VIROLOGY
91,493-495 (1978)
Pl is Required
for Initiation of cRNA Synthesis
in WSN Influenza Virus
S. L. MOWSHOWITZ Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029 Accepted August 24, 1978 TslOl, a mutant of WSN influenza virus, has a temperature-sensitive lesion in Pl, a structural protein of the virion. Pl is required for cRNA synthesis, and no detectable cRNA is synthesized by tslO1 in infected cells at the nonpermissive temperature. In the present study, the virion transcriptsse activity of tslO1 is shown to be temperature-sensitive in vitro, but only at low concentrations of the dinucleotide primer ApG, which is required specifically for initiation. At saturating concentrations of ApG, tslO1 was not distinguishable from ts+. It is suggested that Pl is required for initiation of transcription, and normally functions in uivo in the recognition of the putative physiological primer. Inasmuch as Pl and P3 are known to be the only gene products which are required for the synthesis of viral cRNA, it seems likely that P3 functions in the elongation process.
Mature virions of WSN influenza virus contain an RNA-dependent RNA transcriptase (I). The product of the transcriptase reaction in vitro is complementary to the negative-stranded, segmented viral genome (2, 3) and this activity is presumably responsible for viral cRNA synthesis in uiuo (4). Temperature-sensitive mutants of WSN influenza virus fall into at least seven complementation-recombination groups (5-7). For each group, the specific viral protein which bears the temperature-sensitive lesion has been identified (8, 9). Members of two complementation groups, I and III, corresponding to defects in viral proteins P3 and Pl, respectively (8), do not synthesize cRNA in infected cells at the nonpermissive temperature, 39.5” (10). As would be expected, members of group I were shown to be temperature-sensitive in vitro for virion transcriptase activity (II). In vitro temperature sensitivity of the transcriptase could not be demonstrated, however, for members of group III (11). An important property of the virion-associated transcriptase of influenza is its requirement for a primer. It was first shown that guanosine and related compounds stimulated in vitro RNA synthesis several fold, being incorporated into the 5’ end of the cRNA product in the
process (12). Guanosine was, in fact, used as a primer in the studies with temperaturesensitive mutants. Subsequent to those studies, however, it was demonstrated that the guanosine-primed transcriptase reaction yields incomplete transcripts lacking covalently linked poly(A), and does not initiate properly at the 3’ end of the virion RNA template (13). It was also shown that proper initiation and transcription could be obtained with the use of the dinucleotide primers ApG or GpG. If group III mutants were specifically deficient in the initiation of transcription, this deficiency may not have been evident in a guanosine-primed reaction, in which the authentic physiological initiation process is by-passed. Because the dinucleotide-primed reaction presumably resembles the physiological situation more closely (13), it is possible that a lesion which results in defective initiation in uiuo would be demonstrable in vitro if ApG were used as the primer. Wild-type (ts+) and ts mutants of WSN influenza virus were grown in MDCK cells and purified by sucrose gradient centrifugation as described elsewhere (II). First, the ApG dependence of the transcriptase reaction at 28’ (the optimum temperature) was examined. Figure 1 shows that the reaction was highly dependent on ApG. The
493 0042~3822/78/0912-0493$02.00/O Copyright
0 1978 by Academic
Press, Inc.
All rights of reproduction in any form reserved.
494
SHORT COMMUNICATIONS
I
0.2
I
0.4 mM
I 0.6 ApG
I
0.0
FIG. 1. Dependence of the transcriptase reaction on ApG. The reaction mixture contained 50 mM TrisHCl buffer, pH 8.2, 150 mM KCl, 0.5% Triton X-100, 5 m&f dithiotbreitol, 1.5 mlM ATP, 1.0 mJ4 CTP and GTP, 0.2 m&f r3H]UTP (specific activity; 125 aCi/gmoll, 5 mil4 MgC12, -10 cg of viral protein, and ApG as indicated. The total volume of the final mixture was 0.1 ml. After 60 min of incubation at 23’, the reaction was terminated by the addition of 2 ml of cold 10%trichloroacetic acid (TCA). The acid-precipitable counts were collected of Whatman GF/A glass filters and washed with cold 10% TCA on a filtration apparatus. The filters were dried, transferred to toluenebased scintilla&, and counted. Assays were performed in triplicate and the mean of the three determinations is reported. Unincubated samples were used as background controls.
addition of 0.8 mM ApG to the reaction results in at least an 80-fold stimulation of incorporation of [3H]UTP into trichloroacetic acid-precipitable counts. In Fig. 2, the ApG-dependent activity of the transcriptases of ts+ and tslO1 (group III) at 28” and 35” are compared. At 1 n&f ApG, there is a suggestion of relative temperature sensitivity for tslO1. At lower concentrations of ApG, however, the temperature sensitivity of tslO1 becomes readily apparent. Inasmuch as the relative temperature sensitivity of tslO1 is concentrationdependent with respect to ApG, the principal differences between ts+ and tslO1 at elevated temperature appears to be their differential affinities for the primer. At
I
I
1
a l/mM
I
I
3 ApG
4
FIG. 2. Comparison of ApG-dependent RNA synthesis of ts+ and tslO1 at 28” and 35”. Reaction conditions were sin-&u to those described in the legend to Fig. 1, with the following modifications. Virus suspensions in 50 n&f Tris-HCl buffer, pH 8.2, and a separate cocktail containing the rest of the components of the reaction mixture were equilibrated to the indicated temperatures for 2 min before being mixed. Reaction mixtures without ApG served as background controls. 0, ts+; 4 tslO1.
l/ApG = 0 (theoretically infinite concentration of ApG), the viruses are not distinguishable. The most likely interpretation of the results is that the group III gene product (Pl) is required for initiation of cRNA synthesis. Its role in uiuo may be the recognition of the putative “true” physiological primer (20). Considering that only Pl and P3 appear to be involved in the synthesis of cRNA (8), it is reasonable to assume that P3 functions in the elongation process. ACKNOWLEDGMENTS Tamsen Flanders provided excellent technical assistance. This work was supported by Public Health Service Research Grant Al 09304 from the National Institute of Allergy and Infectious Diseases, and by a generous grant from the American Lung Association. REFERENCES 1. BISHOP, D. H. L., OBIJESKI, J. F., and SIMPSON, R. W., J. Viral. 8, 66-73 (1971).
SHORT
COMMUNICATIONS
2. BISHOP, D. H. L., OBIJESKI, J. F., and SIMPSON, R. W., J. Viral. 8,74-80 (1971). 3. BISHOP, D. H. L., ROY, P., BEAN, W. J., JR., and SIMPSON, R. W., J. Viral. 10,689-697 (1972). 4. BEAN, W. J., JR., and SIMPSON, R. W., Virology 66,656-661 (1973). 5. SUGIURA, A., TOBITA, K., and KILBOURNE, E. D., J. Viral. 10,639~647 (1972). 6. UEDA, M., Arch. Ges. Virusforsch. 39, 360-368 (1972). 7. SUGIURA, A., UEDA, M., TOBITA, K., and ENOMOTO, C., Virology 65, 3663-373 (1975).
495
8. PALESE, P., RITCHEY, M. B., and SCHULMAN, J. L., J. Viral. 21, 1187-1195 (1977). 9. RITCHEY, M. B., and PALESE, P., J. Viral. 21, 1196-1204 (1977). 10. KRUG, R. M., UEDA, M., and PALESE, P., J. Viral. 16, 790-796 (1975). 11. MOWSHOWITZ, S. L., and UEDA, M., Arch. Virol. 52, 135-141 (1976). 12. MCGEOGH, D., and KITRON, N., J. Viral. 15, 686-695 (1975). 13. PLOTCH, S. J., and KRUG, R. M., J. Virol. 21, 24-34 (1977).
91,493-495 (1978)
Pl is Required
for Initiation of cRNA Synthesis
in WSN Influenza Virus
S. L. MOWSHOWITZ Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029 Accepted August 24, 1978 TslOl, a mutant of WSN influenza virus, has a temperature-sensitive lesion in Pl, a structural protein of the virion. Pl is required for cRNA synthesis, and no detectable cRNA is synthesized by tslO1 in infected cells at the nonpermissive temperature. In the present study, the virion transcriptsse activity of tslO1 is shown to be temperature-sensitive in vitro, but only at low concentrations of the dinucleotide primer ApG, which is required specifically for initiation. At saturating concentrations of ApG, tslO1 was not distinguishable from ts+. It is suggested that Pl is required for initiation of transcription, and normally functions in uivo in the recognition of the putative physiological primer. Inasmuch as Pl and P3 are known to be the only gene products which are required for the synthesis of viral cRNA, it seems likely that P3 functions in the elongation process.
Mature virions of WSN influenza virus contain an RNA-dependent RNA transcriptase (I). The product of the transcriptase reaction in vitro is complementary to the negative-stranded, segmented viral genome (2, 3) and this activity is presumably responsible for viral cRNA synthesis in uiuo (4). Temperature-sensitive mutants of WSN influenza virus fall into at least seven complementation-recombination groups (5-7). For each group, the specific viral protein which bears the temperature-sensitive lesion has been identified (8, 9). Members of two complementation groups, I and III, corresponding to defects in viral proteins P3 and Pl, respectively (8), do not synthesize cRNA in infected cells at the nonpermissive temperature, 39.5” (10). As would be expected, members of group I were shown to be temperature-sensitive in vitro for virion transcriptase activity (II). In vitro temperature sensitivity of the transcriptase could not be demonstrated, however, for members of group III (11). An important property of the virion-associated transcriptase of influenza is its requirement for a primer. It was first shown that guanosine and related compounds stimulated in vitro RNA synthesis several fold, being incorporated into the 5’ end of the cRNA product in the
process (12). Guanosine was, in fact, used as a primer in the studies with temperaturesensitive mutants. Subsequent to those studies, however, it was demonstrated that the guanosine-primed transcriptase reaction yields incomplete transcripts lacking covalently linked poly(A), and does not initiate properly at the 3’ end of the virion RNA template (13). It was also shown that proper initiation and transcription could be obtained with the use of the dinucleotide primers ApG or GpG. If group III mutants were specifically deficient in the initiation of transcription, this deficiency may not have been evident in a guanosine-primed reaction, in which the authentic physiological initiation process is by-passed. Because the dinucleotide-primed reaction presumably resembles the physiological situation more closely (13), it is possible that a lesion which results in defective initiation in uiuo would be demonstrable in vitro if ApG were used as the primer. Wild-type (ts+) and ts mutants of WSN influenza virus were grown in MDCK cells and purified by sucrose gradient centrifugation as described elsewhere (II). First, the ApG dependence of the transcriptase reaction at 28’ (the optimum temperature) was examined. Figure 1 shows that the reaction was highly dependent on ApG. The
493 0042~3822/78/0912-0493$02.00/O Copyright
0 1978 by Academic
Press, Inc.
All rights of reproduction in any form reserved.
494
SHORT COMMUNICATIONS
I
0.2
I
0.4 mM
I 0.6 ApG
I
0.0
FIG. 1. Dependence of the transcriptase reaction on ApG. The reaction mixture contained 50 mM TrisHCl buffer, pH 8.2, 150 mM KCl, 0.5% Triton X-100, 5 m&f dithiotbreitol, 1.5 mlM ATP, 1.0 mJ4 CTP and GTP, 0.2 m&f r3H]UTP (specific activity; 125 aCi/gmoll, 5 mil4 MgC12, -10 cg of viral protein, and ApG as indicated. The total volume of the final mixture was 0.1 ml. After 60 min of incubation at 23’, the reaction was terminated by the addition of 2 ml of cold 10%trichloroacetic acid (TCA). The acid-precipitable counts were collected of Whatman GF/A glass filters and washed with cold 10% TCA on a filtration apparatus. The filters were dried, transferred to toluenebased scintilla&, and counted. Assays were performed in triplicate and the mean of the three determinations is reported. Unincubated samples were used as background controls.
addition of 0.8 mM ApG to the reaction results in at least an 80-fold stimulation of incorporation of [3H]UTP into trichloroacetic acid-precipitable counts. In Fig. 2, the ApG-dependent activity of the transcriptases of ts+ and tslO1 (group III) at 28” and 35” are compared. At 1 n&f ApG, there is a suggestion of relative temperature sensitivity for tslO1. At lower concentrations of ApG, however, the temperature sensitivity of tslO1 becomes readily apparent. Inasmuch as the relative temperature sensitivity of tslO1 is concentrationdependent with respect to ApG, the principal differences between ts+ and tslO1 at elevated temperature appears to be their differential affinities for the primer. At
I
I
1
a l/mM
I
I
3 ApG
4
FIG. 2. Comparison of ApG-dependent RNA synthesis of ts+ and tslO1 at 28” and 35”. Reaction conditions were sin-&u to those described in the legend to Fig. 1, with the following modifications. Virus suspensions in 50 n&f Tris-HCl buffer, pH 8.2, and a separate cocktail containing the rest of the components of the reaction mixture were equilibrated to the indicated temperatures for 2 min before being mixed. Reaction mixtures without ApG served as background controls. 0, ts+; 4 tslO1.
l/ApG = 0 (theoretically infinite concentration of ApG), the viruses are not distinguishable. The most likely interpretation of the results is that the group III gene product (Pl) is required for initiation of cRNA synthesis. Its role in uiuo may be the recognition of the putative “true” physiological primer (20). Considering that only Pl and P3 appear to be involved in the synthesis of cRNA (8), it is reasonable to assume that P3 functions in the elongation process. ACKNOWLEDGMENTS Tamsen Flanders provided excellent technical assistance. This work was supported by Public Health Service Research Grant Al 09304 from the National Institute of Allergy and Infectious Diseases, and by a generous grant from the American Lung Association. REFERENCES 1. BISHOP, D. H. L., OBIJESKI, J. F., and SIMPSON, R. W., J. Viral. 8, 66-73 (1971).
SHORT
COMMUNICATIONS
2. BISHOP, D. H. L., OBIJESKI, J. F., and SIMPSON, R. W., J. Viral. 8,74-80 (1971). 3. BISHOP, D. H. L., ROY, P., BEAN, W. J., JR., and SIMPSON, R. W., J. Viral. 10,689-697 (1972). 4. BEAN, W. J., JR., and SIMPSON, R. W., Virology 66,656-661 (1973). 5. SUGIURA, A., TOBITA, K., and KILBOURNE, E. D., J. Viral. 10,639~647 (1972). 6. UEDA, M., Arch. Ges. Virusforsch. 39, 360-368 (1972). 7. SUGIURA, A., UEDA, M., TOBITA, K., and ENOMOTO, C., Virology 65, 3663-373 (1975).
495
8. PALESE, P., RITCHEY, M. B., and SCHULMAN, J. L., J. Viral. 21, 1187-1195 (1977). 9. RITCHEY, M. B., and PALESE, P., J. Viral. 21, 1196-1204 (1977). 10. KRUG, R. M., UEDA, M., and PALESE, P., J. Viral. 16, 790-796 (1975). 11. MOWSHOWITZ, S. L., and UEDA, M., Arch. Virol. 52, 135-141 (1976). 12. MCGEOGH, D., and KITRON, N., J. Viral. 15, 686-695 (1975). 13. PLOTCH, S. J., and KRUG, R. M., J. Virol. 21, 24-34 (1977).
