Proc. Nati. Acad. Sci. USA Vol. 91, pp. 4554-4558, May 1994 Targeted construction of temperature-sensitive mutations in vaccinia virus by replacing clustered charged residues with alanine (viral genetics/in vitro mutagenesis/clustered charge-to-alanine mutgenes /transient dominant selection) DANIEL E. HASSETT AND RICHARD C. CONDIT Department of and Medical , University of Florida, Gainesville, FL 32610-0266 Communicated by Frank Fenner, January 24, 1994 (receivedfor review November IS, 1993)

ABSTRACT The feasibility of using "clustered charge-to- Charged residues that are clustered in the primary se- alanine" mutagenesis (replacement by alanine of two or more quence of a polypeptide cannot be efficiently accommodated charged residues clustered in a five- or six-amino acid se- in the hydrophobic core of the protein; therefore, they are quence) to create temperature-sensitive, conditionally lethal most likely to be found on the surface of the folded protein. mutations in vaccinia virus was examined by creating nine The replacement of multiple, charged residues with alanine mutations in the vaccinia virus gene G2R. G2R was chosen for residues has been shown to disrupt protein-protein interac- this analysis because mutations in this gene confer selectable tions in vitro while not severely altering protein secondary phenotypes. Specifically, vaccinia viruses that contain a wild- structure (1). When this approach was applied to the Sac- type copy ofG2R are sensitive to the effects ofthe anti-poxvirus charomyces cervisiae actin gene (ACT)), of 34 mutations in constructed, 44% had a temperature-sensitive phenotype in drug isatin-3-thiosemicarbazone (IBT), while mutations vivo (2). Likewise, clustered charge-to-alanine mutagenesis G2R that completely abolish the function of the G2R protein ofthe poliovirus 3D polymerase resulted in a high proportion product confer-dependence upon IBT for growth. A previously (37% of 27 mutants) of temperature-sensitive mutants (3). isolated mutant carrying a temperature-sensitive mutation that Thus, alterations of a protein's surface charge may disrupt maps to G2R (Cts56) is resistant to IBT at the permissive interprotein or intraprotein interactions and result in temper- temperature and dependent upon IBT at the restrictive tem- ature sensitivity in vivo. perature. Nine clustered charge-to-alanine mutants were ex- Our laboratory's interest is in the genetic analysis of the amined. Four of these mutants (AS1, AS4, AS6, and AS9) orthopoxvirus vaccinia (4-6). The genome of vaccinia con- display some degree of temperature sensitivity in the function sists ofapproximately 192 kilobases ofdouble-stranded DNA of the G2R gene product. ASi is temperature sensitive for with covalently closed ends. The poxviruses are unique growth in both a plaque assay and in a one-step growth among DNA viruses in that they replicate in the cytoplasm of experiment. AS6 and AS9 form small plaques at the nonper- infected cells. Thus, the virus encodes virtually all of the missive temperature and are temperature sensitive for growth enzymes required for viral RNA and DNA metabolism. in a one-step growth experiment. AS4 manifests its tempera- Vaccinia has proven to be an excellent model system for ture sensitivity as temperature-dependent IBT resistance. Five studying transcription, DNA replication, and the regulation of the mutations (AS2, AS3, AS5, AS7, and AS8) appeared to of gene expression. Numerous enzymatic activities common confer phenotypes inditinguishable from that of wild-type to other eukaryotic organisms have been shown to be en- vaccinia. These results demonstrate that temperature-sensitive coded by the virus. Previous genetic studies in our laboratory conditionally lethal mutants can be created in vaccinia virus by led to the isolation of conditionally lethal temperature- altering the charge characteristics of essential viral proteins. sensitive mutations in 30 separate vaccinia virus complemen- tation groups. These mutants have proven valuable for study- ing all aspects of viral replication. However, vaccinia virus Traditional approaches to viral genetics, which involve ran- has approximately 150 essential genes; therefore, our collec- dom mutagenesis of the virus followed by screening for tion represents mutations in only 20%o of the essential virus phenotypes of interest, have proven to be quite useful in the genes. A directed genetic method that produces temperature- genetic and biochemical characterization of complex DNA- sensitive mutations in specific genes would allow us to containing viruses. However, random mutagenesis methods genetically study many of the viral genes in which we are limited by their inability to target specific open reading presently have no conditionally lethal mutants. frames and by the laborious screening methods required to We have created a series of nine mutations in the vaccinia identify mutants ofinterest. It is for these reasons that a more virus G2R locus by replacing clusters of charged amino acid direct approach for the creation of temperature-sensitive residues with alanines in an attempt to develop a directed virus mutants in specific viral genes must be found. Practical genetic approach for the creation of temperature-sensitive application of directed genetics to a complex DNA virus mutants in specific vaccinia virus genes. requires an in vitro mutagenesis scheme that produces a high proportion of conditionally lethal mutants combined with an efficient method for targeted replacement of the wild-type MATERIALS AND METHODS viral gene with the mutant allele. We report here a directed Cells and Virus. BSC40 cells, wild-type vaccinia virus method for the creation oftemperature-sensitive mutations in strain WR, virus mutants Cts56 and G2A, methods for cell vaccinia virus that combines the techniques of "clustered culture, virus growth, plaque titration, one-step growth, and charge-to-alanine" mutagenesis (replacement by alanine of marker rescue have been described (7-10). Viral stocks ofthe two or more charged residues clustered in a five- or six-amino clustered charge-to-alanine mutants AS1, AS4, AS6, and acid sequence) and transient dominant selection. AS9 were grown on BSC40 cells at 31'C as described for Cts56. CV1 cells were maintained in Dulbecco's modified The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: IBT, isatin-,fthiosemicarbazone; MPA, mycophe- in accordance with 18 U.S.C. §1734 solely to indicate this fact. nolic acid. 4554 Downloaded by guest on October 1, 2021 Genetics: Hassett and Condit Proc. Natl. Acad. Sci. USA 91 (1994) 4555 Eagle's medium (GIBCO) supplemented with nonessential RESULTS amino acids (Sigma), sodium pyruvate (Sigma), and 10% (vol/vol) fetal calf serum (GIBCO). To evaluate the potential of clustered charge-to-alanine mu- Mutagenesis and Cloning. Oligonucleotide-directed muta- tagenesis as a means of creating conditionally lethal vaccinia genesis was performed with the Amersham oligonucleotide virus, a series ofnine mutations were created in the G2R gene mutagenesis kit. Mutagenic oligonucleotides, synthesized by by replacing clusters of charged residues by alanine residues the DNA synthesis core facility of the University of Florida, as described below (Fig. 1). G2R was chosen for the test were designed to include 12 nucleotides 5' and 9 nucleotides because mutations in this gene confer easily selectable phe- 3' of the mismatched region (11). notypes (10), thus circumventing the necessity for screening Mutant G2R-containing plasmids for transient dominant large numbers of individual plaque isolates to determine the selection were constructed by using two similar protocols. phenotype conferred by a particular mutation. The selection For wild-type G2R and for the AS1 and Cts56 mutants, a is based on the sensitivity of the virus to the anti-poxvirus plasmid pUC118G was constructed that consists ofthe phage- drug IBT. Plaque formation by wild-type vaccinia virus was mid vector pUC118 (12), a cassette containing the constitu- inhibited by IBT, deletion mutants in G2R such as G2A were dependent on IBT for growth, and a mutant carrying a tive "7.5K" vaccinia promoter [which drives expression of previously isolated temperature-sensitive mutation that maps the vaccinia virus 7.5-kDa polypeptide (14)] upstream from to G2R (Cts56) was resistant to IBT at the permissive the Escherichia coli guanine phosphoribosyltransferase (gpt) temperature (310C) and dependent upon IBT at the restrictive gene [obtained as a 1.1-kb Hpa I-Dra I fragment from temperature (400C) (Fig. 2 Upper). The function of the G2R pTKgpt-Fls (13)] and a 982-base-pair Pvu II-EcoRV frag- gene and the precise mechanism of action of IBT are un- ment derived from the vaccinia virus HindIII G fragment that known. The predicted amino acid sequence ofthe G2R gene contains the wild-type G2R gene with 82 nucleotides of 5' product is shown in Fig. 1 (10). For the mutagenesis, a charge flanking sequence and 236 nucleotides of3' flanking sequence cluster was defined as two or more charged amino acids (10). The G2R-containing DNA was placed adjacent to and within a five-amino acid window (2). With the exception of upstream from the 7.5K promoter, with the G2R sequences AS2, each ofthe charged residues in the window was changed in the opposite transcriptional orientation relative to the 7.5K to alanine. For the mutant AS1, five charged amino acids in promoter. Single-stranded pUC118G phagemid DNA (12) a six-amino acid window were each mutated to alanines. was used as a template for mutagenesis to construct mutants Mutations were engineered into a cloned copy of G2R by AS1 and Cts56, and transient dominant selections were done using oligonucleotide-directed mutagenesis, and the mutant by using double-stranded plasmid DNA containing either G2R genes were then inserted into the viral chromosome by wild-type or mutant G2R sequences. For AS2 through AS9, using transient dominant selection (14). Specifically, individ- mutagenesis of G2R was done by using single-stranded DNA ual mutant G2R genes were cloned into a plasmid vector from an M13mpl9 clone containing only the coding sequence adjacent to a cassette containing the E. coli gpt gene under of G2R, obtained by PCR amplification. Mutant G2R genes the transcriptional control of a vaccinia-specific promoter. were then subcloned into a plasmid containing pGEM- The plasmids were then recombined into the vaccinia genome 3ZF(-) (Promega) vector DNA and the 7.5K-gpt cassette by transfection of wild-type vaccinia virus-infected cells. described above for conferring resistance to mycophenolic Recombinant viruses resulting from a single crossover event acid (MPA). The G2R gene was placed adjacent to and between plasmid G2R sequences and homologous G2R se- upstream from the 7.5K promoter, with the G2R sequences quences on the viral chromosome contain the E. coli gpt in the same transcriptional orientation relative to the 7.5K cassette flanked by two copies ofG2R, a mutant copy derived promoter. The transcription start site of G2R has been from the plasmid and the wild-type chromosomal copy. mapped to bases 10335 of the vaccinia virus strain WR Expression of gpt provides these viruses with resistance to HindIII G fragment; therefore, none of the cloned G2R the purine biosynthesis inhibitor MPA; thus, recombinants sequences used in this study contains the promoter for the can be selected by plating the lysate from the infected, G2R gene (10). The complete G2R sequence of each mutant was confirmed by DNA sequencing with the Sequenase AS 2 version 2.0 kit (United States Biochemical). 1 MPFRDLILFN LSKFLLTEDE ESLEIVSSLC RGFEISYDDL Transient Dominant Selection and G2R Mutant Screen. Transient dominant selection was performed essentially as AS 1ts AS 9 tS described by Falkner and Moss (14). Briefly, CV1 cells were 41 ITYFPDRKYH KYISKVFEHV DLSEELSMEF HDTTLRDLVY pretreated with MPA, infected with wild-type vaccinia virus G2Ad ta (multiplicity of infection = 0.05 plaque-forming units per KCV IN* AS 6 cell), transfected with 20 ,ug of plasmid DNA using Trans- 81 LRLYKYSKCI RPCYKLGDNL KGIvvIKDRN IYIREANDDL at for 3 Wt AS8" t fectase (Gibco-BRL), and incubated 37°C days. AS 8 Cts56 Infected, transfected cells were harvested and titrated for 12111IEYLLIMYTPELT7 QIYTYSNERV PITGSKLILCI CGRSQGFSQVTFMAY on in the MPA. plaque formation CV1 cells presence of tdr wt Individual MPA-resistant plaques were picked and titrated AS 4 M 5 for plaque formation on BSC40 cells in the presence and 161 ITSHITTNKK VDVLVSKKCI DELVDPINYQ ILQNLF KGS absence of 45 ,M isatin-,B-thiosemicarbazone (IBT) at 31°C and 40°C. To determine their phenotypes, individual IBT- 201 GTINKILRKI FYSVTGGQTP resistant plaques were picked and then replaqued in the presence and absence of IBT at 31°C and 40°C. Individual FIG. 1. Deduced amino acid sequence of the vaccinia virus G2R G2R mutant viruses were then replaque-purified and grown gene product and G2R mutant products. Charged residues are as described above. The G2R gene from mutant viral DNA indicated by boldface type. Enlarged boldface type denotes charged was PCR-amplified, and appropriate selected regions of the residues mutated to alanine in a given mutant. The amino acid changes corresponding to mutants Cts56 (Gly-151 - Arg) and G2A G2R PCR product were sequenced to confirm the presence of (a 10-bp deletion/frameshift starting at residue 90) are designated the desired mutations in Cts56, AS1, AS4, AS6, and AS9 above the appropriate residues. AS2 and AS9 mutations are indicated viruses. Temperature sensitivity in the AS1 virus was by lines above and below the amino acid sequence, respectively. wt, mapped to gene G2R by marker rescue by using a clone Wild type; ts, temperature sensitive; tdr, temperature dependent IBT containing only the wild-type G2R gene. resistance; d, IBT dependent; *, termination of G2R in mutant G2A. Downloaded by guest on October 1, 2021 4556 Genetics: Hassett and Condit Proc. Nad. Acad. Sci. USA 91 (1994)

310-IE3T 310+IBT 4004BT 400+IBT Table 1. Screening MPA-resistant plaques for ..~_- mutant recombinants Plasmid DNA Plaque 310C 400C % wt transfected no. - IBT + IBT - IBT + IBT mutant* Wild type 1 501 0 629 0 0 2 363 0 512 0 0 3 29 0 38 0 0 4 184 0 596 0 0 G2A 5 295 0 326 0 0 Cts56 1 168 5 293 17 10 2 45 9 72 29 63 3 344 17 425 82 24 4 217 6 230 27 12 Cts56 5 367 25 387 104 28 AS1 1 =1500 93 -1500 60 4 2 =1500 116 =1500 70 5 3 =1500 69 =2000 138 9 4 31500 32 2000 56 4 AS 1 5 m1500 258 -2000 268 18 I Transient dominant selection was carried out with plasmids con- taining either a wild type-G2R gene, Cts56, or AS1. Five MPA- resistant plaques from each transfection were titered at 310C and 400C in the presence and absence of IBT. The data shown indicate 310-IBT 310+IBT 400-IBT 400+1 BT the total number of plaques produced per 6-cm dish under each condition. *The percent mutant was calculated by dividing the number of AS 4 plaques at 400C +IBT by the number of plaques at 31°C -IBT. Transfection with aplasmid containing a known temperature- sensitive mutation of G2R (Cts56) generated MPA-resistant plaques, most of which contained a population of IBT- resistant viruses. The percentage of IBT-resistant viruses AS 6 contained in individual MPA-resistant plaques was variable. IO: This variability most likely reflected the efficiency and the timing ofthe recombination event that resolved the G2R gene duplication. For reasons we do not understand, the average -.dLjLwL. percentage of IBT-resistant viruses obtained in this experi- AS 9 ment was somewhat higher than in subsequent experiments with mutants AS1-AS9 (see below). IBT-resistant plaques resulting from the Cts56 transient dominant selection were plaque-purified, grown, subject to DNA sequence analysis, tested for temperature and IBT sensitivity, and shown to be FIG. 2. Plaque formation of G2R mutant viruses. Plaque assays indistinguishable from the original Cts56 virus (Figs. 2 and 3). were incubated in the presence of 45 /AM IBT or in the absence of drug at 31'C and 400C for 1 week and stained with crystal violet. Transient dominant selection was repeated with plasmids containing G2R sequences corresponding to the nine clus- transfected cells in the presence of MPA. During the growth tered charge-to-alanine mutants depicted in Fig. 1. Transient of each MPA-resistant plaque, resolution occurs via recom- dominant selection done with four ofthe mutants (AS1, AS4, bination between the two genomic copies ofG2R, resulting in AS6, and AS9) generated a significant fraction of IBT- viruses that have lost the gpt gene and contain only a single resistant viruses similar to the results obtained with the Cts56 copy ofthe G2R gene. Therefore, each MPA-resistant plaque plasmid. The data from a representative experiment with the contains a mixture ofviruses, some ofwhich contain a mutant AS1 plasmid are shown in Table 1. In this and subsequent copy of G2R derived from the transfected plasmid, while experiments, plaque assays in the presence of MPA were others retain a wild-type copy ofG2R. Approximately 10% of incubated for a longer period of time than in transfections the viruses in each plaque did not resolve (data not shown). with wild type and Cts56; therefore a larger total number of Therefore, these unresolved viruses retain the gpt gene and viruses (columns labeled "-IBT") were recovered from serve a helper function for growth of resolved viruses in the these MPA-resistant plaques. In the course of these experi- presence of MPA. ments, we assayed a total of 70 MPA-resistant plaques from Quantitation of Mutant Progeny in MPA-Resistant Plaques. transient dominant selection with Cts56, AS1, AS4, AS6, or To determine the efficiency of the transient dominant selec- AS9 plasmid. The fraction of IBT-resistant viruses in these tion protocol, experiments were conducted with plasmids MPA-resistant plaques ranged from 0%6 to 63%, and the containing either the wild-type G2R gene or the temperature- average number of IBT-resistant viruses for all 70 MPA- sensitive G2R allele, Cts56. Table 1 shows the results of resistant plaques was 10.6%. Repeated serial plaque purifi- plating five independently isolated MPA-resistant plaques cation ofMPA-resistant plaques did not signifcantly alter the from each transfection in the presence and absence of IBT at average number of IBT-resistant viruses observed. MPA- 31'C and 40'C. MPA-resistant plaques isolated after trans- resistant plaques isolated from five of these transfections fection with a wild-type copy of G2R contained no IBT- (AS2, AS3, ASS, AS7, and AS8) yielded no IBT-resistant resistant viruses, showing that the background level of spon- progeny viruses above background levels. The simplest ex- taneous IBT resistance in these experiments was negligible. planation for this result is that these mutations confer phe- Downloaded by guest on October 1, 2021 Genetics: Hassett and Condit Proc. Natl. Acad. Sci. USA 91 (1994) 4557

ouu1 100 wild 100 Cts56 100 AS1 1 type 10 10 .) 10 0 10 'a 'a- 1 wa 1 t 0.1 0.1 0.1

^ ^4_ 0.01 -I 0.01 l l 0 10 20 30 40 50 60 70 8( ) 10 20 30 40 50 60 70 80 10 20 30 40 50 60 70 S0 hours post infection hours post infection hours post infection

C)

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10 20 30 40 50 60 70 80 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 hours post infection hours post infection hours post infection

FIG. 3. One-step growth experiments of wild type and of G2R mutant viruses. One-step growth experiments were done at a multiplicity of infection of 6 under both permissive and nonpermissive conditions. Samples were taken at various times after infections and titered under permissive conditions. *, 31'C; *, 40TC. notypes indistinguishable from that of wild-type vaccinia. inspection of AS4 and AS9 revealed subtle differences in These mutations were not studied further. plaque size under the various conditions tested. For exam- Plaque Formation by Clustered Charge to Alanine Mu- ple, in the absence of IBT, AS9 produced comparably sized tants. IBT-resistant plaques from transient dominant selec- plaques at 40'C and 31'C; however, at 40TC, it produced tions done with AS1, AS4, AS6, and AS9 were replaque- slightly larger plaques in the presence of IBT than it did in purified, grown, and subjected to additional phenotypic the absence ofdrug. In contrast, in the absence of IBT, AS4 analysis. Plaque assays demonstrating the phenotypes of produced larger plaques at 40'C compared with those at these four mutants are shown in Fig. 2. Our experience with 31TC, and at 40'C it produced smaller plaques in the G2R indicates that subtle differences in vaccinia's response presence of IBT than it did in the absence of drug. These to IBT reflect variation in the activity of the G2R protein results indicate that in both mutants G2R function is normal product. Whereas viruses with wild-type G2R activity are at 310C and compromised at 40'C and that the defect in G2R sensitive to IBT and complete loss of G2R function results function at 40'C is more pronounced in AS9 than in AS4. It in viruses that are IBT dependent, resistance to IBT most is noteworthy that in the absence of IBT at 40TC, plaque likely reflects intermediate levels ofG2R activity. Thus, the formation by AS4 was indistinguishable from wild-type resistance of Cts56 mutant to IBT at 31'C indicates that it virus, whereas plaque formation by AS1, AS6, and AS9 was function even under is somewhat compromised for G2R in case measurably defective relative to wild-type conditions. With this in it is each permissive perspective mind, virus. apparent that AS1, AS4, AS6, and AS9 each displayed One-Step Growth Experiments. One-step growth experi- temperature-sensitive G2R function, as detailed below. ments were performed to further characterize the pheno- of the mutants revealed a gradi- Furthermore, comparison types of the clustered charge-to-alanine mutants ASi, AS4, ent of G2R temperature sensitivity, where AS1 was the most temperature sensitive followed in order by AS6, AS9, AS6, and AS9 and to compare them to wild-type virus and mutant virus grown at 310C or and AS4. AS1 was resistant to IBT at 31'C, and IBT the Cts56 (Fig. 3). Wild-type a and 200 dependent at 40'C, thus G2R function in AS1 was compro- 40'C showed burst size between 10 plaque- mised at 31TC and absent at 40'C, comparable to Cts56. By forming units per cell with maximum yield occurring 24-48 contrast to Cts56, minute plaques became visible after hours after infection. When grown at 31TC, all of the prolonged incubation of AS1 at 40TC in the absence of IBT mutants produced normal amounts of infectious virus with (data not shown). AS6 was IBT resistant at both 31'C and kinetics similar to that of wild-type virus. At 40TC, the 40'C, but the relative plaque sizes in the presence and growth of AS4 was indistinguishable from that of wild-type absence of IBT at the two temperatures were different. At virus. At 40TC, ASi, AS6, and AS9 were unable to produce 31'C, AS6 formed larger plaques in the absence than in the significant levels of infectious progeny virus, identical to presence of IBT, whereas at 40'C AS6 formed smaller Cts56. These data demonstrate that the clustered charge- plaques in the absence of IBT than in the presence of IBT. to-alanine mutants ASi, AS6, and AS9 are clearly temper- Thus, G2R function in AS6 is compromised at both tem- ature sensitive for growth. It is interesting that in this assay, peratures, but the defect is more pronounced at 40'C than AS6 and AS9 appeared to be as temperature sensitive for at 31'C. The mutants AS4 and AS9 both displayed temper- growth as both Cts56 and ASi, even though they formed ature-dependent IBT resistance-that is, they were sensi- plaques at 40'C (Fig. 2). The most likely explanation for this tive to IBT at 31'C and resistant to IBT at 40'C. Close finding is that a very low yield of virus, undetectable in a Downloaded by guest on October 1, 2021 4558 Genetics: Hassett and Condit Proc. Natl. Acad Sci. USA 91 (1994) one-step growth experiment, may be sufficient for plaque The combination of clustered charge to alanine mutagen- formation. esis and transient dominant selection provides us with a powerful tool for the targeted construction of temperature- DISCUSSION sensitive, conditionally lethal vaccinia virus mutants. We are optimistic that this technique will enable us to create condi- We have shown that temperature-sensitive mutants can be tional lethal mutations in genes that have until now proved created in a specific vaccinia virus gene, G2R, with high refractile to standard genetic techniques. efficiency by using clustered-charge-to-alanine mutagenesis and transient dominant selection. Our yield of temperature- We thank David Botstein and Karla Kirkegaard for communica- sensitive mutants is comparable to the yield in experiments tion of unpublished data and helpful discussions and Sue Moyer, done with the yeast actin gene (2) and the poliovirus 3D Chris Bayliss, and David Simpson for comments on the manuscript. polymerase (3). Of nine clustered charge-to-alanine mutants This work was supported by National Institutes of Health Grant tested, four (44%) displayed some degree of temperature AI18094. sensitivity in a plaque assay as evidenced by plaque size or by an altered response to the anti-poxvirus drug IBT. Three 1. Cunningham, B. C. & Wells, J. A. (1989) Science 244, 1081- of the mutants (33%) were clearly temperature sensitive for 1085. growth in a single-step growth experiment. Since biochemical 2. Wertman, K. F., Drubin, D. G. & Botstein, D. (1992) Genetics experiments with temperature-sensitive mutants are usually 132, 337-350. done under conditions of infection identical to those used for 3. Diamond, S. E. & Kirkegaard, K. (1993) J. Virol. 68, 863-876. a one-step growth experiment, the three mutants that are 4. Condit, R. C. & Niles, E. G. (1990) Curr. Top. Microbiol. temperature sensitive in a one-step growth experiment Immunol. 163, 1-40. should be suitable for biochemical analysis of G2R gene 5. Moss, B. (1990) in Fields , eds. Fields, B. N., Knipe, function in vivo. D. M., Chanock, R. M., Hirsch, M. S., Melnick, J. L., Our experience with G2R suggests a protocol for isolation Monath, T. P. & Roizman, B. (Raven, New York), pp. 2079- mutants in genes for which we have 2112. oftemperature-sensitive 6. Goebel, S. J., Johnson, J. P., Perkus, M. E., Davis, S. W., no selection. MPA-resistant plaques isolated after transient Winslow, J. P. & Paoletti, E. (1990) Virology 179, 247-266. dominant selection contain approximately 11% mutant vi- 7. Condit, R. C. & Motyczka, A. (1981) Virology 113, 224-241. ruses with frequencies ranging from 0% to 60% when indi- 8. Condit, R. C., Motyczka, A. & Spizz, G. (1983) Virology 128, vidual MPA-resistant plaques are compared. Therefore, to 429-443. screen for temperature-sensitive mutants by using transient 9. Thompson, C. L. & Condit, R. C. (1986) Virology 150, 10-20. dominant selection, at least 100 total viruses, isolated from 10. Meis, R. J. & Condit, R. C. (1991) Virology 182, 442-454. several different MPA-resistant plaques, should be tested for 11. Bennett, W. F., Paoni, N. F., Keyt, B. A., Botstein, D., Jones, temperature sensitivity in a plaque assay. Any virus that A. J. S., Presta, L., Wurm, F. M. & Zoller, M. J. (1991) J. shows reduced plaque size at 400C has the potential to be Biol. Chem. 266, 5191-5201. temperature sensitive in a one-step growth experiment. We 12. Vieira, J. & Messing, J. (1987) Methods Enzymol. 153, 3-11. have successfully tested this protocol by isolating the AS1 13. Falkner, F. G. & Moss, B. (1988) J. Virol. 62, 1849-1854. virus mutant without using IBT selection (data not shown). 14. Falkner, F. G. & Moss, B. (1990) J. Virol. 64, 3108-3111. Downloaded by guest on October 1, 2021