO. SCHULZE, I. T., PONS, M. W., and HIRST, of Large RNA G. Ii., In “The Biology Viruses” (R. D. Barry and B. W. J. Mahy, eds.), Academic Press, New York, 1970 (in press). ELLEN A. GOLDSTE& MARCEL W. PONS Department of Virology The Public Health Research Insfitute of the City of New York, Inc. New York, New York 10016 Accepted March 30, 1970 2 Submitted in partial fulfillment of the requirements for the degree of Master of Science at New York University.