JOURNAL OF VIROLOGY, Oct. 1978, p. 411-414 0022-538X/78/0028-0411$02.00/0 Copyright © 1978 American Society for Microbiology
Vc0l. 28, No. 1
Printeddin U.S.A.
Identification of Sendai Virus mRNA Species K. JONES, C. PRIDGEN, AND D. W. KINGSBURY* Division of Virology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101
Received for publication 5 June 1978
Cell-free translation of separated Sendai virus mRNA species identified the message for polypeptide M and suggested the identity of the message for polypeptide NP.
Glazier et al. (8) used UV irradiation of in10 fected cells to demonstrate polar transcription of the genome of the paramyxovirus Sendai virus from a single starting point. Similar findings have been made with other paramyxoviruses (L. A. Ball, P. C. Collins, and L. E. Hightower, in B. 10.0 W. J. Mahy and R. D. Barry, ed., Negative Strand Viruses and the Host Cell, in press; W. W. Hall, W. R. Kiessling, and V. ter Meulen, in B. W. J. Mahy and R. D. Barry, ed., Negative Strand Viruses and the Host Cell, in press) and 75 with another model negative-strand virus, vesicular stomatitis virus (1, 2, 3). These data perClT LC) mitted deductions about the order of genes in (-) the viral genomes; in the case of Sendai virus, the gene order deduced was 3'-NP-Fo-M-P-HN50 L-5' (8). However, the Sendai virus gene assignments were made indirectly, by relating the apparent molecular weights of electrophoretically separated virus-specific RNA species to the apparent molecular weights of the viral polypeptides. The present work was undertaken to 25 identify the viral mRNA species directly, by translating them in a cell-free system. Sendai virus-specific RNA labeled with [3H]uridine in the presence of actinomycin D was extracted from infected chicken embyro lung 15 cells, and polyadenylic acid-rich species were 10 selected by oligodeoxythymidylic acid-cellulose FRACTION chromatography (8) and centrifuged in a sucrose gradient (Fig. 1). Although the profile of radioFIG. 1. Sucrose gradient centrifugation of Sendai activity across the gradient appeared to provide virus mRNA species. Polyadenylic acid-rich [3H]urno separation of RNA species, samples taken idine-labeled RNA from oligodeoxythymidylic acidfrom adjacent fractions at the main peak of cellulose chromatography (8) was concentrated by radioactive RNA had different compositions of ethanolprecipitation, dissolved in 0.01 M Tris-hydroRNA species, as revealed by electrophoresis in chloride-0.1 M NaCI-0.001 M EDTA-0.5% sodium formamide-containing polyacrylamide gels (Fig. dodecyl sulfate (pH 7.4), and layered on a linear 15 2). RNA component 1, the most mobile species to 30% sucrose gradient made up in the same solution. in electrophoresis, was nominated previously as After centrifugation for 17 h at 33,000 rpm and 20°C in a Spinco SW41 rotor, 0.5-ml fractions were samthe message for the 34,000-dalton virus poly- pled for acid-insoluble radioactivity ( ), and their peptide M on the basis of its apparent molecular UV absorbance at 254 nm (A2) -) was measured. weight (8). Component 1 was the major RNA The direction of sedimentation (-is from left to right. species in fraction 14 of the sucrose gradient The arrows designate the fractions (14 to 16) exam(Fig. 2a). There was also some heterogeneous ined by gel electrophoresis (see Fig. 2). 411
412
J. VIROL.
NOTES
4 2
a
b
1 (Fig. 2a), programmed the synthesis of a polypeptide that migrated like polypeptide M in the discontinuous polyacrylamide gel system of Laemmli (10) (Fig. 3b). A small amount of a polypeptide that migrated like NP was also made. At the other extreme, a large amount of NP was produced in extracts that received fraction 16, which contained mainly RNA components 2 and 4 (Fig. 2c), but only a scant amount of putative M was made (Fig. 3d). The peak gradient fraction, 15, which contained the major RNA components in a more equivalent mixture (Fig. 2b), directed the synthesis of both polypeptides in roughly similar amounts (Fig. 3c). RNA that sedimented more slowly or more rapidly than the peak fractions yielded cell-free products like those shown in Fig. 3b and d, respectively. Endogenous incorporation of [35S]methionine into
proteins was negligible (Fig. 3e). Peptide maps of the polypeptides made in vitro confirmed their homologies with the authentic M and NP P FoNP F
FIG. 2. Electrophoresis of Sendai virus mRNA species. Fractions from the sucrose gradient (see Fig. 1) were subjected to electrophoresis in formamidecontaining polyacrylamide gels, and radioactive components were detected by autoradiography (8). Shown are densitometer scans of the autoradiograms. RNA components are numbered in order of decreasing electrophoretic mobility (8). (a) Fraction 14; (b) fraction 15; (c) fraction 16.
material of higher molecular weight in this fraction in the positions of viral RNA components 2 and 4 (Fig. 2a). Component 2 was thought to represent the message for nucleocapsid polypeptide NP, the major virus gene product, whereas component 4 was thought to contain a mixture of mRNA species specifying glycopolypeptide Fo and nucleocapsid polypeptide P (8). Both of these components were more abundant than was component 1 in peak fraction 15 (Fig. 2b) and in fraction 16 (Fig. 2c), with component 4 relatively enriched in the latter case. RNA component 3, which may represent' the message for glycopolypeptide HN (8), was not visualized in the present work. Gradient fractions 14 to 16 were tested sepa-
rately for template activity in a cell-free proteinsynthesizing system from rabbit reticulocytes. Endogenous mRNA species in the extract were destroyed by pretreatment with micrococcal nuclease (11). Fraction 14, rich in RNA component
M
b
l
d e
FIG. 3. Polypeptide synthesis directed by Sendai virus mRNA species. Shown are densitometer scans of autoradiograms from electrophoretic separations (1) of [35S]methionine-labeled products from a reticulocyte lysate (11). Migration is depicted from left to right. Polypeptides are designated as described by Scheid and Choppin (12). (a) Sample of radioactive Sendai virions for comparison; glycopolypeptide HN
was not resolved in this sample; (b) product directed by RNA in sucrose gradient fraction 14; (c) product directed by fraction 15; (d) product directed by fraction 16; (e) endogenous product.
I
I
VOL. 28, 1978
. =, 0w
NOTES
polypeptides from virions (Fig. 4). Although we recognize that complete purification of each mRNA species will be necessary for a rigorous identification, we conclude from the present data that RNA component 1 is the message for M, because M was synthesized in proportion to the representation of component 1 in the sucrose gradient fractions tested. The data also assign the message for NP to the remaining pair of major RNA components, but do not decide whether this message resides in component 2 or component 4. Component 2 remains the more likely choice, based on its apparent molecular weight under denaturing conditions, its abundance in extracts from in-
I
2
3
413
fected cells, and the sensitivity of its synthesis to UV irradiation (8). As they are consistent with the data and deductions that generated the genetic map of Sendai virus, the present data support the validity of that map. Small amounts of other polypeptides were also made by the cell-free system, such as one with an electrophoretic mobility corresponding to virus polypeptide F (a cleavage product of Fo; 12) and several others with mobilities greater than that of polypeptide M (Fig. 3b, c, and d). Quantities of these products were insufficient for further analysis, but they are probably cellular polypeptides made under the direction of small amounts of cellular messages which were not
4
5
6
:.m
w.,:w;
IMM
..Mas A*
., e
.5" '
4
.rt->... b-,,>
., :.:
_
*_w
''
n_
_s
FIG. 4. Peptide maps by the method of Cleveland et al. (4) ofpolypeptides from Sendai virions and of cellfree translation products. [3S]methionine-labeled polypeptides were eluted from polyacrylamide gels into 0.125 M Tris-hydrochloride-0.001 M EDTA-0.1% sodium dodecyl sulfate (pH 6.8) and treated with proteolytic enzymes for 15 min at 24°C. Digestions were stopped by diluting samples in electrophoresis sample buffer and boiling them for 2 min. Samples were then subjected to electrophoresis in 15% polyacrylamide gels (4, 10). The direction of migration is from top to bottom. The proteases used and their final concentrations were chymotrypsin (10 pg/ml) for lanes 1 and 2 and staphylococcal protease (200 pg/ml) for lanes 3 to 6. The polypeptides examined were: lane 1, M from virions; lane 2, M made in vitro; lane 3, M from virions; lane 4, M made in vitro; lane 5, NP made in vitro; and lane 6, NP from virions.
414
NOTES
J. VIROL.
labeled in the presence of actinomycin D, but explore its implications for the regulation of viral which were not excluded by our procedure for mRNA transcription (8). preparing viral mRNA. In contrast to previous Betty Ann Lyne provided skillful technical assistance, and results (7, 9), we observed little, if any, synthesis we are grateful to Robert A. Lamb for advice on peptide of a polypeptide that qualified as the large viral mapping. This work was supported by Public Health Service research polypeptide, P (Fig. 3d). In the present case, the grant AI 05343 from the National Institute of Allergy and message for P might have been damaged by Infectious Diseases, by grant RG 1142 from the National nuclease or inadvertently removed by one of our Multiple Sclerosis Society, and by ALSAC. manipulations. There is no reason to believe LITERATURE CITED that the reticulocyte extract discriminates 1. Abraham, G., and A. K. Banerjee. 1976. Sequential against the translation of the P message (9). transcription of the genes of vesicular stomatitis virus. Proc. Natl. Acad. Sci. U.S.A. 73:1504-1508. However, the message for Fo might well be presL. A. 1977. Transcriptional mapping of vesicular ent in RNA component 4, because we have not 2. Ball, stomatis virus in vivo. J. Virol. 21:411-414. previously been able to detect synthesis of either 3. Ball, L. A., and C. N. White. 1976. Order of transcription viral glycopolypeptide when unfractionated viof genes of vesicular stomatitis virus. Proc. Natl. Acad. Sci. U.S.A. 73:442-446. rus 18S mRNA species were tested in memD. W., S. G. Fischer, M. W. Kirschner, brane-free cell-free systems (7, 9). In contrast, 4. Cleveland, and U. K. Laemmli. 1977. Peptide mapping by limited Clinkscales et al. (5) have been able to measure proteolysis in sodium dodecyl sulfate and analysis by the synthesis of all the major Newcastle disease gel electrophoresis. J. Biol. Chem. 252:1102-1106. 5. Clinkscales, C. W., M. A. Bratt, and T. G. Morrison. virus polypeptides in vitro. 1977. Synthesis of Newcastle disease virus polypeptides The ease of recovery of RNA species from in a wheat germ cell-free system. J. Virol. 22:97-101. sucrose gradients makes it an attractive prepar- 6. Collins, B. S., and M. A. Bratt. 1973. Separation of the ative procedure, and our results demonstrate messenger RNAs of Newcastle disease virus by gel electrophoresis. Proc. Natl. Acad. Sci. U.S.A. that the mRNA that specifies Sendai virus poly70:2544-2548. peptide M can easily be isolated for further 7. Davies, J. W., A. Portner, and D. W. Kingsbury. 1976. study in this manner. Despite the fortuitous Synthesis of Sendai virus polypeptides by a cell-free appearance of the separation, because of the extract from wheat germ. J. Gen. Virol. 33:117-123. sharpness of the peak of radioactive viral mes- 8. Glazier, K., R. Raghow, and D. W. Kingsbury. 1977. Regulation of Sendai virus transcription: evidence for a senger RNA species in the gradient (Fig. 1), we single promoter in vivo. J. Virol. 21:863-871. have been able to duplicate the results shown in 9. Kingsbury, D. W. 1973. Cell-free translation of paraFig. 2 without difficulty. Indeed, comparable myxovirus messenger RNA. J. Virol. 12:1020-1027. separations of Newcastle disease virus mRNA 10. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. species were reported earlier by Coffins and Nature (London) 227:680-685. Bratt (6). However, we could not resolve the 11. Pelham, H. R. B., and R. J. Jackson. 1976. An efficient larger Sendai virus mRNA species very well by mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67:247-256. sucrose gradient centrifugation, and other techA., and P. W. Choppin. 1974. Identification of niques, such as preparative gel electrophoresis, 12. Scheid, the biological activities of paramyxovirus glycoproteins. will probably be required to separate them. We Activation of cell fusion, hemolysis, and infectivity by are continuing efforts like these to test the validproteolytic cleavage of an inactive precursor protein of Sendai virus. Virology 57:475-490. ity of our genetic map of Sendai virus and to
Vc0l. 28, No. 1
Printeddin U.S.A.
Identification of Sendai Virus mRNA Species K. JONES, C. PRIDGEN, AND D. W. KINGSBURY* Division of Virology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101
Received for publication 5 June 1978
Cell-free translation of separated Sendai virus mRNA species identified the message for polypeptide M and suggested the identity of the message for polypeptide NP.
Glazier et al. (8) used UV irradiation of in10 fected cells to demonstrate polar transcription of the genome of the paramyxovirus Sendai virus from a single starting point. Similar findings have been made with other paramyxoviruses (L. A. Ball, P. C. Collins, and L. E. Hightower, in B. 10.0 W. J. Mahy and R. D. Barry, ed., Negative Strand Viruses and the Host Cell, in press; W. W. Hall, W. R. Kiessling, and V. ter Meulen, in B. W. J. Mahy and R. D. Barry, ed., Negative Strand Viruses and the Host Cell, in press) and 75 with another model negative-strand virus, vesicular stomatitis virus (1, 2, 3). These data perClT LC) mitted deductions about the order of genes in (-) the viral genomes; in the case of Sendai virus, the gene order deduced was 3'-NP-Fo-M-P-HN50 L-5' (8). However, the Sendai virus gene assignments were made indirectly, by relating the apparent molecular weights of electrophoretically separated virus-specific RNA species to the apparent molecular weights of the viral polypeptides. The present work was undertaken to 25 identify the viral mRNA species directly, by translating them in a cell-free system. Sendai virus-specific RNA labeled with [3H]uridine in the presence of actinomycin D was extracted from infected chicken embyro lung 15 cells, and polyadenylic acid-rich species were 10 selected by oligodeoxythymidylic acid-cellulose FRACTION chromatography (8) and centrifuged in a sucrose gradient (Fig. 1). Although the profile of radioFIG. 1. Sucrose gradient centrifugation of Sendai activity across the gradient appeared to provide virus mRNA species. Polyadenylic acid-rich [3H]urno separation of RNA species, samples taken idine-labeled RNA from oligodeoxythymidylic acidfrom adjacent fractions at the main peak of cellulose chromatography (8) was concentrated by radioactive RNA had different compositions of ethanolprecipitation, dissolved in 0.01 M Tris-hydroRNA species, as revealed by electrophoresis in chloride-0.1 M NaCI-0.001 M EDTA-0.5% sodium formamide-containing polyacrylamide gels (Fig. dodecyl sulfate (pH 7.4), and layered on a linear 15 2). RNA component 1, the most mobile species to 30% sucrose gradient made up in the same solution. in electrophoresis, was nominated previously as After centrifugation for 17 h at 33,000 rpm and 20°C in a Spinco SW41 rotor, 0.5-ml fractions were samthe message for the 34,000-dalton virus poly- pled for acid-insoluble radioactivity ( ), and their peptide M on the basis of its apparent molecular UV absorbance at 254 nm (A2) -) was measured. weight (8). Component 1 was the major RNA The direction of sedimentation (-is from left to right. species in fraction 14 of the sucrose gradient The arrows designate the fractions (14 to 16) exam(Fig. 2a). There was also some heterogeneous ined by gel electrophoresis (see Fig. 2). 411
412
J. VIROL.
NOTES
4 2
a
b
1 (Fig. 2a), programmed the synthesis of a polypeptide that migrated like polypeptide M in the discontinuous polyacrylamide gel system of Laemmli (10) (Fig. 3b). A small amount of a polypeptide that migrated like NP was also made. At the other extreme, a large amount of NP was produced in extracts that received fraction 16, which contained mainly RNA components 2 and 4 (Fig. 2c), but only a scant amount of putative M was made (Fig. 3d). The peak gradient fraction, 15, which contained the major RNA components in a more equivalent mixture (Fig. 2b), directed the synthesis of both polypeptides in roughly similar amounts (Fig. 3c). RNA that sedimented more slowly or more rapidly than the peak fractions yielded cell-free products like those shown in Fig. 3b and d, respectively. Endogenous incorporation of [35S]methionine into
proteins was negligible (Fig. 3e). Peptide maps of the polypeptides made in vitro confirmed their homologies with the authentic M and NP P FoNP F
FIG. 2. Electrophoresis of Sendai virus mRNA species. Fractions from the sucrose gradient (see Fig. 1) were subjected to electrophoresis in formamidecontaining polyacrylamide gels, and radioactive components were detected by autoradiography (8). Shown are densitometer scans of the autoradiograms. RNA components are numbered in order of decreasing electrophoretic mobility (8). (a) Fraction 14; (b) fraction 15; (c) fraction 16.
material of higher molecular weight in this fraction in the positions of viral RNA components 2 and 4 (Fig. 2a). Component 2 was thought to represent the message for nucleocapsid polypeptide NP, the major virus gene product, whereas component 4 was thought to contain a mixture of mRNA species specifying glycopolypeptide Fo and nucleocapsid polypeptide P (8). Both of these components were more abundant than was component 1 in peak fraction 15 (Fig. 2b) and in fraction 16 (Fig. 2c), with component 4 relatively enriched in the latter case. RNA component 3, which may represent' the message for glycopolypeptide HN (8), was not visualized in the present work. Gradient fractions 14 to 16 were tested sepa-
rately for template activity in a cell-free proteinsynthesizing system from rabbit reticulocytes. Endogenous mRNA species in the extract were destroyed by pretreatment with micrococcal nuclease (11). Fraction 14, rich in RNA component
M
b
l
d e
FIG. 3. Polypeptide synthesis directed by Sendai virus mRNA species. Shown are densitometer scans of autoradiograms from electrophoretic separations (1) of [35S]methionine-labeled products from a reticulocyte lysate (11). Migration is depicted from left to right. Polypeptides are designated as described by Scheid and Choppin (12). (a) Sample of radioactive Sendai virions for comparison; glycopolypeptide HN
was not resolved in this sample; (b) product directed by RNA in sucrose gradient fraction 14; (c) product directed by fraction 15; (d) product directed by fraction 16; (e) endogenous product.
I
I
VOL. 28, 1978
. =, 0w
NOTES
polypeptides from virions (Fig. 4). Although we recognize that complete purification of each mRNA species will be necessary for a rigorous identification, we conclude from the present data that RNA component 1 is the message for M, because M was synthesized in proportion to the representation of component 1 in the sucrose gradient fractions tested. The data also assign the message for NP to the remaining pair of major RNA components, but do not decide whether this message resides in component 2 or component 4. Component 2 remains the more likely choice, based on its apparent molecular weight under denaturing conditions, its abundance in extracts from in-
I
2
3
413
fected cells, and the sensitivity of its synthesis to UV irradiation (8). As they are consistent with the data and deductions that generated the genetic map of Sendai virus, the present data support the validity of that map. Small amounts of other polypeptides were also made by the cell-free system, such as one with an electrophoretic mobility corresponding to virus polypeptide F (a cleavage product of Fo; 12) and several others with mobilities greater than that of polypeptide M (Fig. 3b, c, and d). Quantities of these products were insufficient for further analysis, but they are probably cellular polypeptides made under the direction of small amounts of cellular messages which were not
4
5
6
:.m
w.,:w;
IMM
..Mas A*
., e
.5" '
4
.rt->... b-,,>
., :.:
_
*_w
''
n_
_s
FIG. 4. Peptide maps by the method of Cleveland et al. (4) ofpolypeptides from Sendai virions and of cellfree translation products. [3S]methionine-labeled polypeptides were eluted from polyacrylamide gels into 0.125 M Tris-hydrochloride-0.001 M EDTA-0.1% sodium dodecyl sulfate (pH 6.8) and treated with proteolytic enzymes for 15 min at 24°C. Digestions were stopped by diluting samples in electrophoresis sample buffer and boiling them for 2 min. Samples were then subjected to electrophoresis in 15% polyacrylamide gels (4, 10). The direction of migration is from top to bottom. The proteases used and their final concentrations were chymotrypsin (10 pg/ml) for lanes 1 and 2 and staphylococcal protease (200 pg/ml) for lanes 3 to 6. The polypeptides examined were: lane 1, M from virions; lane 2, M made in vitro; lane 3, M from virions; lane 4, M made in vitro; lane 5, NP made in vitro; and lane 6, NP from virions.
414
NOTES
J. VIROL.
labeled in the presence of actinomycin D, but explore its implications for the regulation of viral which were not excluded by our procedure for mRNA transcription (8). preparing viral mRNA. In contrast to previous Betty Ann Lyne provided skillful technical assistance, and results (7, 9), we observed little, if any, synthesis we are grateful to Robert A. Lamb for advice on peptide of a polypeptide that qualified as the large viral mapping. This work was supported by Public Health Service research polypeptide, P (Fig. 3d). In the present case, the grant AI 05343 from the National Institute of Allergy and message for P might have been damaged by Infectious Diseases, by grant RG 1142 from the National nuclease or inadvertently removed by one of our Multiple Sclerosis Society, and by ALSAC. manipulations. There is no reason to believe LITERATURE CITED that the reticulocyte extract discriminates 1. Abraham, G., and A. K. Banerjee. 1976. Sequential against the translation of the P message (9). transcription of the genes of vesicular stomatitis virus. Proc. Natl. Acad. Sci. U.S.A. 73:1504-1508. However, the message for Fo might well be presL. A. 1977. Transcriptional mapping of vesicular ent in RNA component 4, because we have not 2. Ball, stomatis virus in vivo. J. Virol. 21:411-414. previously been able to detect synthesis of either 3. Ball, L. A., and C. N. White. 1976. Order of transcription viral glycopolypeptide when unfractionated viof genes of vesicular stomatitis virus. Proc. Natl. Acad. Sci. U.S.A. 73:442-446. rus 18S mRNA species were tested in memD. W., S. G. Fischer, M. W. Kirschner, brane-free cell-free systems (7, 9). In contrast, 4. Cleveland, and U. K. Laemmli. 1977. Peptide mapping by limited Clinkscales et al. (5) have been able to measure proteolysis in sodium dodecyl sulfate and analysis by the synthesis of all the major Newcastle disease gel electrophoresis. J. Biol. Chem. 252:1102-1106. 5. Clinkscales, C. W., M. A. Bratt, and T. G. Morrison. virus polypeptides in vitro. 1977. Synthesis of Newcastle disease virus polypeptides The ease of recovery of RNA species from in a wheat germ cell-free system. J. Virol. 22:97-101. sucrose gradients makes it an attractive prepar- 6. Collins, B. S., and M. A. Bratt. 1973. Separation of the ative procedure, and our results demonstrate messenger RNAs of Newcastle disease virus by gel electrophoresis. Proc. Natl. Acad. Sci. U.S.A. that the mRNA that specifies Sendai virus poly70:2544-2548. peptide M can easily be isolated for further 7. Davies, J. W., A. Portner, and D. W. Kingsbury. 1976. study in this manner. Despite the fortuitous Synthesis of Sendai virus polypeptides by a cell-free appearance of the separation, because of the extract from wheat germ. J. Gen. Virol. 33:117-123. sharpness of the peak of radioactive viral mes- 8. Glazier, K., R. Raghow, and D. W. Kingsbury. 1977. Regulation of Sendai virus transcription: evidence for a senger RNA species in the gradient (Fig. 1), we single promoter in vivo. J. Virol. 21:863-871. have been able to duplicate the results shown in 9. Kingsbury, D. W. 1973. Cell-free translation of paraFig. 2 without difficulty. Indeed, comparable myxovirus messenger RNA. J. Virol. 12:1020-1027. separations of Newcastle disease virus mRNA 10. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. species were reported earlier by Coffins and Nature (London) 227:680-685. Bratt (6). However, we could not resolve the 11. Pelham, H. R. B., and R. J. Jackson. 1976. An efficient larger Sendai virus mRNA species very well by mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67:247-256. sucrose gradient centrifugation, and other techA., and P. W. Choppin. 1974. Identification of niques, such as preparative gel electrophoresis, 12. Scheid, the biological activities of paramyxovirus glycoproteins. will probably be required to separate them. We Activation of cell fusion, hemolysis, and infectivity by are continuing efforts like these to test the validproteolytic cleavage of an inactive precursor protein of Sendai virus. Virology 57:475-490. ity of our genetic map of Sendai virus and to
