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Analysis of Genetic Variability and Mapping of Point Mutations in Influenza Virus by the Rnase a Mismatch Cleavage Method

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Analysis of Genetic Variability and Mapping of Point Mutations in Influenza Virus by the Rnase a Mismatch Cleavage Method Proc. Nail. Acad. Sci. USA Vol. 85, pp. 3522-3526, May 1988 Genetics Analysis of genetic variability and mapping of point mutations in influenza virus by the RNase A mismatch cleavage method (RNA virus/genetic divergence) CECILIo LOPEZ-GALINDEZ*, JOSE A. LOPEZt, JOSE A. MELEROt, LUIS DE LA FUENTEf, CONCEPCION MARTINEZt, JUAN ORTINt, AND MANUEL PERUCHO*§ *Department of Biochemistry, State University of New York, Stony Brook, NY 11794; tCentro de Biologia Molecular, Universidad Autonoma de Madrid, Canto Blanco, 28049 Madrid, Spain; and tServicio de Biologia Molecular, Centro Nacional de Microbiologia, Majadahonda, 28220 Madrid, Spain Communicated by Severo Ochoa, January 19, 1988 ABSTRACT We have applied the RNase A mismatch However, sequencing of large viral genomes or of multiple cleavage method to analyze genetic variability in RNA viruses viral isolates represents a formidable experimental task. by using influenza virus as a model system. Uniformly labeled Therefore, a simple technique that could detect point muta- RNA probes synthesized from a cloned hemagglutinin gene of tions in viral RNA genomes would be very useful for this type a given viral strain were hybridized to RNA isolated from other of study. strains of characterized or uncharacterized genetic composi- We developed a method to detect single base substitutions tion. The RNARNA heteroduplexes containing a variable in transcribed genes that is based on the ability of RNase A number of base mismatches were digested with RNase A, and to cleave single base mismatches in RNA-RNA heterodu- the resistant products were analyzed by denaturing polyacryl- plexes (13). The method has been successfully applied to amide gel electrophoresis. We show that many of these single detect mutated cellular KRAS oncogenes in human tumors base mismatches are cleaved by RNase A, generating unique (13, 14) and single mutations in the human hypoxanthine- and characteristic patterns ofresistant RNA fragments specific guanine phosphoribosyltransferase gene (15) and in the for each of the different viral strains. Comparative analysis of mouse ornithine carbamroyltransferase gene (16). In addition, the cleavage patterns allows a qualitative estimation of the single base substitutions in the j3-globin gene have been genetic relatedness and evolution of field strains. We also show detected in 8-thalassemias by RNase A cleavage of that cleavage by RNase A at single base mismatches can readily DNA*RNA heteroduplexes containing single base mis- detect and localize point mutations present in monoclonal matches (17). antibody-resistant variants. This method should have wide Using influenza virus as a model system, we show that the applications in the study of RNA viruses, not only for epide- RNase A mismatch cleavage method provides an alternative miological analysis but also in some diagnostic problems, such and simple approach for analysis of genetic variability in as characterization of phenotypic mutants. RNA viruses. We also show that cleavage by RNase A at single base mismatches in viral RNA hybrids can readily RNA viruses show a high spontaneous mutation rate, which detect and localize single point mutations in monoclonal allows the rapid evolution of viral populations (1) and the antibody-resistant variants. existence of extensive genetic heterogeneity with important biological consequences (2). In many instances, one or a few MATERIALS AND METHODS point mutations have been shown to lead to essential phe- notypic changes. Some relevant examples include the out- Viral Strains and RNAs. The viral strains used throughout come of epidemic variants of influenza or foot and mouth this work (with their abbreviated names in parenthesis) are as disease viruses (antigenic drift) (3), changes in tropism (4) or follows: A/Hong Kong/8/68 (HK68), A/England/42/72 in pathogenicity (5) of viruses, or isolation of variants (EN72), A/Victoria/3/75 (VA75), A/Texas/i/77 (TX77), resistant to neutralizing monoclonal antibodies (6). A/Philipines/2/82 (FP82), A/Brazil/11/78 (Hi), and A variety of techniques have been used for the detection A/England/12/64 (H2). Strains A/Madrid/9688/80 (MA80) and study of genetic variation among RNA viruses. Some of and A/Madrid/716/81 (MA81) were isolated 2 months apart them are based on migration differences that under certain during the same influenza outbreak. Both isolates have been electrophoretic conditions can be observed with single- (7) or characterized by Ti fingerprinting (18). Mutants RM1 (Mi), double-stranded (8) RNAs differing in one or a few nucleo- RM2 (M2), and RM4 (M4) are viruses resistant to neutral- tides. Similarly, conditions of partial denaturation can be ization by M58/p7/C anti-hemagglutinin monoclonal anti- used to distinguish RNA-RNA or DNA-RNA heteroduplexes body (19). These monoclonal antibody-resistant variants from the corresponding homoduplexes (9, 10). However, were obtained from the VA75 strain after selection by two these techniques do not discriminate between single or consecutive plaque isolation steps in the presence of anti- multiple nucleotide changes and also require experimental body. conditions that have to be empirically determined in each Viruses were grown in either 10-day-old embryonated case. RNase T1 oligonucleotide fingerprinting has been chicken eggs or in cultures of Madin-Darby canine kidney useful in many studies of viral heterogeneity and evolution (MDCK) cells. They were partially purified by precipitation (ii). This technique yields quantitative data but becomes with 10% PEG-6000 followed by centrifugation through a 30%o cumbersome in the analysis of multiple samples. Nucleotide sucrose gradient (19). Viral RNAs were obtained from sequencing is widely used for genetic analysis of RNA purified viruses as described (20). Total cellular RNA from viruses and has opened insights into variation studies (12). MDCK-infected cells was isolated from subconfluent cul- tures infected at high multiplicity. Twenty-four hours after infection, cell monolayers were harvested and RNA was The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 3522 Downloaded by guest on October 2, 2021 Genetics: Lopez-Galindez et al. Proc. Natl. Acad. Sci. USA 85 (1988) 3523 prepared by the guanidine hydrochloride method as de- RNase Ti was added to achieve a more extensive digestion scribed (13). of small fragments of the nonhybridized RNA probes. The Plasmids. Plasmid pSVa970 contains a full-length cDNA products were analyzed by electrophoresis in 8% denaturing sequence of the influenza virus hemagglutinin gene from the polyacrylamide gels and autoradiography as described (13). VA75 strain cloned into the pBSV9 vector (21). pSVa970 was digested with Stu I (which cuts in the early promoter ofsimian virus 40, 20 nucleotides from the 5' end of the viral HA) and RESULTS EcoRI (at position 1272 in the hemagglutinin gene). The Stu RNase A Mismatch Cleavage Analysis of Influenza Virus I/EcoRI fragment was subcloned into the Sma I and EcoRI Genetic Variants. We selected a fragment of the influenza sites of pGEM3 (Promega Biotec, Madison, WI), generating virus hemagglutinin gene spanning positions 1-1272. This pIVAV1. From this recombinant, deletion plasmids were region encodes the major antigenic determinants of the obtained by digestion with HindIII (pIVAV3), Xba I hemagglutinin molecule and exhibits considerable sequence (pIVAV4), and Pst I (pIVAV5), and ligation with T4 DNA heterogeneity between different viral strains (Fig. 1 Upper). ligase. The 523-nucleotide Taq I fragment from the BK79 HA RNA probes complementary to this region ofthe VA75 strain sequence (positions 676-1199) was isolated from plasmid genome (Fig. 1 Upper) were hybridized to viral RNA isolated pBK4 (22) and was cloned into the Acc I site of pGEM3, from the BK79 or from the MA80 strains. These viral strains generating pIVAB1. are genetically similar as determined by Ti fingerprinting Hybridizations and RNase Digestions. Plasmid DNA (1,ug), (18). The predicted mismatches present in heteroduplexes digested with the appropriate enzyme, was used for the between the VA75 complementary RNA probe and the BK79 preparation ofuniformly labeled RNA probes with either SP6 viral RNA was inferred from the known nucleotide sequence or T7 RNA polymerases as described (13) using 32P-labeled of these viruses (23). As shown in Fig. 1, most of these CTP (Amersham). For short probes (<400 nucleotides), 24 mismatches accumulate in the region between the HindIII ,uM unlabeled CTP was used in the reaction mixture and and Pst I sites encoding the major antigenic area of the incubation was for 1 hr, whereas for longer probes the influenza virus hemagglutinin. RNAs prepared from three amount of unlabeled CTP was raised to 500 ,M and incuba- different VA75 subclones isolated by their resistance to tion was for 2 hr. Uniformly labeled RNA probes were neutralization by a monoclonal antibody raised against the purified by preparative electrophoresis in 5% polyacrylamide viral hemagglutinin were also used (Ml, M2, and M4). Only gels and hybridized (7 x 104 cpm) to 1-5 ,ug of either viral a few nucleotide differences were expected between these RNA or total cellular RNA from infected cells, for 3.5 hr at last viral RNAs and the RNA from the parental VA75 virus. 57°C, as described (13). Hybridization with viral RNA from The RNA hybrids were digested with RNase A and the HiN1 and H3N2 reference strains was done at 40°C. The resistant RNA fragments were analyzed by denaturing gel hybrids were digested with RNase A (60 ,ug/ml) (P-L Bio- electrophoresis. Due to the presence of multiple mismatches chemicals) and RNase T1 (2 ,ug/ml) (Sigma) for 1 hr at 30°C. in VA75-BK79 hybrids (Fig. 1 Upper), a complex band ?IT " Iff 17?~~(4. A-C O U.G e 77 I 4 0 + 11 II A-A A U-U A + - A-G O U.C K BK4- I + _- I + + + W ±+++ + + + BK- it IT t '*T r Tf:T ftT T ff T T .1 II] fit.
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