Vol. 64, No. 5
JOURNAL OF VIROLOGY, May 1990, p. 2208-2216
0022-538X/90/052208-09$02.00/0 Copyright © 1990, American Society for Microbiology
Isolation and Characterization of Herpes Simplex Virus Mutants Containing Engineered Mutations at the DNA Polymerase Locus ALICE I. MARCY, DORNE R. YAGER, AND DONALD M. COEN* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
Received 1 November 1989/Accepted 25 January 1990 We have derived Vero cell lines containing the herpes simplex virus DNA polymerase (pol) gene that complement temperature-sensitive pol mutants. These cell lines were used to recover viruses containing new mutations at the pol locus. Two spontaneously arising host-range mutants, 6C4 and 7E4, were isolated. These mutants did not grow efficiently on Vero cells or synthesize late polypeptides but formed plaques on a cell line containing the pol gene (DP6 cells). Whereas mutant 6C4 specified a wild-type-size Pol protein, we detected no full-length Pol protein in 7E4-infected cell extracts. Complementation studies demonstrated that 6C4 and 7E4 contain different mutations and indicated that 6C4 is in a complementation group different from that of pol temperature-sensitive mutant tsC7 or tsD9. A mutant in which 2.2 kilobases of pol sequences were replaced with the Escherichia coli lacZ gene under the control of the herpes simplex virus thymidine kinase promoter was constructed. This mutant formed blue plaques on DP6 cells in the presence of 5-bromo-4-chloro-3-indolyl-o-Dgalactoside. Using this virus in marker rescue experiments, we engineered three mutants containing deletions in the pol coding region which grew efficiently on DP6 cells but not on Vero cells and which differed in their synthesis of Pol polypeptides. The lacZ insertion virus was also used to introduce a deletion in the region upstream of the pol long open reading frame, which removes a short open reading frame that could encode a 10-amino-acid peptide. This mutant grew to similar titers on Vero and DP6 cells, indicating that these sequences are not essential for growth of the virus in tissue culture.
The herpes simplex virus (HSV) DNA polymerase (Pol) is essential for viral DNA replication (1, 35, 40), serves as a target for antiviral drugs (9), and provides an excellent model for studies of eucaryotic replicative DNA polymerase in part because it is amenable to both biochemical and genetic analysis. Understanding Pol functions will be greatly facilitated by mutants defining protein domains that contribute to catalysis, interactions with other proteins, stability, and intracellular localization. Such a combined approach has already contributed to efforts to correlate Pol structure with its functions. Genetic mapping and sequencing of pol mutants exhibiting altered sensitivities to nucleoside and PPi analogs and aphidicolin indicate that amino acids lying in discrete regions between residues 597 and 961 contribute to substrate binding (17, 18, 27, 31, 32, 45). Further insights into Pol function have been provided by temperature-sensitive (ts) pol mutants (6, 10). Four pol ts mutants map within a 4-kilobase region that specifies a single major mRNA and long open reading frame (18, 23, 48). Genetic analysis of these mutants has identified three complementation groups (groups 1-3, 1-4, and 1-14), with complex overlapping patterns of complementation, consistent with the enzyme having multiple functions (6, 23, 39, 46). The prototypic mutant in complementation group 1-4, tsD9, exhibits altered drug sensitivity and contains a mutation in a domain thought to participate in substrate recognition (17, 18). Mutants in complementation group 1-3 specify thermolabile Pol activity in vivo (1) and in vitro (37) and map to sequences that include the left end of the gene (10, 23), suggesting that the N-terminal portion of the protein includes regions contributing to stability. The limits of sequences to which certain of these ts mutations have been mapped also include a short open reading frame which could *
encode a 10-amino-acid peptide (18). This short open reading frame lies 75 base pairs (bp) downstream of the pol transcriptional start site and ends 93 bp upstream of the long open reading frame (18, 48). Its role is unknown. The studies described here were initiated to facilitate our understanding of Pol functions by increasing the number and type of pol mutants available for biochemical and genetic analysis and to examine the importance of the 10-amino-acid upstream open reading frame for viral growth. We wished to be able to introduce site-specific mutations in the pol gene into the HSV genome to allow mutant Pol proteins to be analyzed in the context of a viral infection. Since we expected to recover viruses containing lethal pol mutations, we isolated a cell line able to support the growth of these viruses. As these mutants would not necessarily have a selectable phenotype, we also needed to develop a system for screening pol mutants. We demonstrate the use of a complementing cell line and a pol mutant that produces blue plaques to provide a system for the efficient recovery of pol mutants and describe the isolation and preliminary characterization of six new pol mutants, one of which lacks the 10-amino-acid open reading frame. MATERIALS AND METHODS Cells and viruses. Vero cells were grown and maintained as described previously (46). Nero cells are a cloned line of G418-resistant cells derived by transforming Vero cells with pSV2neo (42). HSV type 1 mutants tsC4, tsC7, and tsD9 were derived from wild-type strain KOS and have mutations conferring temperature sensitivity at the pol locus (10). Mutant hrR3, kindly provided by Sandra Weller, has a lacZ insert in the ICP6 gene (20). Plasmids. The plasmids used in this study are depicted in Fig. 1. Plasmid pDP1 contains the 153 bp upstream of the
Corresponding author. 2208
ENGINEERED HSV pol MUTANTS
VOL. 64, 1990
UL 0.0
MIap Units Restriction Sites
0.1
0.407
0.2
EJ 0.4
0.3
0.413
0.8
0.7
0.6
0.5
0.9
0.422
1.0 0.439
III
-I
Ss Sm Sm Bt
G SmSK
Bt
Sf B Sm
Bg
Sp
E
X
Sm
Ss
K
X SP B
K
DNA POLYMERASE
AAA
ATG
TGA
U LSORFF
Open Reading Fraimes
B
N L2Kbp
ICPB
Transcripts
2209
tsC7
ts Mutants
tsD9
tsC4
i
I
pKOS 29 Q
BamH pKEF-P4 ----
pSGI7 JF--
-
I.
EcoRl M
BamHI V pDP1
Plasmids
pDP2A
pDPaX pDP&S IpDPtkcBG
tk PROMOTER
lac Z
Bc pDP4
pDPtB
-
FIG. 1. Map location of HSV pol. The locations of pol transcript, open reading frames, and relevant restriction sites and the mapped limits of ts mutations are shown. The bottom portion of the figure represents structures of plasmids used in this study. Restriction enzymes are abbreviated as follows: B, BamHI; Bc, BcII; Bg, Bg!I; Bt, BstII; E, EcoRI; G, BgIII; K, KpnI; P, PstI; S, SaII; Sf, SfiI; Ss, SstI; Sm, SmaI; and X, XhoI. UL, Unique long sequence; Us, unique short sequence; SORF, short upstream open reading frame.
first ATG of the HSV pol open reading frame, the entire pol open reading frame, and 156 bp downstream of the termination TGA codon (HSV map coordinates 0.413 to 0.439). The plasmid was constructed by treating pKOS29 (10) with BamHI, filling in the overhanging ends by using Klenow fragment, ligating on HindlIl linkers, and digesting with HindIII and PstI. The 3.2-kbp HindIII-PstI fragment was then ligated to HindIII-PstI-digested pUC19 DNA (50) to give rise to plasmid p5'DP. Plasmid pSG17 (19) was treated with KpnI and then with T4 DNA polymerase to remove overhanging ends, ligated to XbaI linkers, and digested with PstI and XbaI. The 0.8-kbp PstI-XbaI fragment from this plasmid was recovered and ligated to PstI-XbaI-digested p5'DP to create pDP1. pDP1 still has the original BamHI site at 0.413 map units adjacent to the unique HindIII site. Plasmid pDP2A, which contains the pol open reading frame and promoter sequences, was made as follows. The 363-bp BstII-BamHI fragment containing the pol promoter and 51 bp of 5' untranslated sequences was removed from pKEFP4 (47), treated with Klenow fragment, and ligated to HindIIl linkers. The resulting fragment was digested with HindIII and ligated to HindIII-treated pDP1 to create pDP2A. Viral sequences in this plasmid differ from the viral genome by the presence of the HindIII site and a deletion starting 95 bp upstream of the mRNA start site and extending upstream into oriL for 62 bp (present in pKEFP4; 47, 48). This deletion does not remove the binding sites for transcription factors
Spl and TFIID upstream of the pol mRNA cap site (48), and the pol promoter in pDP2A retains activity, as judged by its ability to express pol in transfected cells upon HSV superinfection. Plasmid pDPtkBG was made by first inserting the 800-bp BamHI-BglII fragment containing the HSV thymidine kinase (tk) promoter from plasmid pKG3.6 (26) into BglII-digested pDPl. The resulting plasmid was digested with Sail, treated with Klenow fragment, ligated to BglII linkers, and then digested with BgIH. This DNA was then ligated to a 3.0-kbp BamHI fragment containing the coding sequences for Escherichia coli 3-galactosidase from pDP500, kindly provided by Dennis Panicali (34). Plasmids pDPAS and pDPAX were made by treating pDP1 with Sail or XhoI, respectively, and ligase. Site-specific mutagenesis was used to introduce a unique BclI site (TGATCA) 11 bp upstream of the pol open reading frame in pDP1 by a procedure developed by Taylor et al. (43). Mutagenesis was performed by using the oligonucleotide 5'-TCGGGTGIGAICAACCGC-3' and a 636-bp HindIII-SphI fragment of pDP1 cloned into the bacteriophage vector M13mpl9. The oligonucleotide hybridized to the noncoding pol strand, and the altered bases are underlined. Reagents for this procedure were provided in a commercial kit (Amersham Corp.). Clones containing the mutation were identified directly by DNA sequencing, using previously described procedures (18). Double-stranded rep-
2210
J. VIROL.
MARCY ET AL. TABLE 1. Efficiency of plating of HSV ts mutants on Vero and DP6 cells Mutation site
KOS tsD9 tsC4 tsC7 tsJ12
pol pol
Titer at 340C x 107 x 106 x 106
po/
4.2 9.3 3.8 9.5
gB
5.5 x 106
X 105
increase in
Titer at 390C
x 107
EOPa
Titer at 34 C
Titer at 39 C
EOP
titer at 39oCb
3.8 x 107 1.1 X 107 1.1 X 107
2.4 x 107 3.4 x 106 2.6 x 105
0.63 0.31 0.02
1.1 5.6 x 104 1.4 x 103
0.03
5.7 x 102 NDC
101
0.55
JOURNAL OF VIROLOGY, May 1990, p. 2208-2216
0022-538X/90/052208-09$02.00/0 Copyright © 1990, American Society for Microbiology
Isolation and Characterization of Herpes Simplex Virus Mutants Containing Engineered Mutations at the DNA Polymerase Locus ALICE I. MARCY, DORNE R. YAGER, AND DONALD M. COEN* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
Received 1 November 1989/Accepted 25 January 1990 We have derived Vero cell lines containing the herpes simplex virus DNA polymerase (pol) gene that complement temperature-sensitive pol mutants. These cell lines were used to recover viruses containing new mutations at the pol locus. Two spontaneously arising host-range mutants, 6C4 and 7E4, were isolated. These mutants did not grow efficiently on Vero cells or synthesize late polypeptides but formed plaques on a cell line containing the pol gene (DP6 cells). Whereas mutant 6C4 specified a wild-type-size Pol protein, we detected no full-length Pol protein in 7E4-infected cell extracts. Complementation studies demonstrated that 6C4 and 7E4 contain different mutations and indicated that 6C4 is in a complementation group different from that of pol temperature-sensitive mutant tsC7 or tsD9. A mutant in which 2.2 kilobases of pol sequences were replaced with the Escherichia coli lacZ gene under the control of the herpes simplex virus thymidine kinase promoter was constructed. This mutant formed blue plaques on DP6 cells in the presence of 5-bromo-4-chloro-3-indolyl-o-Dgalactoside. Using this virus in marker rescue experiments, we engineered three mutants containing deletions in the pol coding region which grew efficiently on DP6 cells but not on Vero cells and which differed in their synthesis of Pol polypeptides. The lacZ insertion virus was also used to introduce a deletion in the region upstream of the pol long open reading frame, which removes a short open reading frame that could encode a 10-amino-acid peptide. This mutant grew to similar titers on Vero and DP6 cells, indicating that these sequences are not essential for growth of the virus in tissue culture.
The herpes simplex virus (HSV) DNA polymerase (Pol) is essential for viral DNA replication (1, 35, 40), serves as a target for antiviral drugs (9), and provides an excellent model for studies of eucaryotic replicative DNA polymerase in part because it is amenable to both biochemical and genetic analysis. Understanding Pol functions will be greatly facilitated by mutants defining protein domains that contribute to catalysis, interactions with other proteins, stability, and intracellular localization. Such a combined approach has already contributed to efforts to correlate Pol structure with its functions. Genetic mapping and sequencing of pol mutants exhibiting altered sensitivities to nucleoside and PPi analogs and aphidicolin indicate that amino acids lying in discrete regions between residues 597 and 961 contribute to substrate binding (17, 18, 27, 31, 32, 45). Further insights into Pol function have been provided by temperature-sensitive (ts) pol mutants (6, 10). Four pol ts mutants map within a 4-kilobase region that specifies a single major mRNA and long open reading frame (18, 23, 48). Genetic analysis of these mutants has identified three complementation groups (groups 1-3, 1-4, and 1-14), with complex overlapping patterns of complementation, consistent with the enzyme having multiple functions (6, 23, 39, 46). The prototypic mutant in complementation group 1-4, tsD9, exhibits altered drug sensitivity and contains a mutation in a domain thought to participate in substrate recognition (17, 18). Mutants in complementation group 1-3 specify thermolabile Pol activity in vivo (1) and in vitro (37) and map to sequences that include the left end of the gene (10, 23), suggesting that the N-terminal portion of the protein includes regions contributing to stability. The limits of sequences to which certain of these ts mutations have been mapped also include a short open reading frame which could *
encode a 10-amino-acid peptide (18). This short open reading frame lies 75 base pairs (bp) downstream of the pol transcriptional start site and ends 93 bp upstream of the long open reading frame (18, 48). Its role is unknown. The studies described here were initiated to facilitate our understanding of Pol functions by increasing the number and type of pol mutants available for biochemical and genetic analysis and to examine the importance of the 10-amino-acid upstream open reading frame for viral growth. We wished to be able to introduce site-specific mutations in the pol gene into the HSV genome to allow mutant Pol proteins to be analyzed in the context of a viral infection. Since we expected to recover viruses containing lethal pol mutations, we isolated a cell line able to support the growth of these viruses. As these mutants would not necessarily have a selectable phenotype, we also needed to develop a system for screening pol mutants. We demonstrate the use of a complementing cell line and a pol mutant that produces blue plaques to provide a system for the efficient recovery of pol mutants and describe the isolation and preliminary characterization of six new pol mutants, one of which lacks the 10-amino-acid open reading frame. MATERIALS AND METHODS Cells and viruses. Vero cells were grown and maintained as described previously (46). Nero cells are a cloned line of G418-resistant cells derived by transforming Vero cells with pSV2neo (42). HSV type 1 mutants tsC4, tsC7, and tsD9 were derived from wild-type strain KOS and have mutations conferring temperature sensitivity at the pol locus (10). Mutant hrR3, kindly provided by Sandra Weller, has a lacZ insert in the ICP6 gene (20). Plasmids. The plasmids used in this study are depicted in Fig. 1. Plasmid pDP1 contains the 153 bp upstream of the
Corresponding author. 2208
ENGINEERED HSV pol MUTANTS
VOL. 64, 1990
UL 0.0
MIap Units Restriction Sites
0.1
0.407
0.2
EJ 0.4
0.3
0.413
0.8
0.7
0.6
0.5
0.9
0.422
1.0 0.439
III
-I
Ss Sm Sm Bt
G SmSK
Bt
Sf B Sm
Bg
Sp
E
X
Sm
Ss
K
X SP B
K
DNA POLYMERASE
AAA
ATG
TGA
U LSORFF
Open Reading Fraimes
B
N L2Kbp
ICPB
Transcripts
2209
tsC7
ts Mutants
tsD9
tsC4
i
I
pKOS 29 Q
BamH pKEF-P4 ----
pSGI7 JF--
-
I.
EcoRl M
BamHI V pDP1
Plasmids
pDP2A
pDPaX pDP&S IpDPtkcBG
tk PROMOTER
lac Z
Bc pDP4
pDPtB
-
FIG. 1. Map location of HSV pol. The locations of pol transcript, open reading frames, and relevant restriction sites and the mapped limits of ts mutations are shown. The bottom portion of the figure represents structures of plasmids used in this study. Restriction enzymes are abbreviated as follows: B, BamHI; Bc, BcII; Bg, Bg!I; Bt, BstII; E, EcoRI; G, BgIII; K, KpnI; P, PstI; S, SaII; Sf, SfiI; Ss, SstI; Sm, SmaI; and X, XhoI. UL, Unique long sequence; Us, unique short sequence; SORF, short upstream open reading frame.
first ATG of the HSV pol open reading frame, the entire pol open reading frame, and 156 bp downstream of the termination TGA codon (HSV map coordinates 0.413 to 0.439). The plasmid was constructed by treating pKOS29 (10) with BamHI, filling in the overhanging ends by using Klenow fragment, ligating on HindlIl linkers, and digesting with HindIII and PstI. The 3.2-kbp HindIII-PstI fragment was then ligated to HindIII-PstI-digested pUC19 DNA (50) to give rise to plasmid p5'DP. Plasmid pSG17 (19) was treated with KpnI and then with T4 DNA polymerase to remove overhanging ends, ligated to XbaI linkers, and digested with PstI and XbaI. The 0.8-kbp PstI-XbaI fragment from this plasmid was recovered and ligated to PstI-XbaI-digested p5'DP to create pDP1. pDP1 still has the original BamHI site at 0.413 map units adjacent to the unique HindIII site. Plasmid pDP2A, which contains the pol open reading frame and promoter sequences, was made as follows. The 363-bp BstII-BamHI fragment containing the pol promoter and 51 bp of 5' untranslated sequences was removed from pKEFP4 (47), treated with Klenow fragment, and ligated to HindIIl linkers. The resulting fragment was digested with HindIII and ligated to HindIII-treated pDP1 to create pDP2A. Viral sequences in this plasmid differ from the viral genome by the presence of the HindIII site and a deletion starting 95 bp upstream of the mRNA start site and extending upstream into oriL for 62 bp (present in pKEFP4; 47, 48). This deletion does not remove the binding sites for transcription factors
Spl and TFIID upstream of the pol mRNA cap site (48), and the pol promoter in pDP2A retains activity, as judged by its ability to express pol in transfected cells upon HSV superinfection. Plasmid pDPtkBG was made by first inserting the 800-bp BamHI-BglII fragment containing the HSV thymidine kinase (tk) promoter from plasmid pKG3.6 (26) into BglII-digested pDPl. The resulting plasmid was digested with Sail, treated with Klenow fragment, ligated to BglII linkers, and then digested with BgIH. This DNA was then ligated to a 3.0-kbp BamHI fragment containing the coding sequences for Escherichia coli 3-galactosidase from pDP500, kindly provided by Dennis Panicali (34). Plasmids pDPAS and pDPAX were made by treating pDP1 with Sail or XhoI, respectively, and ligase. Site-specific mutagenesis was used to introduce a unique BclI site (TGATCA) 11 bp upstream of the pol open reading frame in pDP1 by a procedure developed by Taylor et al. (43). Mutagenesis was performed by using the oligonucleotide 5'-TCGGGTGIGAICAACCGC-3' and a 636-bp HindIII-SphI fragment of pDP1 cloned into the bacteriophage vector M13mpl9. The oligonucleotide hybridized to the noncoding pol strand, and the altered bases are underlined. Reagents for this procedure were provided in a commercial kit (Amersham Corp.). Clones containing the mutation were identified directly by DNA sequencing, using previously described procedures (18). Double-stranded rep-
2210
J. VIROL.
MARCY ET AL. TABLE 1. Efficiency of plating of HSV ts mutants on Vero and DP6 cells Mutation site
KOS tsD9 tsC4 tsC7 tsJ12
pol pol
Titer at 340C x 107 x 106 x 106
po/
4.2 9.3 3.8 9.5
gB
5.5 x 106
X 105
increase in
Titer at 390C
x 107
EOPa
Titer at 34 C
Titer at 39 C
EOP
titer at 39oCb
3.8 x 107 1.1 X 107 1.1 X 107
2.4 x 107 3.4 x 106 2.6 x 105
0.63 0.31 0.02
1.1 5.6 x 104 1.4 x 103
0.03
5.7 x 102 NDC
101
0.55
