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The gene organisation of a small RNA-containing insect virus: Comparison with that of mammalian picornaviruses
VIHOI.0C.Y131, 551-554 (19k3)
The Gene Organisation Comparison
of a Small RNA-Containing
with That of Mammalian
BRIAN REAVY’
AND NORMAN
Insect
Virus:
Picomaviruses F. MOORE”
The coding regions of an insect virus, cricket paralysis virus, have hesn mapped using pactamyrin. The results suggest that. the genome of this virus functions as a polycistronic mKNA, the structural proteins being encoded by the 5’end of the RNA in an order similar to those of mammalian picornaviruses. High-molecular-weight proteins of unknown function map at the 3’ end of the genome.
A large number of small RNA-containing viruses have been isolated from insects (I). Two of these viruses, cricket, paralysis virus (CrPV) and Lh-osophila C virus (UCV), undergo replicative events in Drosophila wwlunoyuskr tissue culture cells reminiscent of those found with mammalian picornaviruses (2). While CrPV has many characteristics in common with the enterovirus genus of the PticrrnuviW it has been excluded from that genus on the basis of the low GC content in its RNA (S, 4). The genome of picornaviruses is a single piece of single-stranded RNA which functions as a mRNA. Initiation of protein synthesis occurs at one site near the 5’ end of the genome and functional proteins are produced by post-translational cleavages of a polypeptide which represents the complete translation product of the RNA (5). This mode of protein synthesis has made picornaviruses amenable to gene mapping by the use of inhibitors of the initiation step of protein synlhesis. An inhibitor of initiation will lead to an apparent increase in the synthesis of proteins which are encoded by regions furthest from the initiation site, as ribosomes which have initiated protein synthesis will continue synthesising until the end of the RNA. Thus,
the amount of synthesis of a protein after inhibition of initiation is proportional to the distance of its coding region from the initiation site. The coding regions on the genomes of a number of picornaviruses have been mapped using pactamyein to inhibit initiation and a common gene order has been found (G-K?). We have mapped the order of the coding regions on the RNA of CrPV and have determined that the gene organisation of CrPV is similar to that of mammalian picornaviruses. Optimum condit.ions for the USCof pactamycin (supplied by Upjohn Co.) were determined by the incorporation of ?%Imethionine into TCA-prccipitablc material by CrPV-infected Drosophila eells. Pactamycin (1O--6 M) caused a marked inhibition of protein synthesis while 10e7 M caused only a slight decrease and the cells were totally unaffected by lo-’ M pactamycin. Since high levels of paetamycin inhibit elongation the drug was used at a concentration of 5 X 1O-‘7 M to map the CrPV-specified proteins. The differential effect of pactamycin on the synthesis of CrPV-specified proteins is shown in Fig. 1. In the absence of pactamycin the major proteins synthesised were the capsid proteins VP1 and VP3 with lesser amounts of VP0 (the precursor of the third capsid protein, VP‘4 (14,15) and the two comigrating proteins, E and F. There were even smaller amounts of higher-molecular-weight proteins: protein A, the comigrating proteins
’ Present address: Genetics Dcpnrtment, Animal Virus Research Institute, Pirbright, Woking, Surrey GU24 ONF, U. K. ‘To whom reprint requests should be addressed. 551
552
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FIG. 1. The effect of pactamycin on the proteins synthesised in CrPV-infected Drosophila cells. W-cm2 monolayers of cells were infected with 100 plaqueforming units/cell of CrPV as previously described (14). Proteins were labelled by pulsing with 100 rCi [“SSlmethionine (1500 Ci/mmol) at 4-4.5 hr postinfection in untreated cells (A) or in cells to which pactamycin was added at the time of pulsing (B), or which were pretreated with pactamycin for 5 min before the pulse and during the pulse (C). Pactamycin was added at a final concentration of 5 X 10.’ M. Cells were solubilised and proteins separated on a 12.5% polyacrylamide gel as previously described (14). Densitometer scans were made of an autoradiograph of the gel. Virus-specific proteins are indicated and the capsid proteins and precursor are numbered 0, 1, and 3.
B and C, and a group of proteins labeled D. as well as low-molecular weight proteins P; Q, R, and S (Fig. 1A). Markeddifferences
were observed in the CrPV-specified proteins when pactamycin was added at the time of pulsing. The relative amounts of proteins A and B/C increased and there was a reduction in the amounts of VP0 and VP3 (Fig. 1B). Pretreatment of cells with pactamycin depletes the 5’ end of the RNA of ribosomes before pulsing, thus leading to a greater decrease in the amount of label incorporated into proteins coded for at that end of the RNA. When pretreatment with pactamycin was performed a greater decrease was observed in the amounts of VP0 and VP3, and proteins A and B/C appeared to increase in relative amount. This suggests A, B, and C are coded for by regions at the 3’end of the RNA and furthest from the initiation site (Fig. 1C). The relative amounts of proteins were determined as a percentage of the total radioactivity incorporated into proteins. The ratio of this percentage in the pactamycin-treated samples relative to the value of the corresponding protein in the untreated (control) cells was then determined. This pactamycin-control ratio is related to the position of the polypeptides on the virus RNA. A low pactamycin-control ratio indicates that the relative amount of a protein decreased upon pactamycin treatment and that it is coded for by regions near the initiation site at the 5’end of the RNA. Conversely, a large pactamycin-control ratio indicates that the relative amount of a protein increased upon pactamycin treatment, showing that it is coded for by regions near the 3’ end of the RNA. Genetic maps constructed using the pactamycin-control ratios of CrPV-specified proteins are shown in Fig. 2. Mapping positions of proteins were reproducible. No attempt was made to map proteins in region D where a complicated sequence of precursor-product relationships exists. The virus proteins mapped as VPO-VP& VP1 at the 5’ end. As VP0 is a precursor of VP2 (14,15) this would give a gene order of 5’ VPB-VPS-VP1 3’ for the capsid proteins which is similar to that observed with mammalian picornaviruses. Proteins S, P, and Q mapped with the capsid proteins, suggesting that they were produced during cleavages of capsid protein precursors. However, the small amount of radioactivity
SHORT
A. Addition Precursors
jf
Stable proteins
5’
of pwztamycin
at time of puking
-VP0 0.55
E/F 1.33
-VP2
-VP3 0.75
-VP1 1.24
S o.Fj2
P 0.85
Q 0.89
B. Pretreatment Precursors
553
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& 3’ B/C 3.2 R iis
with pwtamycin
before pulsing -E/F 1.42
g!j (VP2)
-VP3 0.37
VP1 1.06
P
& 1.24
B/C 3.4
Stable S is
i
R 2.36
FIG. 2. Pactamycin maps of CrPV-specified proteins. The relative amounts of proteins were determined by excision and weighing of peaks from the densitometer scans shown in Fig. 1. The relative amount of each peak was determined as a percentage of the total radioactivity incorporated into proteins (as determined by the cumulative weight of the peaks). Pactamycin-control ratios were determined as described in the text. VP2 has been positioned with VP0 because of the precursor-product relationship which exists. Proteins are assigned as precursors or stable products according to Moore et al (14).
incorporated into proteins S, P, and Q makes accurate quantitation difficult and there must be some uncertainty about the map positions of these proteins. If S, P, and Q do represent bona fide cleavage products synthesised during capsid protein production, CrPV will differ fundamentally from mammalian picornaviruses where such products are not seen. Protein A mapped at the 3’ end of the RNA and proteins B and C also mapped towards that end of the genome. Proteins B and C did not map directly with protein A as would be expected if both proteins were cleavage products of protein A. Also, pretreatment with pactamycin increased the difference in mapping positions of the proteins. It is possible that only one protein of the B/C doublet was the cleavage product of A and that the other was a separate protein mapping in the centre of the genome. This would then result in both proteins mapping at a position intermediate between the centre of the genome and the 5’ end, Furthermore, the presence of a low-molecularweight protein, R, which mapped in the centre of the genome suggests that there
may be a higher-molecular-weight protein mapping in that region. Proteins E and F map immediately to the right of VP1 and may also be involved in cleavages occurring in the proteins coded for by the sentral region of the genome. The gene organisation of CrPV has similarities to that of mammalian picornaviruses. The structural proteins map at the 5’ end in the “correct” order, but no structural protein equivalent to VP4 has been found in CrPV. It is possible that VP4 of CrPV is readily lost during purification of the virus and has not been identified for that reason. We have previously reported that A, B, and C are precipitated with antiserum to CrPV (16), but these proteins map at the 3’ end of the CrPV genome and not with the capsid proteins. It is possible that proteins A, B, and C are present in preparations of CrPV and elicit an immune response in the same way that the replicase of aphthovirus functions as a “virus-infection-associated antigen” (17’). The molecular weights of proteins B and C are ~100 X lo” (14) and proteins of a similar size have been identified in the translation
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products of a number of small RNA viruses SUMMERS, D. F., and MAIZEL, J. V., Proc NatL Amd sci. USA 66, 2852-2856 (1971). of insects (18~20), and it has been suggested BUTTERWORTH, B. E., and RUECKERT. R. R., Viwith Nodamura virus that such a protein T-obgg 50, 535-549 (1972). functions as a replicase (21). It is probable MCLEAN, C., and RIXJCKERT, R. R., J. Viral 11. that, one of the high-molecular-weight 34-344 (1973). proteins mapping at the 3’ end of CrPV 10. BUTIXRWORTH, B. E., Virology 56,439-453 (1973). RNA functions as an RNA-dependent RNA 11. PAUCI~A, E., SCHAFER, J., and ‘ALTER, J. S., Vipolymerase. A molecular weight of 100 ro109y 61, 315-326 (1974). X lo3 is considerably larger than that pro12. SANGAR, D. V., BLACK, D. N., ROWLANDS, D. J., posed for the replicases of mammalian piand BROWN, F., J. Gen. Vim! 35,281-29’7 (1977). cornaviruses which is ~60 X lo3 (2%~2424) 13. DOEL, T. R., SANC.AR, D. V., ROWLANDS, D. J., and BROWN, F., J. Gm ViroL 41. 395-404 (1978). although it should be noted that the replicase of aphthovirus is synthesized as a 14 MOORE, N. F., KEAKNS, A., and PULLW, J. S. K., J. Vi&. 33, 1-9 (1980). 100 X lo3 molecular weight precursor 15. REAW, B., and MOORE, N. F., J. Gm Viral 55, (~100) which is subsequently cleaved to 429-438 (1981). give the mature form (p56a) (25). ACKNOWLEDGMENTS We thank Gail Davies and Chris Hatton for typing and photography, respectively. Brian Reavy was funded by a NERC research studentship during the course of this work. REFERENCES 1. MOORE, N. F., and TINSLEY, T. W., Arch
Viral. 72, 2‘2-245 (1982). 2. REAVY, B., and MOORE, N. F., Microbiologica 5,W 84 (1982). .?. LONGWORTH, J. F., Advan Virus Res 23,103-157 (197% .& MATTHEWS, R. E. F., IntcrvirdoDy 17, 131 (1982). 5. RIIECKERT, R. R., In “Comprehensive Virology” (H. Fraenkel-anrat, and R. R. Wagner, eds.), Vol. 6, pp. 131-211. Plenum, New York, 1976. 6. TAREK, R., REKOSH, D., and BALTIMORE, D.,J. Vim! 8. 395-401 (1971).
16. Mooa~, N. F., REAVY, B., and PULLIN, J. S. K., Arch ViroL 68, 1-8 (1981). 17. POI,ATNICK, J., ARLINGHAIJS, R. B., GRAVES, J. H., and CQXEN, K. M., Virology 31.609-615 (1967). 18. GUARINO, L. A., HRUBY, D. E., BALL, L. A., and KAESBERG, P., J. Vird 37, W-505 (1981). 1.9. FRIESEN, P. D., and RUECKEKT, R. R., J. ViroL 37, 876-886 (1981). 20. CRUMP, W. A. L., and MOORE, N. F., Arch ViroL 69, 131-139 (1981). 21. NEWMAN, J. F. E., MAITHEWS, T., OMILIANOWSKI, D. R., SALERNO, T., KAESBERB, P., and RUECKERT, R., J. Viral 25, 78-85 (1978). 22. LOWE, P. A., and BROWN, F., Virology 111, 23-32 (1981). 23. FLANEGAN, J. B., and BALTIMORE, D., J. Vi& 29. 352-360 (1979). 24 PALMEXRERG, A. C., PALLANSCH, M. A., and RUE~KERT, R. R., J. ViroL 32, 770-778 (1979). 25. SANGAR, D. V., BLACK, D. N., ROWLANDS, D. J., HARRIS, T. J. R., and Bnowx, F., J. ViroL 33, 59-68 (1980).
The Gene Organisation Comparison
of a Small RNA-Containing
with That of Mammalian
BRIAN REAVY’
AND NORMAN
Insect
Virus:
Picomaviruses F. MOORE”
The coding regions of an insect virus, cricket paralysis virus, have hesn mapped using pactamyrin. The results suggest that. the genome of this virus functions as a polycistronic mKNA, the structural proteins being encoded by the 5’end of the RNA in an order similar to those of mammalian picornaviruses. High-molecular-weight proteins of unknown function map at the 3’ end of the genome.
A large number of small RNA-containing viruses have been isolated from insects (I). Two of these viruses, cricket, paralysis virus (CrPV) and Lh-osophila C virus (UCV), undergo replicative events in Drosophila wwlunoyuskr tissue culture cells reminiscent of those found with mammalian picornaviruses (2). While CrPV has many characteristics in common with the enterovirus genus of the PticrrnuviW it has been excluded from that genus on the basis of the low GC content in its RNA (S, 4). The genome of picornaviruses is a single piece of single-stranded RNA which functions as a mRNA. Initiation of protein synthesis occurs at one site near the 5’ end of the genome and functional proteins are produced by post-translational cleavages of a polypeptide which represents the complete translation product of the RNA (5). This mode of protein synthesis has made picornaviruses amenable to gene mapping by the use of inhibitors of the initiation step of protein synlhesis. An inhibitor of initiation will lead to an apparent increase in the synthesis of proteins which are encoded by regions furthest from the initiation site, as ribosomes which have initiated protein synthesis will continue synthesising until the end of the RNA. Thus,
the amount of synthesis of a protein after inhibition of initiation is proportional to the distance of its coding region from the initiation site. The coding regions on the genomes of a number of picornaviruses have been mapped using pactamyein to inhibit initiation and a common gene order has been found (G-K?). We have mapped the order of the coding regions on the RNA of CrPV and have determined that the gene organisation of CrPV is similar to that of mammalian picornaviruses. Optimum condit.ions for the USCof pactamycin (supplied by Upjohn Co.) were determined by the incorporation of ?%Imethionine into TCA-prccipitablc material by CrPV-infected Drosophila eells. Pactamycin (1O--6 M) caused a marked inhibition of protein synthesis while 10e7 M caused only a slight decrease and the cells were totally unaffected by lo-’ M pactamycin. Since high levels of paetamycin inhibit elongation the drug was used at a concentration of 5 X 1O-‘7 M to map the CrPV-specified proteins. The differential effect of pactamycin on the synthesis of CrPV-specified proteins is shown in Fig. 1. In the absence of pactamycin the major proteins synthesised were the capsid proteins VP1 and VP3 with lesser amounts of VP0 (the precursor of the third capsid protein, VP‘4 (14,15) and the two comigrating proteins, E and F. There were even smaller amounts of higher-molecular-weight proteins: protein A, the comigrating proteins
’ Present address: Genetics Dcpnrtment, Animal Virus Research Institute, Pirbright, Woking, Surrey GU24 ONF, U. K. ‘To whom reprint requests should be addressed. 551
552
SHORT
3
0
Migration
Y
COMMUNICATIONS
IZ
km)
FIG. 1. The effect of pactamycin on the proteins synthesised in CrPV-infected Drosophila cells. W-cm2 monolayers of cells were infected with 100 plaqueforming units/cell of CrPV as previously described (14). Proteins were labelled by pulsing with 100 rCi [“SSlmethionine (1500 Ci/mmol) at 4-4.5 hr postinfection in untreated cells (A) or in cells to which pactamycin was added at the time of pulsing (B), or which were pretreated with pactamycin for 5 min before the pulse and during the pulse (C). Pactamycin was added at a final concentration of 5 X 10.’ M. Cells were solubilised and proteins separated on a 12.5% polyacrylamide gel as previously described (14). Densitometer scans were made of an autoradiograph of the gel. Virus-specific proteins are indicated and the capsid proteins and precursor are numbered 0, 1, and 3.
B and C, and a group of proteins labeled D. as well as low-molecular weight proteins P; Q, R, and S (Fig. 1A). Markeddifferences
were observed in the CrPV-specified proteins when pactamycin was added at the time of pulsing. The relative amounts of proteins A and B/C increased and there was a reduction in the amounts of VP0 and VP3 (Fig. 1B). Pretreatment of cells with pactamycin depletes the 5’ end of the RNA of ribosomes before pulsing, thus leading to a greater decrease in the amount of label incorporated into proteins coded for at that end of the RNA. When pretreatment with pactamycin was performed a greater decrease was observed in the amounts of VP0 and VP3, and proteins A and B/C appeared to increase in relative amount. This suggests A, B, and C are coded for by regions at the 3’end of the RNA and furthest from the initiation site (Fig. 1C). The relative amounts of proteins were determined as a percentage of the total radioactivity incorporated into proteins. The ratio of this percentage in the pactamycin-treated samples relative to the value of the corresponding protein in the untreated (control) cells was then determined. This pactamycin-control ratio is related to the position of the polypeptides on the virus RNA. A low pactamycin-control ratio indicates that the relative amount of a protein decreased upon pactamycin treatment and that it is coded for by regions near the initiation site at the 5’end of the RNA. Conversely, a large pactamycin-control ratio indicates that the relative amount of a protein increased upon pactamycin treatment, showing that it is coded for by regions near the 3’ end of the RNA. Genetic maps constructed using the pactamycin-control ratios of CrPV-specified proteins are shown in Fig. 2. Mapping positions of proteins were reproducible. No attempt was made to map proteins in region D where a complicated sequence of precursor-product relationships exists. The virus proteins mapped as VPO-VP& VP1 at the 5’ end. As VP0 is a precursor of VP2 (14,15) this would give a gene order of 5’ VPB-VPS-VP1 3’ for the capsid proteins which is similar to that observed with mammalian picornaviruses. Proteins S, P, and Q mapped with the capsid proteins, suggesting that they were produced during cleavages of capsid protein precursors. However, the small amount of radioactivity
SHORT
A. Addition Precursors
jf
Stable proteins
5’
of pwztamycin
at time of puking
-VP0 0.55
E/F 1.33
-VP2
-VP3 0.75
-VP1 1.24
S o.Fj2
P 0.85
Q 0.89
B. Pretreatment Precursors
553
COMMUNICATIONS
& 3’ B/C 3.2 R iis
with pwtamycin
before pulsing -E/F 1.42
g!j (VP2)
-VP3 0.37
VP1 1.06
P
& 1.24
B/C 3.4
Stable S is
i
R 2.36
FIG. 2. Pactamycin maps of CrPV-specified proteins. The relative amounts of proteins were determined by excision and weighing of peaks from the densitometer scans shown in Fig. 1. The relative amount of each peak was determined as a percentage of the total radioactivity incorporated into proteins (as determined by the cumulative weight of the peaks). Pactamycin-control ratios were determined as described in the text. VP2 has been positioned with VP0 because of the precursor-product relationship which exists. Proteins are assigned as precursors or stable products according to Moore et al (14).
incorporated into proteins S, P, and Q makes accurate quantitation difficult and there must be some uncertainty about the map positions of these proteins. If S, P, and Q do represent bona fide cleavage products synthesised during capsid protein production, CrPV will differ fundamentally from mammalian picornaviruses where such products are not seen. Protein A mapped at the 3’ end of the RNA and proteins B and C also mapped towards that end of the genome. Proteins B and C did not map directly with protein A as would be expected if both proteins were cleavage products of protein A. Also, pretreatment with pactamycin increased the difference in mapping positions of the proteins. It is possible that only one protein of the B/C doublet was the cleavage product of A and that the other was a separate protein mapping in the centre of the genome. This would then result in both proteins mapping at a position intermediate between the centre of the genome and the 5’ end, Furthermore, the presence of a low-molecularweight protein, R, which mapped in the centre of the genome suggests that there
may be a higher-molecular-weight protein mapping in that region. Proteins E and F map immediately to the right of VP1 and may also be involved in cleavages occurring in the proteins coded for by the sentral region of the genome. The gene organisation of CrPV has similarities to that of mammalian picornaviruses. The structural proteins map at the 5’ end in the “correct” order, but no structural protein equivalent to VP4 has been found in CrPV. It is possible that VP4 of CrPV is readily lost during purification of the virus and has not been identified for that reason. We have previously reported that A, B, and C are precipitated with antiserum to CrPV (16), but these proteins map at the 3’ end of the CrPV genome and not with the capsid proteins. It is possible that proteins A, B, and C are present in preparations of CrPV and elicit an immune response in the same way that the replicase of aphthovirus functions as a “virus-infection-associated antigen” (17’). The molecular weights of proteins B and C are ~100 X lo” (14) and proteins of a similar size have been identified in the translation
554
SHORT
COMMUNICATIONS
products of a number of small RNA viruses SUMMERS, D. F., and MAIZEL, J. V., Proc NatL Amd sci. USA 66, 2852-2856 (1971). of insects (18~20), and it has been suggested BUTTERWORTH, B. E., and RUECKERT. R. R., Viwith Nodamura virus that such a protein T-obgg 50, 535-549 (1972). functions as a replicase (21). It is probable MCLEAN, C., and RIXJCKERT, R. R., J. Viral 11. that, one of the high-molecular-weight 34-344 (1973). proteins mapping at the 3’ end of CrPV 10. BUTIXRWORTH, B. E., Virology 56,439-453 (1973). RNA functions as an RNA-dependent RNA 11. PAUCI~A, E., SCHAFER, J., and ‘ALTER, J. S., Vipolymerase. A molecular weight of 100 ro109y 61, 315-326 (1974). X lo3 is considerably larger than that pro12. SANGAR, D. V., BLACK, D. N., ROWLANDS, D. J., posed for the replicases of mammalian piand BROWN, F., J. Gen. Vim! 35,281-29’7 (1977). cornaviruses which is ~60 X lo3 (2%~2424) 13. DOEL, T. R., SANC.AR, D. V., ROWLANDS, D. J., and BROWN, F., J. Gm ViroL 41. 395-404 (1978). although it should be noted that the replicase of aphthovirus is synthesized as a 14 MOORE, N. F., KEAKNS, A., and PULLW, J. S. K., J. Vi&. 33, 1-9 (1980). 100 X lo3 molecular weight precursor 15. REAW, B., and MOORE, N. F., J. Gm Viral 55, (~100) which is subsequently cleaved to 429-438 (1981). give the mature form (p56a) (25). ACKNOWLEDGMENTS We thank Gail Davies and Chris Hatton for typing and photography, respectively. Brian Reavy was funded by a NERC research studentship during the course of this work. REFERENCES 1. MOORE, N. F., and TINSLEY, T. W., Arch
Viral. 72, 2‘2-245 (1982). 2. REAVY, B., and MOORE, N. F., Microbiologica 5,W 84 (1982). .?. LONGWORTH, J. F., Advan Virus Res 23,103-157 (197% .& MATTHEWS, R. E. F., IntcrvirdoDy 17, 131 (1982). 5. RIIECKERT, R. R., In “Comprehensive Virology” (H. Fraenkel-anrat, and R. R. Wagner, eds.), Vol. 6, pp. 131-211. Plenum, New York, 1976. 6. TAREK, R., REKOSH, D., and BALTIMORE, D.,J. Vim! 8. 395-401 (1971).
16. Mooa~, N. F., REAVY, B., and PULLIN, J. S. K., Arch ViroL 68, 1-8 (1981). 17. POI,ATNICK, J., ARLINGHAIJS, R. B., GRAVES, J. H., and CQXEN, K. M., Virology 31.609-615 (1967). 18. GUARINO, L. A., HRUBY, D. E., BALL, L. A., and KAESBERG, P., J. Vird 37, W-505 (1981). 1.9. FRIESEN, P. D., and RUECKEKT, R. R., J. ViroL 37, 876-886 (1981). 20. CRUMP, W. A. L., and MOORE, N. F., Arch ViroL 69, 131-139 (1981). 21. NEWMAN, J. F. E., MAITHEWS, T., OMILIANOWSKI, D. R., SALERNO, T., KAESBERB, P., and RUECKERT, R., J. Viral 25, 78-85 (1978). 22. LOWE, P. A., and BROWN, F., Virology 111, 23-32 (1981). 23. FLANEGAN, J. B., and BALTIMORE, D., J. Vi& 29. 352-360 (1979). 24 PALMEXRERG, A. C., PALLANSCH, M. A., and RUE~KERT, R. R., J. ViroL 32, 770-778 (1979). 25. SANGAR, D. V., BLACK, D. N., ROWLANDS, D. J., HARRIS, T. J. R., and Bnowx, F., J. ViroL 33, 59-68 (1980).