Vaccinia Virus by Replacing Clustered Charged Residues with Alanine
Total Page：16
File Type：pdf, Size：1020Kb
Proc. Nati. Acad. Sci. USA Vol. 91, pp. 4554-4558, May 1994 Genetics 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 Immunology and Medical Microbiology, 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.