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Vaccinia virus gene D7R encodes a 20,000-dalton subunit of the viral DNA-dependent RNA polymerase
VIROLOGY
178,603W305
Vaccinia
(1990)
Virus Gene D7R Encodes
a 20,000-Dalton STEVEN
Department
D.
QUICK
of Biochemistry, Received
April
Subunit AND
Purdue
STEVEN
University,
7 7, 1990; accepted
of the Viral DNA-Dependent S.
BROYLES’
West Lafayette,
47907
synthesized in bacteria. Immunization a virion polypeptide of 20,000 daltons. with the virion-associated DNA-depengradient sedimentation. These results c IssoAcademic viral RNA polymerase.
Inc.
Poxviruses are large DNA viruses that replicate in the host-cell cytoplasm. Because of their cytoplasmic habitat, poxviruses have evolved autonomous replication and transcription machinery [reviewed in (I)]. including the viral DNA-dependent RNA polymerase responsible for the synthesis of viral mRNA. The RNA polymerase has been purified to apparent homogeneity from vaccinia. the prototypal poxvirus, and has been shown to be a large, multisubunit complex of at least seven polypeptlde subunits (Z-4). Mapping of the RNA polymerase subunit genes with antibodies directed against the purified enzyme has demonstrated that all subunits are encoded by the viral genome (5). The gene sequences for three poxvirus RNA polymerase subunits have been reported (6, 7). The 147,000-dalton (Da) subunit of the vaccinia RNA polymerase and the 135 kDa subunit of the cowpox virus RNA polymerase are predicted to share considerable sequence homology with their respective counterparts from prokaryotes and eukaryotes, suggesting that these two subunits provide functions basic to the DNA-dependent polymerization of RNA. Because the genes for most of the smaller subunits have not yet been identified, it has not been possible to assess their respective roles in the RNA polymerase complex or their potential relatedness to the subunits of cellular polymerases. To this end, we have undertaken the identification of the genes for smaller RNA polymerase subunits. It was reported previously that a 20-kDa subunit of vaccinia RNA polymerase is encoded within the HindIll D fragment of the viral genome (5). The 16,060-base patr D fragment encodes 13 major open reading frames (ORFs), 4 of which predict proteins in the range of 17 to 25 kDa (8) and thus could conceivably encode the
’ To whom
Indiana
June 7 1, 7990
The polypeptide encoded by the vaccinia virus open reading frame D7R was of rabbits with the polypeptide resulted in antibodies that specifically recognized The immunoreactivity with the 20,000-dalton polypeptide was found to coincide dent RNA polymerase through DEAE-cellulose chromatography and glycerol argue that the product of the vaccinia open reading frame D7R is a subunit of the Press.
RNA Polymerase
requests
for reprints
should
RNA polymerase subunit. Appraisal of the extensive knowledge of the genetic organization of the HindIll D fragment suggests that the ORF D7R gene product is the most likely candidate for the 20.kDa RNA polymerase subunit. The D7R ORF predicts a 17,892-Da polypeptide (8), closest to that of the RNA polymerase subunit, and the gene is transcribed as an early viral gene (9). as is the RNA polymerase gene mapped to this segment of the genome (5). In order to determine if ORF D7R encodes an RNA polymerase subunit, we sought to produce antibodies specific to the D7R protein, synthesized in bacteria, and determine their reactlvtty toward purified vacclnia RNA polymerase. For this purpose, the D7R gene was placed under the control of a bacteriophage T7 promoter. The D7R gene was subcloned from the vaccinia HindIll D segment, and a Ndel endonuciease cleavage site was incorporated at the D7R initiation codon by oligonucleotide-directed mutagenesis (10). The gene was then inserted into the T7 expression vector PET3b (7 I), such that full-length D7R polypeptide devoid of any fusion sequences could be produced upon induction. D7R protein synthesis was induced upon infection with bacteriophage X CE6 (data not shown), which harbors the T7 RNA polymerase gene (12). An insoluble fraction was prepared from induced cells, and the D7R protein was purified by preparative SDS-polyacryamide gel electrophoresis as previously described ( 13). Immunization of rabbits with purified D7R protean resulted in antibody that reacted with a virion-associated polypeptide of about 20 kDa that was not detected with serum from rabbits not immunized (Fig. 1). The size of this polypeptide is very close to the 18 kDa predicted by the D7R ORF and is similar to that of the RNA polymerase subunit reportedly encoded in the HindIll D segment (5). The reactivity of the antr-D-/R antibody to the RNA polymerase was assessed by analyzing RNA polymer-
be addressed. 603
0042.6822190
$3.00
Copyright ic\ 1990 by Academic Press. Inc All rights of reproduction I” any form resewed
604
SHORT
COMMUNICATIONS
A
z30
E. coli
Pol
200 97 69 45
0
4
6
12
Fraction
D
2
4
6
6
10
12
16
20
24
Number
14
16
16
20
22
24
FIG. 1. Specificity of the antiD7R antiserum. Total vaccinia virion proteins were subjected to immunoblot analysis using preimmune serum (lane 1) or serum from a rabbit immunized with the D7R protein (lane 2). The mobility of protein standards in kDa is indicated on the right.
C
Fraction D
2
4
6
6
10
Number 12
14
16
16
20
22
24 -97 -66
-43
-31
Fraction
-21
Number
-14
B
V
U
10
12
14
16
16
20
22
24 -
97
- 67 -
46
-
31
-
14
FIG. 2. DEAE-cellulose chromatography of vaccinia RNA polymerase. An extract of vaccinia virions was chromatographed on DEAEcellulose. Protein was applied to the column in 0.05 M NaCI. 20 mM Tris-HCI, pH 8.0, 0.1 mM EDTA, 1 mM dithiothreitol, 0.01% NonidetP40, and 10% glycerol and developed with a 40.ml gradient of 0.05 to 0.40 M NaCl in the same buffer. Column fractions were assayed for RNA polymerase activity (A) as described previously (13) and for the D7R polypeptide by immunoblotting (B). Lane V is total vaccinia virion proteins, and lane U is unbound protein eluting as the flowthrough fraction,
FIG. 3. Glycerol gradient sedimentation of vaccinia RNA polymerase. DEAE-cellulose-purified RNA polymerase was sedimented on a 15-35% glycerol gradient containing 1 M NaCI, 20 mM HEPES. pH 7.9, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.01% NonidetP40 in a SW41Ti rotor at 40,000 rpm for 24 hr. Fractions were collected from tube bottoms and were assayed for RNA polymerase activity (A), total polypeptides by SDS-polyacrylamide gel electrophoresis on a 12% acrylamide gel and silver staining (B), and D7R polypeptide by immunoblotting (C). Lane D is RNA polymerase prior to sedimentation. The sedimentation position of E. co/i RNA polymerase in a parallel gradient is indicated in (A).
ase purified by two techniques. RNA polymerase was solubilized from virion cores (74) and purified by chromatography on DEAE-cellulose. As previously observed (3, 73) the RNA polymerase was bound quantitatively to the column and released with 0.2 M NaCl (Fig. 2). lmmunoblot analysis of the column fractions using the anti-D7R antiserum revealed that the 20-kDa
SHORT
605
COMMUNICATIONS
D7R polypeptide was also bound to DEAE-cellulose and eluted with the same general profile as the RNA polymerase. The RNA polymerase was purified further by sedimentation through a glycerol gradient containing 1 M NaCI. This method takes advantage of the extraordinarily large size of the RNA polymerase (about 500 kDa) and its correspondingly rapid sedimentation rate. Sodium chloride was included in the gradients to minimize nonspecific ionic association of other proteins with the RNA polymerase. Under these conditions, the vaccinia RNA polymerase sedimented at a rate almost identical with that of the 15 S fscherichia co/i RNA polymerase (15) (Fig. 3). SDS-polyactylamide gel electrophoresis of proteins in the glycerol gradient fractions (76) revealed several polypeptides cosedimenting with the RNA polymerase. Polypeptides with masses of 140, 130, 37, 34, 31, 24, 22, 20, and 19 kDa appear to be part of a large complex cosedimenting with the RNA polymerase. These sizes are consistent with previous descriptions of highly purified vaccinia RNA polymerase (2, 3). Polypeptides of 84 and 33 kDa also appear to cosediment with the RNA polymerase, but stain as though they are present in less than molar quantities. lmmunoblot analysis of the glycerol gradient fractions with anti-D7R antiserum revealed an immunoreactive 20-kDa polypeptide whose sedimentation coincided with that of the RNA polymerase. The results presented here argue that the polypeptide encoded by the vaccinia ORF D7R is a subunit of the viral RNA polymerase. The D7R poiypeptide copurified with the RNA polymerase through the DEAE-cellulose chromatography. Even more compelling is the cosedimentation of the D7R polypeptide with RNA polymerase on a high ionic strength glycerol gradient. Many proteins present in the DEAE-purified RNA polymerase preparation were observed to have sedimentation rates less than that of the polymerase itself and thus are not part of the RNA polymerase complex. The D7R protein sedimented at a rate consistent with that of a protein in excess of 400 kDa, identical with that of the RNA poiymerase, and therefore must be a subunit of the RNA polymerase. The protein encoded by the D7R ORF is known to be essential for virus growth as evidenced by temperature-sensitive mutations mapping within the confines of the D7R ORF (17). The phenotype of these mutants is a defective expression of late viral proteins at the nonpermissive temperature. This is similar to the phenotype of other temperature-sensitive mutants with lesions localized to the 22 and 147 kDa subunits of the vaccinia RNA polymerase (18). The defects in late pro-
tein expression are presumably due to defective late mRNA synthesis. This phenotype has been attributed to a requirement for synthesis of RNA polymerase subunits during the early phase that subsequently participate in the synthesis of late messages. The assignment of the D7R gene to the family of vaccinia RNA polymerase subunit genes should facilitate further studies on the assembly of the RNA polymerase complex and the determination of the roles of the individual subunits in the synthesis of viral mRNA. ACKNOWLEDGMENTS We thank Michael Merchllnsky for the clone of the vacclnla H/ndllI D segment, Stewart Shuman for valuable dIscussIons, and Bntta Feslerfortechnical assistance. This work was supported by Al28432 from the Natlonal lnstltute of Allergy and lnfectlous Diseases to S.S.B. and by a David Ross FellowshIp to S.D.C. This IS Paper 12588 from the Purdue Unlverslty Agricultural Experiment Station. /Vore added in proof. The D7R gene product an RNA polymerase subunit by another group and B. Moss, 1990. /. Viral. 64, 3019 -3024)
has been ldenttfled as (B.-Y Ahn. E. V. Jones,
REFERENCES 1. Moss, B., In “Virology” (B N. Fields and D. N. Knipe, Eds.), 2nd ed.. pp. 2079-21 11. Raven, New York, 1990. 2. NEVINS, J R., and JOKLIK, W. K., / B/o/ Chem. 252, 6930-6938 (1977). 3. BAROUDY. B. M., and Moss, B.. / B/o/ Chem. 255, 4372-4380 (1980) 4. SPENCER, E., SHUMAN, S., and HURWITZ, J / Viol. Chem. 255, 5388-5395 (1980). 5. JONES, E. V., PUCKETT, C., and Moss, 5.. i. Viral. 61, 1765-l 77 1 (1987). 6. BROYLES, S S., and Moss, B., Proc. Nat/. Acad. Sci. USA 83, 3141-3145(1986). 7. PATEL, D. D., and PICKUP, D. J., J. tirol. 63, 1076-1086 (1989). 8. NILES, E. G., CONDIT, R. C., CARO, P.. DAVIDSON, K., MATUSICK. L.. and SETO, J., virology 153, 96-l 12 (1986). 9. LEECHEN, G.-J., and NILES, E. G., Virology 163, 52-63 (1988). 10. KUNKEL, T. A., ROBERTS. J. D.. and ZAKOUR, R. A., in “Methods In Enzymology” (R. Wu and L. Grossman, Eds.), Vol. 154, pp. 367-382. Academic Press, San Diego, CA, 1987. 11. ROSENBERG, A. H., LADE, B. N., CHUI, D., LIN. H.-W., DUNN, 1. J., and STUDIER, F. W., Gene 56, 125--l 35 (1987). 12 STUDIER, F. W., and MOFFATT, B. A /. Mol. R/o/. 189, 113-~130 (1986). 13. BROYLES, S. S., and FESLER, B. S.. /. Viral. 64, 1523-l 529 (1990). 14. SHUMAN, S., SURKS, M., FURNEAUX. H., and HURWITZ, J., J. Biol. Chem. 255, 1 1,588-l 1,598. 15. BERG, D., BARRETT, K., and CHAMBERLIN, M., ic “Methods in Enzymology” (J. M. Lowensteln, Ed.), Vol. 2 1, pp. 506-519. Academic Press, San Diego, CA, 197 1. 16. LAEMMLI. U K., Nature (London) 227, 680.-685 (1970). 17 SETO, J., CELENZA, M., CONDIT, R C., and NIIES, E. G., Vkology 160,110~119(1987). 18. HOODA-DHINGRA, V., THOMPSON, C. L and CONDIT, R. C., Viroi 63,714.729 (1989).
178,603W305
Vaccinia
(1990)
Virus Gene D7R Encodes
a 20,000-Dalton STEVEN
Department
D.
QUICK
of Biochemistry, Received
April
Subunit AND
Purdue
STEVEN
University,
7 7, 1990; accepted
of the Viral DNA-Dependent S.
BROYLES’
West Lafayette,
47907
synthesized in bacteria. Immunization a virion polypeptide of 20,000 daltons. with the virion-associated DNA-depengradient sedimentation. These results c IssoAcademic viral RNA polymerase.
Inc.
Poxviruses are large DNA viruses that replicate in the host-cell cytoplasm. Because of their cytoplasmic habitat, poxviruses have evolved autonomous replication and transcription machinery [reviewed in (I)]. including the viral DNA-dependent RNA polymerase responsible for the synthesis of viral mRNA. The RNA polymerase has been purified to apparent homogeneity from vaccinia. the prototypal poxvirus, and has been shown to be a large, multisubunit complex of at least seven polypeptlde subunits (Z-4). Mapping of the RNA polymerase subunit genes with antibodies directed against the purified enzyme has demonstrated that all subunits are encoded by the viral genome (5). The gene sequences for three poxvirus RNA polymerase subunits have been reported (6, 7). The 147,000-dalton (Da) subunit of the vaccinia RNA polymerase and the 135 kDa subunit of the cowpox virus RNA polymerase are predicted to share considerable sequence homology with their respective counterparts from prokaryotes and eukaryotes, suggesting that these two subunits provide functions basic to the DNA-dependent polymerization of RNA. Because the genes for most of the smaller subunits have not yet been identified, it has not been possible to assess their respective roles in the RNA polymerase complex or their potential relatedness to the subunits of cellular polymerases. To this end, we have undertaken the identification of the genes for smaller RNA polymerase subunits. It was reported previously that a 20-kDa subunit of vaccinia RNA polymerase is encoded within the HindIll D fragment of the viral genome (5). The 16,060-base patr D fragment encodes 13 major open reading frames (ORFs), 4 of which predict proteins in the range of 17 to 25 kDa (8) and thus could conceivably encode the
’ To whom
Indiana
June 7 1, 7990
The polypeptide encoded by the vaccinia virus open reading frame D7R was of rabbits with the polypeptide resulted in antibodies that specifically recognized The immunoreactivity with the 20,000-dalton polypeptide was found to coincide dent RNA polymerase through DEAE-cellulose chromatography and glycerol argue that the product of the vaccinia open reading frame D7R is a subunit of the Press.
RNA Polymerase
requests
for reprints
should
RNA polymerase subunit. Appraisal of the extensive knowledge of the genetic organization of the HindIll D fragment suggests that the ORF D7R gene product is the most likely candidate for the 20.kDa RNA polymerase subunit. The D7R ORF predicts a 17,892-Da polypeptide (8), closest to that of the RNA polymerase subunit, and the gene is transcribed as an early viral gene (9). as is the RNA polymerase gene mapped to this segment of the genome (5). In order to determine if ORF D7R encodes an RNA polymerase subunit, we sought to produce antibodies specific to the D7R protein, synthesized in bacteria, and determine their reactlvtty toward purified vacclnia RNA polymerase. For this purpose, the D7R gene was placed under the control of a bacteriophage T7 promoter. The D7R gene was subcloned from the vaccinia HindIll D segment, and a Ndel endonuciease cleavage site was incorporated at the D7R initiation codon by oligonucleotide-directed mutagenesis (10). The gene was then inserted into the T7 expression vector PET3b (7 I), such that full-length D7R polypeptide devoid of any fusion sequences could be produced upon induction. D7R protein synthesis was induced upon infection with bacteriophage X CE6 (data not shown), which harbors the T7 RNA polymerase gene (12). An insoluble fraction was prepared from induced cells, and the D7R protein was purified by preparative SDS-polyacryamide gel electrophoresis as previously described ( 13). Immunization of rabbits with purified D7R protean resulted in antibody that reacted with a virion-associated polypeptide of about 20 kDa that was not detected with serum from rabbits not immunized (Fig. 1). The size of this polypeptide is very close to the 18 kDa predicted by the D7R ORF and is similar to that of the RNA polymerase subunit reportedly encoded in the HindIll D segment (5). The reactivity of the antr-D-/R antibody to the RNA polymerase was assessed by analyzing RNA polymer-
be addressed. 603
0042.6822190
$3.00
Copyright ic\ 1990 by Academic Press. Inc All rights of reproduction I” any form resewed
604
SHORT
COMMUNICATIONS
A
z30
E. coli
Pol
200 97 69 45
0
4
6
12
Fraction
D
2
4
6
6
10
12
16
20
24
Number
14
16
16
20
22
24
FIG. 1. Specificity of the antiD7R antiserum. Total vaccinia virion proteins were subjected to immunoblot analysis using preimmune serum (lane 1) or serum from a rabbit immunized with the D7R protein (lane 2). The mobility of protein standards in kDa is indicated on the right.
C
Fraction D
2
4
6
6
10
Number 12
14
16
16
20
22
24 -97 -66
-43
-31
Fraction
-21
Number
-14
B
V
U
10
12
14
16
16
20
22
24 -
97
- 67 -
46
-
31
-
14
FIG. 2. DEAE-cellulose chromatography of vaccinia RNA polymerase. An extract of vaccinia virions was chromatographed on DEAEcellulose. Protein was applied to the column in 0.05 M NaCI. 20 mM Tris-HCI, pH 8.0, 0.1 mM EDTA, 1 mM dithiothreitol, 0.01% NonidetP40, and 10% glycerol and developed with a 40.ml gradient of 0.05 to 0.40 M NaCl in the same buffer. Column fractions were assayed for RNA polymerase activity (A) as described previously (13) and for the D7R polypeptide by immunoblotting (B). Lane V is total vaccinia virion proteins, and lane U is unbound protein eluting as the flowthrough fraction,
FIG. 3. Glycerol gradient sedimentation of vaccinia RNA polymerase. DEAE-cellulose-purified RNA polymerase was sedimented on a 15-35% glycerol gradient containing 1 M NaCI, 20 mM HEPES. pH 7.9, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.01% NonidetP40 in a SW41Ti rotor at 40,000 rpm for 24 hr. Fractions were collected from tube bottoms and were assayed for RNA polymerase activity (A), total polypeptides by SDS-polyacrylamide gel electrophoresis on a 12% acrylamide gel and silver staining (B), and D7R polypeptide by immunoblotting (C). Lane D is RNA polymerase prior to sedimentation. The sedimentation position of E. co/i RNA polymerase in a parallel gradient is indicated in (A).
ase purified by two techniques. RNA polymerase was solubilized from virion cores (74) and purified by chromatography on DEAE-cellulose. As previously observed (3, 73) the RNA polymerase was bound quantitatively to the column and released with 0.2 M NaCl (Fig. 2). lmmunoblot analysis of the column fractions using the anti-D7R antiserum revealed that the 20-kDa
SHORT
605
COMMUNICATIONS
D7R polypeptide was also bound to DEAE-cellulose and eluted with the same general profile as the RNA polymerase. The RNA polymerase was purified further by sedimentation through a glycerol gradient containing 1 M NaCI. This method takes advantage of the extraordinarily large size of the RNA polymerase (about 500 kDa) and its correspondingly rapid sedimentation rate. Sodium chloride was included in the gradients to minimize nonspecific ionic association of other proteins with the RNA polymerase. Under these conditions, the vaccinia RNA polymerase sedimented at a rate almost identical with that of the 15 S fscherichia co/i RNA polymerase (15) (Fig. 3). SDS-polyactylamide gel electrophoresis of proteins in the glycerol gradient fractions (76) revealed several polypeptides cosedimenting with the RNA polymerase. Polypeptides with masses of 140, 130, 37, 34, 31, 24, 22, 20, and 19 kDa appear to be part of a large complex cosedimenting with the RNA polymerase. These sizes are consistent with previous descriptions of highly purified vaccinia RNA polymerase (2, 3). Polypeptides of 84 and 33 kDa also appear to cosediment with the RNA polymerase, but stain as though they are present in less than molar quantities. lmmunoblot analysis of the glycerol gradient fractions with anti-D7R antiserum revealed an immunoreactive 20-kDa polypeptide whose sedimentation coincided with that of the RNA polymerase. The results presented here argue that the polypeptide encoded by the vaccinia ORF D7R is a subunit of the viral RNA polymerase. The D7R poiypeptide copurified with the RNA polymerase through the DEAE-cellulose chromatography. Even more compelling is the cosedimentation of the D7R polypeptide with RNA polymerase on a high ionic strength glycerol gradient. Many proteins present in the DEAE-purified RNA polymerase preparation were observed to have sedimentation rates less than that of the polymerase itself and thus are not part of the RNA polymerase complex. The D7R protein sedimented at a rate consistent with that of a protein in excess of 400 kDa, identical with that of the RNA poiymerase, and therefore must be a subunit of the RNA polymerase. The protein encoded by the D7R ORF is known to be essential for virus growth as evidenced by temperature-sensitive mutations mapping within the confines of the D7R ORF (17). The phenotype of these mutants is a defective expression of late viral proteins at the nonpermissive temperature. This is similar to the phenotype of other temperature-sensitive mutants with lesions localized to the 22 and 147 kDa subunits of the vaccinia RNA polymerase (18). The defects in late pro-
tein expression are presumably due to defective late mRNA synthesis. This phenotype has been attributed to a requirement for synthesis of RNA polymerase subunits during the early phase that subsequently participate in the synthesis of late messages. The assignment of the D7R gene to the family of vaccinia RNA polymerase subunit genes should facilitate further studies on the assembly of the RNA polymerase complex and the determination of the roles of the individual subunits in the synthesis of viral mRNA. ACKNOWLEDGMENTS We thank Michael Merchllnsky for the clone of the vacclnla H/ndllI D segment, Stewart Shuman for valuable dIscussIons, and Bntta Feslerfortechnical assistance. This work was supported by Al28432 from the Natlonal lnstltute of Allergy and lnfectlous Diseases to S.S.B. and by a David Ross FellowshIp to S.D.C. This IS Paper 12588 from the Purdue Unlverslty Agricultural Experiment Station. /Vore added in proof. The D7R gene product an RNA polymerase subunit by another group and B. Moss, 1990. /. Viral. 64, 3019 -3024)
has been ldenttfled as (B.-Y Ahn. E. V. Jones,
REFERENCES 1. Moss, B., In “Virology” (B N. Fields and D. N. Knipe, Eds.), 2nd ed.. pp. 2079-21 11. Raven, New York, 1990. 2. NEVINS, J R., and JOKLIK, W. K., / B/o/ Chem. 252, 6930-6938 (1977). 3. BAROUDY. B. M., and Moss, B.. / B/o/ Chem. 255, 4372-4380 (1980) 4. SPENCER, E., SHUMAN, S., and HURWITZ, J / Viol. Chem. 255, 5388-5395 (1980). 5. JONES, E. V., PUCKETT, C., and Moss, 5.. i. Viral. 61, 1765-l 77 1 (1987). 6. BROYLES, S S., and Moss, B., Proc. Nat/. Acad. Sci. USA 83, 3141-3145(1986). 7. PATEL, D. D., and PICKUP, D. J., J. tirol. 63, 1076-1086 (1989). 8. NILES, E. G., CONDIT, R. C., CARO, P.. DAVIDSON, K., MATUSICK. L.. and SETO, J., virology 153, 96-l 12 (1986). 9. LEECHEN, G.-J., and NILES, E. G., Virology 163, 52-63 (1988). 10. KUNKEL, T. A., ROBERTS. J. D.. and ZAKOUR, R. A., in “Methods In Enzymology” (R. Wu and L. Grossman, Eds.), Vol. 154, pp. 367-382. Academic Press, San Diego, CA, 1987. 11. ROSENBERG, A. H., LADE, B. N., CHUI, D., LIN. H.-W., DUNN, 1. J., and STUDIER, F. W., Gene 56, 125--l 35 (1987). 12 STUDIER, F. W., and MOFFATT, B. A /. Mol. R/o/. 189, 113-~130 (1986). 13. BROYLES, S. S., and FESLER, B. S.. /. Viral. 64, 1523-l 529 (1990). 14. SHUMAN, S., SURKS, M., FURNEAUX. H., and HURWITZ, J., J. Biol. Chem. 255, 1 1,588-l 1,598. 15. BERG, D., BARRETT, K., and CHAMBERLIN, M., ic “Methods in Enzymology” (J. M. Lowensteln, Ed.), Vol. 2 1, pp. 506-519. Academic Press, San Diego, CA, 197 1. 16. LAEMMLI. U K., Nature (London) 227, 680.-685 (1970). 17 SETO, J., CELENZA, M., CONDIT, R C., and NIIES, E. G., Vkology 160,110~119(1987). 18. HOODA-DHINGRA, V., THOMPSON, C. L and CONDIT, R. C., Viroi 63,714.729 (1989).