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Mapping of the tobacco vein mottling virus VPg cistron
VIROLOGY
163, 635-637
(1988)
Mapping MUHAMMAD Departmenfs
of the Tobacco SHAHABUDDIN,*
of *Biochemistry
and tP/ant
Received
Vein Mottling
JOHN
G.
Pathology,
SHAw,t” University
July 20, 1987; accepted
Virus VPg Cistron AND
ROBERT
of Kentucky,
December
E.
Lexington,
RHOADS* Kentucky
40546
15. 1987
The location of the cistron encoding the genome-linked protein (VPg) in the potyvirus tobacco vein mottling virus (TVMV) was investigated. Precipitation of ‘251-labeled VPg with anti-tobacco etch virus 49K nuclear inclusion protein antiserum (which reacts with the NI, nuclear inclusion protein of TVMV) indicated that the TVMV VPg is immunologically related to NI.. Lysyl residues were found to be present at positions 2, 11, and 16 of the amino-terminal region of the VPg. A search of the TVMV polyprotein sequence for this distribution of lysyl residues revealed a unique location beginning at amino acid residue 1801, the proposed amino-terminus of the NI, protein. o is88 Academic PWSS. I~C.
Many viruses contain a protein covalently linked to the 5’-terminus of their genomic nucleic acid [reviewed in (1, 2)]. This protein (VPg) is thought to serve as a primer for viral nucleic acid synthesis. RNA of the potyvirus tobacco etch virus (TEV) has been shown to be linked to a VPg (3), and, more recently, the site of attachmenl of the VPg of another potyvirus, tobacco vein mottling virus (TVMV), has been shown to be the 5’-terminus of the single-stranded viral RNA (4). Of the six proteins ,which have been found to be associated with potyvirus infections, five have been mapped on the viral genome (5- 11). The potyviral VPg, however, has not been positively identified as a virusencoded protein, nor has its cistron been mapped on the viral genome. To address these questions, we have performed an immunological analysis of lz51-labeled TVMV VPg using antibodies against various potyviral proteins and have determined the distribution of lysyl residues at the amino-terminus of the VPg. TVMV RzlA was isolated from purified virus as previously reported (6) except that it was subjected to an additional cycle of sucrose gradient centrifugation prior to radioiodination. This procedure removed all detectable amounts of contaminating coat and other proteins, leaving the VPg as the only detectable protein (C. S. Luciano and M. F. E. Siaw, unpublished observations). The VPg-like nature of this protein was confirmed by demonstrating that it could be separated from the viral RNA by digestion with ribonuclease or proteinasel K but not by treatment with 59/o sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol at 1OO”, 8 A/I urea and 1% SDS at 65’, phenol-chloroform, or 5 M NaC104 (4; C. S. Luciano, unpublished observations).
’ To whom
requests
for reprints
should
Lysyl residues in the TVMV VPg were labeled by reacting TVMV RNA with ‘251-Bolton-Hunter reagent (4); substoichiometric amounts of the reagent were used in order to minimize blocking of the amino-terminus. Radioiodinated VPg was removed from the RNA by digestion with ribonuclease and was purified by gel filtration. Analysis of the VPg by polyacrylamide gel electrophoresis (PAGE) (Fig. 1, lane A) revealed a single radioactive band corresponding to the position reported previously for TVMV VPg (4), thus confirming that the VPg was the only labeled protein present. To determine whether it was immunologically related to other known potyviral proteins, the VPg was incubated with antisera against the helper component (HC) and cylindrical inclusion (Cl) proteins of TVMV (Fig. 1, lanes C and D) and the 54K and 49K nuclear inclusion proteins (NI) of TEV (lanes E and F). Analysis of immunoprecipitates by PAGE showed that radioiodinated VPg was precipitated only by antiserum against the TEV 49K NI protein, shown previously to cross-react with TVMV NI, (7). The addition of nonradioactive TEV 49K NI protein (lane G) or TVMV VPg (lane H) to immunoprecipitation reactions resulted in a substantial reduction in the amount of VPg in the immunoprecipitates. The effect of addition of comparable amounts of bovine serum albumin (lane I) was much less pronounced. These results suggested that the TVMV VPg is antigenically related to the NI, protein. In a previous study we presented evidence, consisting of immunoprecipitation of “‘1-TVMV VPg with antisera against various viral proteins, that the VPg was not related to other TVMV proteins (4). In that study, antisera against intact TVMV, TVMV coat protein (CP), or TEV 49K NI protein precipitated approximately four times as much radioactivity as did antisera against TVMV HC, TVMV Cl, TEV 54K NI, or preimmune sera.
be addressed. 635
0042-6822188
$3.00
CopyrIght Q 1999 by Academkc Press. Inc All rights of reproduction I” any form reserved.
636
SHORT
COMMUNICATIONS
ABCDEFGHI Origin
-
92 -
18 12 FIG. 1. lmmunoprectpitation of TVMV VPg. TVMV RNA (250 pg) reagent as prevrously was reacted with 5 &i of ‘251-Bolton-Hunter described (I). RNA was removed by drgestion with 10 units each of RNase A and RNase Tl. The VPg was passed through a Sephadex G-25 column and precipitated with 10% trichloroacetic acid. The precipitate was washed twice with 80% acetone and the gel filtration procedure was repeated. The protein (typrcally 10,000 cpm per microgram of the original RNA preparation) was dissolved in 50 miZ/I ammonium bicarbonate (pH 9.0). ‘251-labeled VPg was reacted with antibodies against TVMV HC and Cl (6, 7) and against TEV 49K NI and 54K NI (6). Immunoprecipitates, obtained in the presence or absence of nonradioactive, competitor proteins, were analyzed by PAGE in presence of SDS (6). Lane A, ‘251-labeled TVMV VPg; lanes B-l, resuspended precipitates after treatment of labeled TVMV VPg with no antiserum (lane 8) or with antisera against TVMV HC (lane C), TVMV Cl (lane D), TEV 54K NI (lane E), TEV 49K NI (lanes F-l). The following competitor proteins were added prior to immunoprecipitation: none (lane F), TEV 49K NI (lane G), TVMV VPg (lane H), or bovine serum albumin (lane I). Mobilities of standard proteins of the indicated molecular weights are shown on the left. The arrow rndrcates the position of TVMV VPg.
activity predominantly in cycles 1, 2, 1 1, and 16 (Fig. 2A). The radioactivity released in step 1 is typically observed and is the result of elution of labeled protein not tightly bound to the glass fiber disk. The radioactivity present in cycles 12 and 17 is attributed to carryover from the previous cycle (T. C. Vanaman, University of Kentucky, personal communication). Thus, lysyl residues appear to be present at positions 2, 11, and 16 of the VPg. The amino acid sequence of the TVMV polyprotein (12) was searched for the sequence XKX8KX4K (Fig. .?‘A), where X represents any amino acid residue except lysine. This sequence was found to be uniquely located at positions 1801-l 816; the sequences KaX8K2X3Kand K2X8KX4Kdid not occur in the polyprotein. Amino acid residue position 1801 in the TVMV polyprotein is one of the positions which we have previously suggested might correspond to the amino-terminus of NI, (Fig. 2B). Originally, we proposed that the amino-terminus of this protein was at amino acid position 1748, based on the size of a polypeptide detected
EDMAN
CYCLE
NUMBER
B
However, the precipitated radioactivity was not analyzed by SDS-PAGE to determine which proteins were present. Due to the low amounts of radioactivity, it was suggested (4) that the immunoprecipitation was nonspecific. The preparation of RNA used in that study contained a small amount of contaminating CP and, during iodination, both the VPg and the CP were labeled with 1251.This explains why antisera against CP and intact virus precipitated radioactivity. From the present study, it is clear that radioactivity precipitated by antisera to TEV 49K NI protein was due to reaction with the VPg. Partial amino acid sequence data and the availability of the nucleotide sequence of TVMV RNA (12) were used to map the TVMV VPg cistron in more detail. Automated Edman degradation of VPg labeled with ‘251-Bolton-Hunter reagent led to the release of radio-
j28K/
HC
j42K)
Cl
M
Nlo
j
Nlb
jCP/
/\ 1748
1801
FIG. 2. Drstnbution of lysyl residues at the amino-terminus of TVMV VPg. ‘251-labeled VPg (prepared as described in Fig. 1) was subjected to Edman degradation, using the trifluoroacetic acid conversion method, with a Model 470A gas-phase sequencer (Applied Biosystems). Approximately 1 x lo6 cpm of labeled VPg was injected. (A) Radioactivity (determined by gamma counting) in the eluate from each Edman cycle. The sequence of 21 amino acids in the NI, region of the polyprotein (12) beginning with residue 1801, is also indicated. (B) Location in the TVMV polyprotein of the amino acid sequence XKX,KX.+K (X, any amino acid except lysine). The locations of potential protease cleavage sites at polyprotein amino acid residues 1746 and 1801 (72, 13) are also shown; the numbers represent the positrons of the first amino acid after the cleavage site. ?, location in the polyprotein of a 5.5kDa polypeptide which would be produced by cleavages at both sites.
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by Western blot analysis of infected plant extracts, which reacted with anti-TEV 49K NI protein antibodies (Z. Xu, unpublished observations), and on the occurrence of a putative consensus sequence, VRFQ/S, at that position (12). Subsequently, data on the production of TVMV proteins in tobacco protoplasts (G. M. Hellmann and C. S. Luciano, unpublished observations) made it appear more likely that the NI, protein begins at residue 1801, the location of another putative consensus cleavage site, VKFQ/G (13). Cleavage at each of these sites (amino acid positions 1748 and 1801) would yield a polypeptide of 5.5 kDa located between the Cl and NI, cistrons (Fig. 2B). Such a polypeptide would have the small size and cistron location characteristic of the picorna- and comoviral VPgs (14). However, the data presented here indicate that it is the second of the two sites, that at amino acid position 1801, which corresponds to the amino-terminus of the VPg and, therefore, that there may be an additional cistron preceding that for the VPg. To firmly establish whether the VPg and [VI, share amino-termini at amino acid position 1801, it will be necessary to determine the amino acid sequence at the amino-terminus of NI,. In the absence of amino acid sequence information from the carboxy-terminus of the VPg, the exact size of the VPg cistron could not be determined. Our previous studies suggested that the molecular weight of TVMV VPg is approximately 24,000, based solely on its electrophoretic mobility (L?). However, VPgs of other viruses migrate more slowly in SDS-polyacrylamide gels than would be predicted from their molecular weights (15- 18). ACKNOWLEDGMENTS The authors thank Leslie Domler, Gary Hellmann, and Shivanand Hiremath for much helpful advlce and dlscussion, Steve Gathy for his assistance in radiochemical sequencing of the VPg, and E. Hiebert (University of Florida) fclr providing TEV NI proteins and anti-TEV NI antisera. This work was supported by Grant 4E021 from the
637
University of Kentucky Grant 85CRCR-1-1536 gram.
Tobacco and Health Research from the USDA Competitive
Institute Grants
and Pro-
REFERENCES
5. 6. 7.
8.
9. 10.
11. 12.
13. 14. 15. 16. 17.
18.
WIMMER, E., Cell 28, 199-201 (1982). DAUBERT, S. D., and BRUENING, G., Methods Viral. 8, 347-379 (1984). HARI, V., Virology 112, 391-399 (1981). SIAW, M. F. E., SHAHABUDDIN, M.. BALLARD, S., SHAW. I. G., and RHOADS, R. E.. V,ro/ogy 142, 134-143 (1985). DOUGHERTY, W. G., and HIEBERT, E., l/iro/ogy 104, 183-194 (1980). HELLMANN, G. M., SHAW. 1. G., LESNAW, J. A., CHU, L.-Y., PIRONE, T. P., and RHOADS, R. E., Virology 106, 207-216 (1980). HELLMANN. G. M., THORNBURY. D. W., HIEBERT. E., SHAW, J. G., PIRONE, T. P., and RHOADS, R. E., Virology 124, 434-444 (1983). ALLISON, R. F., SORENSEN, 1. C., KELLY, M. E., ARMSTRONG, F. B., and DOUGHERTY. W. G., Proc. Nat/. Acad. SC;. USA 82, 3969-3972(1985). NAGEL. J.. and HIEBERT. E., Virology 143, 435-441 (1985). DOUGHERTY, W. G.. ALLISON, R. F., PARKS, T. D., JOHNSTON, R. E., FEILD, M. J., and ARMSTRONG, F. B., wrology 146, 282-292 (1985). HELLMANN, G. M., HIREMATH, S. T., SHAW, J. G.. and RHOADS, R. E.. virology 151, 159-171 (1986). DOMIER, L. L., FRANKLIN, K. M., SHAHABUDDIN, M., HELLMANN, G. M., OVERMEYER. J. H., HIREMATH. S. T., SIAW, M. F. E., LOMONOSSOFF, G. P., SHAW, J. G.. and RHOADS, R. E., /Vuc/eic Acids Res. 14, 5417-5430 (1986). DOMIER, L. L., SHAW, I. G., and RHOADS, R. E., Virology 158, 20-27 (1987). ARGO% P., KAMER, G., NICKLIN, M. 1. H., and WIMMER, E., Nucleic Acids Res. 12,7251-7267 (1984). AMEROS, V., and BALTIMORE, D., J. Biol. Chem. 253, 5263-5266 (1978). DAUBERT, S. D., BRUENING, G., and NAJARIAN. R. C., Eur. 1. Biochem.92,45-51(1978). KITAMURA, N., SEMLER, B., ROTHBERG, P. G., LARSON, G. P., ADLER, C. J., DORNER. A. I., EMINI. E. A., HANECAK, R., LEE, J. J., VAN DER VERFS, S., ANDERSON, C. W., and WIMMER, E., Nature (London) 291,547-553(1981). WELLINK, J., REZEKMAN. G., GOLDBACH, R., and BEYREUTHER, K.,/. Viral. 59, 50-58 (1986).
163, 635-637
(1988)
Mapping MUHAMMAD Departmenfs
of the Tobacco SHAHABUDDIN,*
of *Biochemistry
and tP/ant
Received
Vein Mottling
JOHN
G.
Pathology,
SHAw,t” University
July 20, 1987; accepted
Virus VPg Cistron AND
ROBERT
of Kentucky,
December
E.
Lexington,
RHOADS* Kentucky
40546
15. 1987
The location of the cistron encoding the genome-linked protein (VPg) in the potyvirus tobacco vein mottling virus (TVMV) was investigated. Precipitation of ‘251-labeled VPg with anti-tobacco etch virus 49K nuclear inclusion protein antiserum (which reacts with the NI, nuclear inclusion protein of TVMV) indicated that the TVMV VPg is immunologically related to NI.. Lysyl residues were found to be present at positions 2, 11, and 16 of the amino-terminal region of the VPg. A search of the TVMV polyprotein sequence for this distribution of lysyl residues revealed a unique location beginning at amino acid residue 1801, the proposed amino-terminus of the NI, protein. o is88 Academic PWSS. I~C.
Many viruses contain a protein covalently linked to the 5’-terminus of their genomic nucleic acid [reviewed in (1, 2)]. This protein (VPg) is thought to serve as a primer for viral nucleic acid synthesis. RNA of the potyvirus tobacco etch virus (TEV) has been shown to be linked to a VPg (3), and, more recently, the site of attachmenl of the VPg of another potyvirus, tobacco vein mottling virus (TVMV), has been shown to be the 5’-terminus of the single-stranded viral RNA (4). Of the six proteins ,which have been found to be associated with potyvirus infections, five have been mapped on the viral genome (5- 11). The potyviral VPg, however, has not been positively identified as a virusencoded protein, nor has its cistron been mapped on the viral genome. To address these questions, we have performed an immunological analysis of lz51-labeled TVMV VPg using antibodies against various potyviral proteins and have determined the distribution of lysyl residues at the amino-terminus of the VPg. TVMV RzlA was isolated from purified virus as previously reported (6) except that it was subjected to an additional cycle of sucrose gradient centrifugation prior to radioiodination. This procedure removed all detectable amounts of contaminating coat and other proteins, leaving the VPg as the only detectable protein (C. S. Luciano and M. F. E. Siaw, unpublished observations). The VPg-like nature of this protein was confirmed by demonstrating that it could be separated from the viral RNA by digestion with ribonuclease or proteinasel K but not by treatment with 59/o sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol at 1OO”, 8 A/I urea and 1% SDS at 65’, phenol-chloroform, or 5 M NaC104 (4; C. S. Luciano, unpublished observations).
’ To whom
requests
for reprints
should
Lysyl residues in the TVMV VPg were labeled by reacting TVMV RNA with ‘251-Bolton-Hunter reagent (4); substoichiometric amounts of the reagent were used in order to minimize blocking of the amino-terminus. Radioiodinated VPg was removed from the RNA by digestion with ribonuclease and was purified by gel filtration. Analysis of the VPg by polyacrylamide gel electrophoresis (PAGE) (Fig. 1, lane A) revealed a single radioactive band corresponding to the position reported previously for TVMV VPg (4), thus confirming that the VPg was the only labeled protein present. To determine whether it was immunologically related to other known potyviral proteins, the VPg was incubated with antisera against the helper component (HC) and cylindrical inclusion (Cl) proteins of TVMV (Fig. 1, lanes C and D) and the 54K and 49K nuclear inclusion proteins (NI) of TEV (lanes E and F). Analysis of immunoprecipitates by PAGE showed that radioiodinated VPg was precipitated only by antiserum against the TEV 49K NI protein, shown previously to cross-react with TVMV NI, (7). The addition of nonradioactive TEV 49K NI protein (lane G) or TVMV VPg (lane H) to immunoprecipitation reactions resulted in a substantial reduction in the amount of VPg in the immunoprecipitates. The effect of addition of comparable amounts of bovine serum albumin (lane I) was much less pronounced. These results suggested that the TVMV VPg is antigenically related to the NI, protein. In a previous study we presented evidence, consisting of immunoprecipitation of “‘1-TVMV VPg with antisera against various viral proteins, that the VPg was not related to other TVMV proteins (4). In that study, antisera against intact TVMV, TVMV coat protein (CP), or TEV 49K NI protein precipitated approximately four times as much radioactivity as did antisera against TVMV HC, TVMV Cl, TEV 54K NI, or preimmune sera.
be addressed. 635
0042-6822188
$3.00
CopyrIght Q 1999 by Academkc Press. Inc All rights of reproduction I” any form reserved.
636
SHORT
COMMUNICATIONS
ABCDEFGHI Origin
-
92 -
18 12 FIG. 1. lmmunoprectpitation of TVMV VPg. TVMV RNA (250 pg) reagent as prevrously was reacted with 5 &i of ‘251-Bolton-Hunter described (I). RNA was removed by drgestion with 10 units each of RNase A and RNase Tl. The VPg was passed through a Sephadex G-25 column and precipitated with 10% trichloroacetic acid. The precipitate was washed twice with 80% acetone and the gel filtration procedure was repeated. The protein (typrcally 10,000 cpm per microgram of the original RNA preparation) was dissolved in 50 miZ/I ammonium bicarbonate (pH 9.0). ‘251-labeled VPg was reacted with antibodies against TVMV HC and Cl (6, 7) and against TEV 49K NI and 54K NI (6). Immunoprecipitates, obtained in the presence or absence of nonradioactive, competitor proteins, were analyzed by PAGE in presence of SDS (6). Lane A, ‘251-labeled TVMV VPg; lanes B-l, resuspended precipitates after treatment of labeled TVMV VPg with no antiserum (lane 8) or with antisera against TVMV HC (lane C), TVMV Cl (lane D), TEV 54K NI (lane E), TEV 49K NI (lanes F-l). The following competitor proteins were added prior to immunoprecipitation: none (lane F), TEV 49K NI (lane G), TVMV VPg (lane H), or bovine serum albumin (lane I). Mobilities of standard proteins of the indicated molecular weights are shown on the left. The arrow rndrcates the position of TVMV VPg.
activity predominantly in cycles 1, 2, 1 1, and 16 (Fig. 2A). The radioactivity released in step 1 is typically observed and is the result of elution of labeled protein not tightly bound to the glass fiber disk. The radioactivity present in cycles 12 and 17 is attributed to carryover from the previous cycle (T. C. Vanaman, University of Kentucky, personal communication). Thus, lysyl residues appear to be present at positions 2, 11, and 16 of the VPg. The amino acid sequence of the TVMV polyprotein (12) was searched for the sequence XKX8KX4K (Fig. .?‘A), where X represents any amino acid residue except lysine. This sequence was found to be uniquely located at positions 1801-l 816; the sequences KaX8K2X3Kand K2X8KX4Kdid not occur in the polyprotein. Amino acid residue position 1801 in the TVMV polyprotein is one of the positions which we have previously suggested might correspond to the amino-terminus of NI, (Fig. 2B). Originally, we proposed that the amino-terminus of this protein was at amino acid position 1748, based on the size of a polypeptide detected
EDMAN
CYCLE
NUMBER
B
However, the precipitated radioactivity was not analyzed by SDS-PAGE to determine which proteins were present. Due to the low amounts of radioactivity, it was suggested (4) that the immunoprecipitation was nonspecific. The preparation of RNA used in that study contained a small amount of contaminating CP and, during iodination, both the VPg and the CP were labeled with 1251.This explains why antisera against CP and intact virus precipitated radioactivity. From the present study, it is clear that radioactivity precipitated by antisera to TEV 49K NI protein was due to reaction with the VPg. Partial amino acid sequence data and the availability of the nucleotide sequence of TVMV RNA (12) were used to map the TVMV VPg cistron in more detail. Automated Edman degradation of VPg labeled with ‘251-Bolton-Hunter reagent led to the release of radio-
j28K/
HC
j42K)
Cl
M
Nlo
j
Nlb
jCP/
/\ 1748
1801
FIG. 2. Drstnbution of lysyl residues at the amino-terminus of TVMV VPg. ‘251-labeled VPg (prepared as described in Fig. 1) was subjected to Edman degradation, using the trifluoroacetic acid conversion method, with a Model 470A gas-phase sequencer (Applied Biosystems). Approximately 1 x lo6 cpm of labeled VPg was injected. (A) Radioactivity (determined by gamma counting) in the eluate from each Edman cycle. The sequence of 21 amino acids in the NI, region of the polyprotein (12) beginning with residue 1801, is also indicated. (B) Location in the TVMV polyprotein of the amino acid sequence XKX,KX.+K (X, any amino acid except lysine). The locations of potential protease cleavage sites at polyprotein amino acid residues 1746 and 1801 (72, 13) are also shown; the numbers represent the positrons of the first amino acid after the cleavage site. ?, location in the polyprotein of a 5.5kDa polypeptide which would be produced by cleavages at both sites.
SHORT
COMMUNICATIONS
by Western blot analysis of infected plant extracts, which reacted with anti-TEV 49K NI protein antibodies (Z. Xu, unpublished observations), and on the occurrence of a putative consensus sequence, VRFQ/S, at that position (12). Subsequently, data on the production of TVMV proteins in tobacco protoplasts (G. M. Hellmann and C. S. Luciano, unpublished observations) made it appear more likely that the NI, protein begins at residue 1801, the location of another putative consensus cleavage site, VKFQ/G (13). Cleavage at each of these sites (amino acid positions 1748 and 1801) would yield a polypeptide of 5.5 kDa located between the Cl and NI, cistrons (Fig. 2B). Such a polypeptide would have the small size and cistron location characteristic of the picorna- and comoviral VPgs (14). However, the data presented here indicate that it is the second of the two sites, that at amino acid position 1801, which corresponds to the amino-terminus of the VPg and, therefore, that there may be an additional cistron preceding that for the VPg. To firmly establish whether the VPg and [VI, share amino-termini at amino acid position 1801, it will be necessary to determine the amino acid sequence at the amino-terminus of NI,. In the absence of amino acid sequence information from the carboxy-terminus of the VPg, the exact size of the VPg cistron could not be determined. Our previous studies suggested that the molecular weight of TVMV VPg is approximately 24,000, based solely on its electrophoretic mobility (L?). However, VPgs of other viruses migrate more slowly in SDS-polyacrylamide gels than would be predicted from their molecular weights (15- 18). ACKNOWLEDGMENTS The authors thank Leslie Domler, Gary Hellmann, and Shivanand Hiremath for much helpful advlce and dlscussion, Steve Gathy for his assistance in radiochemical sequencing of the VPg, and E. Hiebert (University of Florida) fclr providing TEV NI proteins and anti-TEV NI antisera. This work was supported by Grant 4E021 from the
637
University of Kentucky Grant 85CRCR-1-1536 gram.
Tobacco and Health Research from the USDA Competitive
Institute Grants
and Pro-
REFERENCES
5. 6. 7.
8.
9. 10.
11. 12.
13. 14. 15. 16. 17.
18.
WIMMER, E., Cell 28, 199-201 (1982). DAUBERT, S. D., and BRUENING, G., Methods Viral. 8, 347-379 (1984). HARI, V., Virology 112, 391-399 (1981). SIAW, M. F. E., SHAHABUDDIN, M.. BALLARD, S., SHAW. I. G., and RHOADS, R. E.. V,ro/ogy 142, 134-143 (1985). DOUGHERTY, W. G., and HIEBERT, E., l/iro/ogy 104, 183-194 (1980). HELLMANN, G. M., SHAW. 1. G., LESNAW, J. A., CHU, L.-Y., PIRONE, T. P., and RHOADS, R. E., Virology 106, 207-216 (1980). HELLMANN. G. M., THORNBURY. D. W., HIEBERT. E., SHAW, J. G., PIRONE, T. P., and RHOADS, R. E., Virology 124, 434-444 (1983). ALLISON, R. F., SORENSEN, 1. C., KELLY, M. E., ARMSTRONG, F. B., and DOUGHERTY. W. G., Proc. Nat/. Acad. SC;. USA 82, 3969-3972(1985). NAGEL. J.. and HIEBERT. E., Virology 143, 435-441 (1985). DOUGHERTY, W. G.. ALLISON, R. F., PARKS, T. D., JOHNSTON, R. E., FEILD, M. J., and ARMSTRONG, F. B., wrology 146, 282-292 (1985). HELLMANN, G. M., HIREMATH, S. T., SHAW, J. G.. and RHOADS, R. E.. virology 151, 159-171 (1986). DOMIER, L. L., FRANKLIN, K. M., SHAHABUDDIN, M., HELLMANN, G. M., OVERMEYER. J. H., HIREMATH. S. T., SIAW, M. F. E., LOMONOSSOFF, G. P., SHAW, J. G.. and RHOADS, R. E., /Vuc/eic Acids Res. 14, 5417-5430 (1986). DOMIER, L. L., SHAW, I. G., and RHOADS, R. E., Virology 158, 20-27 (1987). ARGO% P., KAMER, G., NICKLIN, M. 1. H., and WIMMER, E., Nucleic Acids Res. 12,7251-7267 (1984). AMEROS, V., and BALTIMORE, D., J. Biol. Chem. 253, 5263-5266 (1978). DAUBERT, S. D., BRUENING, G., and NAJARIAN. R. C., Eur. 1. Biochem.92,45-51(1978). KITAMURA, N., SEMLER, B., ROTHBERG, P. G., LARSON, G. P., ADLER, C. J., DORNER. A. I., EMINI. E. A., HANECAK, R., LEE, J. J., VAN DER VERFS, S., ANDERSON, C. W., and WIMMER, E., Nature (London) 291,547-553(1981). WELLINK, J., REZEKMAN. G., GOLDBACH, R., and BEYREUTHER, K.,/. Viral. 59, 50-58 (1986).