Proc. Nati. Acad. Sci. USA Vol. 86, pp. 3539-3543, May 1989 Biochemistry Ribonuclease H activities associated with viral reverse transcriptases are endonucleases (human immunodeficiency virus/avian myeloblastosis virus) MARC S. KRUG AND SHELBY L. BERGER* Division of Cancer Biology and Diagnosis, National Cancer Institute, Bethesda, MD 20892 Communicated by Gilbert Ashwell, January 27, 1989 (received for review December 9, 1988) ABSTRACT A series of test substrates have been synthe- hybrids with blocked termini and circles. Both were cleaved sized to establish the effect of termini on the putative exoribo- by the RNase H isolated either from Escherichia coli or from nuclease H activity of reverse transcriptase. Recombinant mammalian tissues, but neither served as a substrate for the reverse transcriptase from human immunodeficiency virus, viral enzymes. Clearly, an AIDS drug might be designed to natural enzyme from avian myeloblastosis virus, and a known exploit this difference. endonuclease, Escherichia coli ribonuclease H, cleaved re- We report here a comparative study of the effect of termini laxed, circular, covalently closed plasmids in which 770 con- on a recombinant HIV reverse transcriptase, the natural secutive residues of one strand were ribonucleotides. The avian enzyme from avian myeloblastosis virus (AMV), and E. coli enzyme also deadenylated capped globin mRNA with a cova- RNase H. Because these enzymes cleaved our constructs, lently attached oligo(dT) tail at the 3' end. These results resolve including covalently closed circular DNA with 770 consec- a long-standing controversy-that the viral enzymes are oblig- utive ribonucleotide substitutions, the conclusion must be atory exonucleases in vitro, based on their failure to cleave drawn that the RNase H activities of reverse transcriptases certain substrates for E. coli ribonuclease H. including circular are endonucleases. poly(A)-linear poly(T) and ribonucleotide-substituted super- coiled plasmids, but resemble endonucleases in vivo, based on EXPERIMENTAL PROCEDURES their ability to degrade RNA in complex DNARNA hybrids. The data strongly suggest that the viral enzymes are endonu- Materials. The sources of enzymes, nucleic acids, and cleases with exquisite sensitivity to the conformation of hetero- accessories were as follows: AMV reverse transcriptase, duplexes. Inhibition of viral, but not cellular, ribonuclease H Molecular Genetic Resources (Tampa, FL); recombinant with ribonucleoside-vanadyl complexes further distinguishes HIV reverse transcriptase, Genetics Institute (Cambridge, these enzymes. MA); T4 RNA ligase, T4 polynucleotide kinase, E. coli RNase H, E. coli poly(A) polymerase, and rabbit globin The functions encoded in the mRNA containing 30% rRNA on a molar basis (13), BRL; genomes of retroviruses have endonuclease-free E. coli DNA polymerase I (Pol I), Boeh- come under increasingly intense scrutiny since the human ringer Mannheim; Micrococcus luteus polynucleotide phos- immunodeficiency virus (HIV) was isolated and recognized phorylase (type 15), T4 DNA ligase and oligomers, Pharma- as the causative agent of AIDS. However, the most prom- cia; DNase I, T7 RNA polymerase, pGEM-4, and RNasin, ising drugs or vaccines target only two HIV functions; Promega; pancreatic RNase A, Sigma; restriction enzymes 3'-azido-3'-deoxythymidine (AZT) acts predominantly as an and mung bean nuclease, New England Biolabs; and Blue- inhibitor of the polymerase activity of reverse transcriptase script, Stratagene. Radiolabeled compounds came from Am- (1) and recombinant CD4 inhibits viral attachment by binding ersham. to products of the env gene (reviewed in ref. 2). Preparation of 3'-End-Modified Globin mRNA. Globin A retroviral function that has attracted relatively little mRNA with one labeled adenylate residue at the 3' end was attention, despite its being carried out by one of the better synthesized as described (13). Subsequently, reactions cat- characterized proteins, is the ribonuclease H (RNase H) alyzed by RNA ligase were performed with 27 pmol (1.4 x 106 activity of reverse transcriptase. An RNase H cleaves RNA dpm) of mRNA and 125 pmol of cytidine 5',3'-bisphosphate in a DNARNA hybrid. The viral enzyme is responsible for (pCp) in 25 Al, or with 12.5 pmol (107 dpm) of mRNA and 370 degradation of genomic viral RNA after synthesis of minus- pmol of p(dT)17 in 10 ,1u, under conditions specified by strand DNA has occurred. Upon removal of the RNA moiety England et al. (14) for large viral RNAs but without dimethyl of the DNARNA hybrid, minus-strand DNA becomes avail- sulfoxide. Unreacted globin mRNA was removed from able as a template for synthesis of plus-strand DNA (3). The mRNA-p(dT)17 by diluting 1:2 and treating with polynucle- RNase H activity of reverse transcriptase has also been otide phosphorylase (15). Both modified mRNAs were puri- implicated in releasing the tRNA primer that is covalently fied by phenol/chloroform extractions (16) and drop dialysis bound to minus-strand DNA (4), in nicking viral RNA to against H20 (17); this procedure completely eliminated ex- generate the polypurine-rich oligoribonucleotide that primes cess pCp and p(dT)17. Similar reactions were also carried out synthesis of plus-strand DNA, and in removing the polypu- with unlabeled reagents, and the stated specific activities rine tract from the DNA extension to which it is linked (5-9). were achieved by mixing labeled and unlabeled materials. The RNase H activities of reverse transcriptases are Yields of mRNA-pCp and mRNA-p(dT)17 were -95% and believed to be exonucleases that can attack RNA from either 100%, respectively. They were ascertained by performing the 5' or the 3' end, whereas the normal cellular RNase H ligations of measured amounts of unlabeled globin mRNA activities are endonucleases (10-12). These conclusions were with either [32P]pCp or of known based on experiments with molecules of two types: synthetic [32P]p(dT)17 specific activ- Abbreviations: AMV, avian myeloblastosis virus; HIV, human The publication costs of this article were defrayed in part by page charge immunodeficiency virus; Pol I, polymerase I. payment. This article must therefore be hereby marked "advertisement" *To whom reprint requests should be addressed at: National Insti- in accordance with 18 U.S.C. §1734 solely to indicate this fact. tutes of Health, Building 10, Room B1B38, Bethesda, MD 20892. 3539 Downloaded by guest on September 24, 2021 3540 Biochemistry: Krug and Berger Proc. Natl. Acad. Sci. USA 86 (1989) ity, under the conditions above, and determining the amount 37 GBq) and 40 ng of the gel-purified Pvu II-Sma I fragment of radioactivity rendered Cl3CCOOH precipitable. of pGEM-4.723. DNA was digested with DNase I and the Preparation of Unligated and Ligated Hybrids. Both ligated 5'-triphosphate was removed (19). The RNA was phenol/ and unligated hybrids consist of three molecules: a 770-base chloroform extracted and drop-dialyzed as described above; labeled RNA, covalently closed circular single-stranded afterwards, it was phosphorylated (19), ethanol-precipitated, Bluescript DNA containing 770 bases complementary to the and dissolved in 10 dul of water. The product was predomi- RNA ("innertube" DNA), and single-stranded linear Blue- nantly 0.77 kilobase'in denaturing gels (data not shown) but script with sequences complementary to the entire circular a component at 1.5 kilobases was also observed. The latter Bluescript DNA, excepting the RNA-derived region ("out- molecule is ribonuclease-resistant and is probably a double- ertube" DNA). The construction ofboth hybrids is illustrated stranded RNA hairpin. in Fig. 1. Covalently closed circular single-stranded DNA contain- The plasmid, pGEM-4.723, was generated from pGEM-4 ing an insert complementary to the RNA transcript was by the insertion of a 723-base-pair Pst I fragment ofa porcine constructed from double-stranded Bluescript KS(+) and two major histocompatibility complex gene into the Pst I site (18). pieces of DNA: a double-stranded adaptor, 5'-GGGAGAC- Using Promega's instructions, RNA was transcribed with T7 CGGA-3', hybridized to 3'-CCCTCTGGCCTTCGA-5', rep- RNA polymerase in a 20-,1d reaction mixture, containing 150 resenting the region from the transcriptional start site to the UTP (including 50 ofRNA-grade [a-32P]UTP; 1 Ci = HindIII site; and the HindIII-Sma I fragment of pGEM- AM ktCi 4.723. Adaptors, in 100-fold excess, were ligated to the Hind III HindIII-Sma I fragment and the excess was removed with Sma I. This construct was ligated into Sma I-cleaved Blue- Pvu I 0c script. After selecting the correct orientation, the desired strand of Bluescript KS(+)770 was rescued using the Strat- agene procedure. Linear Bluescript was obtained by cleaving double- stranded Bluescript KS(+) with Sma I; strands were sepa- rated by denaturation as described below. To assemble unligated hybrids, 5 4g of circular single- stranded innertube DNA and 12.5 ,zg of linear Bluescript KS(+) DNA, one strand of which is outertube, were boiled for 2 min in 130 kkI of 65% (vol/vol) deionized formamide/40 1) T7 RNA Polym erase mM Pipes-NaOH, pH 6.5/0.4 M NaCl. After cooling to 70TC, 2) DNase 1.5 i&g (5 x 107 dpm) of RNA was allowed to anneal for 1 hr pp_- as the mixture was cooled to 55°C. Hybrids were ethanol- 770 base RNA precipitated, dissolved in 50 ,ul of H20, and drop-dialyzed. Bacterial Ligated hybrids were obtained by incubating 45 ,ul of Alkaline unligated hybrids overnight at 15°C with 160 units ofRNasin, Phiospliatasey 35 units ofT4 DNA ligase, and 16 ,ug ofT4 RNA ligase in 150 HO ,ul of buffer composed of 50 mM Tris HCl (pH 7.6), 10 mM 770 base RNA MgCl2, 5 mM dithiothreitol, and 250 mM ATP. After several phenol/chloroform extractions, hybrids were drop-dialyzed against H20. Where indicated, nicked circular molecules were removed by treatment with endonuclease-free Pol I for 75 min under the conditions detailed in the legend to Fig.