The Sequence Features Important for Plus Strand

Vol. 268, No. 9, Ieeue of March 25, pp. 62214227,1993 Printed in U. S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biochemietry and Molecular Biology, Inc.

The Sequence Features Important for Plus Strand Priming by Human Immunodeficiency Virus Type 1 Reverse Transcriptase* (Received for publication, December 7, 1992)

Katherine A. Pullen, Alison J. Rattray, and James J. Champoux$ From the Department of Mkrobiology, School of Medicine, University of Washington, Seattle, Washington 98195

boa et al., 1980; Varmus, 1982; Varmus and Swanstrom, 1982, 1985). Furthermore, sequences at the LTR ends are recognized during integration of the viral DNA into the host cell chromosome (Colicelli and Goff, 1985,1988; Panganiban and Temin, 1983). The integrated provirus of all known retroviruses begins with 5‘-TG.. . and ends with . . .CA-3’, and in all cases studied thus far, a number of bases (usually 2) are lost from each end of the linear double-stranded DNA intermediate to generate these ends (Varmus and Brown, 1989). The left and right ends of the LTRs before integration are defined by the sites of initiation of the plus and the minus DNA strands, respectively (Gilboa et al., 1980; Varmus and Brown, 1989). It is known that both the generation and the removal of the primers for plus strands and the removal of the primers for minus strands are precise events carried out by the RNase H activity of reverse transcriptase. The primer for minus strand DNA synthesis is a tRNA molecule that is packaged in the virion and base-paired to the plus RNA genomeadjacent to theU5 sequences (Varmus and Brown, 1989). Synthesis of the minus strand initiates from the 3‘ end of the tRNA molecule,which is subsequently removed bya precise RNase H cleavage that defines the right end of the right LTR (Furfine and Reardon, 1991; Omer and Faras, 1982; Pullen and Champoux, 1992). The primer for plus strand DNA synthesis is derived from the RNA genome at a highly conserved region called the polypurine tract (PPT), which is located adjacent to the U3 sequences (Varmus and Brown, 1989). After the minus strand DNA has been synthesized,cleavage by RNase H within the PPT creates this primer, anda second cleavage event removes the primer The replication cycle of a retrovirus begins with reverse following the initiation of plus DNA synthesis (Champoux et transcription of the single-stranded plus RNA genome bythe al., 1984; Huber and Richardson, 1990). combined DNA polymerase and RNase H activities of the The plus strand priming site has been mapped in uitro for virion-associated reverse transcriptase to produce a double- several different retroviruses, including Moloney murine leustranded DNA copyof the genome (Varmus andBrown, 1989). kemia virus (M-MuLV) and human immunodeficiency virus During this process, unique sequences from the 3‘ and 5‘ ends type 1 (HIV-1) (Finstonand Champoux, 1984; Huber and of the RNA genome (U3 and U5, respectively) and a direct Richardson, 1990; Pullen and Champoux, 1990; Resnick et repeat (R) found at both ends areduplicated to form the long al., 1984). For M-MuLV, the plus strand origin has also been terminal repeats (LTR)’ present at each end of the resulting mapped in the endogenous reaction, and the initiation site is double-stranded DNA molecule. The LTRs, which have the identical with that observed invitro (Mitra et al., 1982; structure U3-R-U5, are important for continuation of the Rattray and Champoux, 1987). In all cases studied thus far, replication cycle because they contain promoter, enhancer, the start site is located 2 bases before the conserved TG and termination signals essential for the transcription of the dinucleotide observed at the5‘ end of the provirus. Since the provirus to produce both genomic and messenger RNAs (Gilretroviral RNase H does not generally exhibit any particular * This work was supported by National Institutes of Health Grant sequence preference when it cleaves RNA-DNA hybrids (BalCA51605. The costs of publication of this article were defrayed in timore and Smoler, 1972; Keller and Crouch, 1972; Leis et al. part by the payment of page charges. This article must therefore be 1973; Starnes and Cheng, 1989), we have been interested in hereby marked “advertisement” in accordance with 18 U.S.C. Section determining what causes RNase H to cleave within the PPT 1734 solely to indicate this fact. with such specificity. To address this question for M-MuLV, $ To whom correspondence should be addressed. ’The abbreviations used are: LTR, long terminal repeat; PPT, we generated a series of point mutations in or near the PPT polypurine tract; M-MuLV, Moloney murine leukemia virus; HIV-I, and examined their effects on the specificity of the priming human immunodeficiency virus type 1; DTT, dithiothreitol; NTP, reaction in uitro. The results indicate that the junction beunspecified nucleoside triphosphate. tween the strings of 5 A residues and 6 G residues within the

A specific cleavage by the reverse transcriptase-associated RNase H activity generates the RNA primer for plus strand DNA synthesis during reverse transcription. Previously, we used site-directed mutagenesis to define the sequence features of the polypurine tract (PPT) required for correct plus strand priming by the Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Rattray, A. J., and Champoux, J. J. (1989) J. Mol. Biol. 208, 445-456). Although the sequences of human immunodeficiency virus type 1 (HIV-1) and M-MuLV diverge completely outside a 20base region encompassing the PPT, within thisregion there are only three differences between the two viruses. Here we show that the HIV-1 reverse transcriptase will utilizethe M-MuLV PPT asan origin for plus strand initiation in vitro. This finding enabled us to use the set of PPT mutants previously generated in MMuLV, in conjunction with a small set of newly derived mutations within the HIV-1 PPT, to study plus strand priming by the HIV-1 reverse transcriptase. Despite the similarity between the two PPT regions, the sequence features important for positioning RNase H for the cleavage reaction that generates the plus strand primer are different for the two viruses. For M-MuLV, the -7A residue is a critical specificity determinant in the priming reaction, whereas for HIV- 1, the -2G and -4G residues play key roles in determining the specificity of priming.

6221

6222

Plus Strand Priming by HIV-1 RNase H

PPT (see Fig. 1) plays a critical role in positioning RNase H for the plus strand primer-cleavage reaction (Rattray and Champoux, 1989). T h e sequence of t h e HIV-1 PPT is almost identical with the M-MuLV PPT, with the only exceptions occurringat t h e +2 position (a C instead of an A) and at t h e -11 and -12 positions (the cleavage reaction is defined as occurring between the -1 and +l residues) (Fig. l ) . We show here that the HIV-1 reversetranscriptase will initiateplus strands properly using M-MuLV DNA-RNA hybrids. This observation enabledus to study plus strand priming byHIV-1 reverse transcriptase by employing the same set of mutants previously used in the studyof the M-MuLV priming reaction. In addition, several selected mutations were made in the HIV-1 PPT and used to further analyzethe priming reaction. Surprisingly, the specific bases within the PPT that are most important for positioningRNase H for the cleavage eventthat generates the plus strand primer are different for HIV-1 and M-MuLV

plus strand of the insert, and M13mp7HXEV(-) for the one that results in packaging of the minus strand of the insert. The XhoI to SacI fragment (positions 8896 to 9571) of HIV-1 was cloned in pBSM13(+) as previously described (Pullen and Champoux, 1992)to generate pBHXS, which produces plus strand RNA whentranscribed in vitro by T7 RNA polymerase.

Construction of PPT Mutants M-MuLV Mutants-The +1C+5C mutant was constructed by oligonucleotide-directed mutagenesis of the M13p190 clone using the method of Zoller and Smith(1984).All other mutationswere produced using the dut- ung- technique of Kunkel et al. (1987) as modified by Geisselsoder et al. (1987). A detailed description of the isolation and characterization of these mutants has been previously described (Rattray and Champoux, 1989). HIV-I Mutants-All HIV-1 mutants were generated by oligonucleotide-directed mutagenesis using the polymerase chain reaction method of Nelson and Long (1989). Table I shows the oligonucleotide primers used. The primers are labeled A through D, according to the convention of Nelson and Long (1989), with primer A containing the nucleotide change. Briefly, step 1 reactions (final volume, 100 pl) reverse transcriptases. contained 1 fmol of the pBHXS DNA linearized with HindIII, 100 pmol of primer A, 100 pmol of primer B, 200 p~ dNTPs, 10 mM Tris/ hydrochloride (pH 7.5),50 mM KC1, 1.5 mM MgC12,0.001%w/v EXPERIMENTALPROCEDURES gelatin, and 0.025 units/pl Amplitaq DNA polymerase, and were incubated for 1 min at 94 "C, 1 min at 37 "C, and 1 min at 72 "C for Materials 30 cycleson a Perkin-Elmer CetusInstruments DNA thermal cycler. All restriction enzymes were purchased from New England Biolabs. The resulting 213-218-base pair product (the exact size was dependCloned phage T4 polynucleotide kinase, phage T4 DNA ligase, phage ent on the particular mutant) was gel-purified, and approximately 0.6 T4 DNA polymerase, clonedphage T7 RNA polymerase, and sequenc- pmol was added to the step 2 reaction (final volume, 100 pl), which ing reagents (as partof a Sequenase kit) were purchased from U. S. contained 1fmol of linearized pBHXS DNA, 200 pM dNTPs, 10 mM Biochemical Corp. Superscript RNase H- reverse transcriptase was Tris/hydrochloride (pH 7.5), 50mM KCI, 1.5 mMMgC12, and 0.001% purchased from GIBCO-BRL, Amplitaq DNA polymerase from Per- gelatin. This mixture was boiled for 5 min and annealed at 37 "C for kin-Elmer Cetus Instruments, and RNasin and RQ1 DNase from 3 min before addition of Amplitaq DNA polymerase to a concentraPromega Biotec. The plasmid vectors pBSM13(+) and M13mp7 were tion of 0.025 units/pl and incubation at 72 "C for 10 min. For step 3, purchased from Stratagene. OligonucleotideM7878(-) waspurchased 100 pmol of primer C and primer D were added to the reaction, and from Immunex Corp., and all other oligonucleotides were synthesized the mixture was cycled 30 times on the DNA thermal cycler as in on a Biosearch 8600DNA synthesizer. Each oligonucleotide was step 1. The resulting 576-base pair product was cut with EcoRV (in purified by electrophoresis in a 20% polyacrylamide gel containing 8 the primer B sequences) and ScaI (position 9397) to yield a 511-base M urea followed by elution from a Waters Sep-Pak C-18 column with pair fragment that was gel-purified and cloned in pBSM13(+) that 60% methanol, 4 mM triethylammonium carbonate. [-y-32P]ATPwas had been cut with EcoRV and SmI. These mutant clones are desigpurchased from Du Pont-New England Nuclear. nated pBHES and are also named according to the base change and the position of the mutation in relation to the normal cleavage site General Methods for plus strand priming (between -1 and +l).For example, the clone All cloning was carried out using standard techniques (Ausubel et with a G to A substitution at the +2A position is named pBHES+2A. al., 1987). The recombinant pBHES plasmids, for which the inserts All mutations were verified by dideoxy sequence analysis using 32Pwere generated by PCR, were isolated from Escherichia coli strain labeled H9119(-) as theoligonucleotide primer. TGl, and all other recombinant DNAs were isolated from E. coli In Vitro Plus RNA Synthesis strain GW5180, a recA- derivative of JMlOl (G. Walker, MIT). All restriction enzyme reactions were carried out using conditions recThe templates for in uitro run-off transcription reactions were ommended by the supplier. Primer extension and sequencing reaction generated by linearizing B190(-) DNA with PuuII, pBHXS DNA products were analyzed by electrophoresis in 8%polyacrylamide gels with ScaI, and the pBHESDNAs with HindIII. Transcription reaccontaining 8 M urea. In all cases, the samples were boiled for 2 min tions contained 40 mM Tris/hydrochloride (pH 8.0), 6 mMMgC12, 10 in 50% formamide, 0.05% xylene cyanol, and 0.05% bromphenol blue mM DTT, 500 p~ NTPs, 60 ng/pl DNA template, 3 unitslpl T7RNA before loading. The gels were dried on Whatman 3MM paper and polymerase, 1.6 units/pl RNasin, and were incubated for 90 min at exposed to x-ray film at -80 "C with an enhancing screen. 37 'C. DNase was added to a final concentrationof 0.05 unitslpl, and incubation was continued for another 30 min at 37 "C. The reactions Construction of PPT-containing Clones were terminated with excess EDTA, and the RNA was purified by M-MuLV Clones"M13mp7-Al5 containsthe Hind11 SacI to frag- extraction with phenol and CHCl3:isoamyl alcohol (241, v/v) and ment (positions 4992-8231) corresponding to the right third of the stored at -20 "C as a precipitate in ethanol.The RNA wasquantitated M-MuLV genome (Shinnick et al., 1981) cloned in the orientation by ultraviolet spectroscopy at 260 nm. such that the insert in the single-strand phage DNA is the same Reverse Transcription Reactions polarity as the genomicRNA.M13p190 is an M13mp7 derivative RNA-DNA Hybrids as the Templates-Single-stranded minus containing the 190-base PuuII fragment (positions 7745-7935) of MMuLV DNA cloned such that the single-stranded phage DNA con- DNA fragments were excised from M13mp7HXEV(-) and ssB19O(-) tains theinsert in the minus polarity (Finstonand Champoux, 1984). wild-type DNAs with EcoRI and freed from vector sequences by sedimentation in a 5-20% alkaline sucrose gradient as described by Clone B190(-) contains the same 190-base pair PuuII fragment in the EcoRI site of pBSM13(+), and itsconstruction hasbeen described Been and Champoux (1983). The fragments were quantitated and previously (Rattray and Champoux, 1987, 1989). For this plasmid, annealed to an equimolar amount of the corresponding RNA synthethe single-stranded DNA that is packaged in the presence of helper sized in uitro using T7 RNA polymerase (see above) as described by Rattray and Champoux (1989). Reverse transcriptase reactions conphage contains a minus strand insert and is designated ssB19O(-). HIV-1 Clones-Two clones were constructed containing the 224- tained 50 mM Tris/hy&ochloride (pH 8.0), 10 mM MgClz, 50 mM base HIV-1 XhoI to EcoRV fragment (positions 8896-9119) (Ratner KCI, 0.8 mM DTT, 1.7 ng/pl RNA-DNA hybrid, 200 p M dNTPs, 2 et al., 1985)in M13mp7 in the two possible orientations. Construction unitslpl RNasin, and 0.1-1 units/pl of the appropriate reverse tranof these clones has been described previously (Pullen and Champoux, scriptase. After incubation at 37 "C for 30 min and termination with 1990), and they have since been designated M13mp7HXEV(+) for excess EDTA, the products were treated with 0.3 M NaOH at 65 "C the orientation that packages single-stranded DNA containing the for 15 min to hydrolyze the RNA. The samples were neutralized with

Plus Strand Priming by HIV-1 RNase H

6223

TABLEI Oligonucleotides Name”

H9119(-) M7878(-) H9094(-) +2A (primer A) H9093(-) +lC (primer A) H9091(-) -2A (primer A) H9090(-) -3A (primer A) H9089(-) -4A (primer A) E20H8896(+) (primer B) H9452(-) (primer C) (primer E20H(+) D) The polarities of the oligonucleotidesin relation to

Sequence

5‘-TAGAACAGAAGAAACCCTC-3’ 5’-TCCATGCCTTGCAAAAT-3’

5’-AGCCCTTCCATTCCCCCCTTT-3’ 5’-GCCCTTCCAGGCCCCCCTTTT-3’ 5’-CCTTCCAGTCTCCCCTTTTCT-3’ 5’-CTTCCAGTCCTCCCTTTTCTT-3’ 5’-TTCCAGTCCCTCCTTTTCTTT-3’ 5‘-GGCCGATCAGAATTCCCGCCTCGAGACCTGGAAAAACATG-3’ 5”GTCCCAGCGGAAAGTCC-3’

5”GGCCGATCAGAATTCCCGCC-3’ theplus viral genome are given in parenthesesas partof the name of the oligonucleotide.

acetic acid, ethanol-precipitated, and analyzed by an oligonucleotide extension assay (see below). Two-step Reactions Using RNA Alone as the Template-Reactions contained 50 mM Tris/hydrochloride (pH 8.3), 6 mMMgC12, 40 mM KCI, 1 mM DTT, and 6 ng/pl of the appropriate RNA template and were heated to 70 “C for 5 min. After cooling to room temperature, dNTPs were added to a final concentration of 200 p ~ RNasin , to 2 units/pl, and Superscript RNaseH- reverse transcriptase to 10 units/ pl. The reactions were incubated at 37 “C for 20 min to convert the RNA template to a DNA-RNA hybrid by a self-priming mechanism (Rattrayand Champoux, 1989), and M-MuLV orHIV-1 reverse transcriptase was added to a concentration of 0.1-1 unitslpl. Reactions were terminated and treated with alkali as described above. OligonucleotideExtemion Assays Oligonucleotides M7878(-) and H9119(-) (Table I) were labeled to a specific activity of 5-10 X lo’ cpm/pg by phosphorylation with T4 polynucleotide kinase in the presence of [y-32P]ATPusing conditions recommended by the supplier. Products of HIV-1 and MMuLV reverse transcriptase reactions were boiled in the presence of the appropriate oligonucleotide for 3 min and chilled on ice for 5 min, and theoligonucleotides were extended to theend of the template in reactions containing 50 mM Tris/hydrochloride (pH 8.0), 5 mM DTT, 5 mM MgCl,,280 p~ all four dNTPs, and 0.15 units/pl T4 DNA polymerase. After incubation at 37 “C for 20 min, the reactions were terminated by the addition of excess EDTA. Sequencing Reactions Sequencing reactions were carried out by the dideoxy method of Sanger et al. (1977). Briefly, 200 ng/pl of the single-stranded templates M13mp7HXEV(+) and M13mp7-Al5 were boiled in 20 mM Tris/hydrochloride (pH 7.5), 10 mM MgCL, and 25 mM NaCl in the presence of0.75 ng/pl “P-labeled oligonucleotides H9119(-) and M7878(-), respectively (Table I). After cooling to room temperature, dNTPs were added to a final concentration of 33 nM each, DTT to7 mM, and Sequenase T7 DNA polymerase to 0.5 units/& and the reactions were incubated for 5 min at room temperature. From this mixture, four termination reactions were prepared, each of which contained all four dNTPs ata concentration of 33 PM and one of the four ddNTPs ata concentration of 3 p ~Incubation . was at 37 “C for 5 min, after which the reactions were terminated by addition of excess EDTA. For sequencing of double-stranded templates (used to verify the presence of mutations after site-directed mutagenesis), the same procedure was followed, except that the templates were first denatured by treatment with 0.2 M NaOH at room temperature for 5 min and precipitated with absoluteethanolin the presence of 5.4 M ammonium acetate before being annealed to the oligonucleotide. RESULTS

HIV-1 Reverse Transcriptase Can Initiate Plus Strands Correctly Using the M-MuLVPPT Sequence-In vitro studies have previously shown that HIV-1 reverse transcriptase initiates plus DNA synthesis 2 bases before the conserved proviral TG dinucleotide when incubated with an RNA-DNA hybrid containing the HIV-1 PPT (Huber and Richardson, 1990; Pullen and Champoux, 1990). Furthermore, consistent with the similarities in the PPT sequences of the two viruses

(Fig. l), theM-MuLV reverse transcriptase can utilize RNADNA hybrids containing the HIV-1 PPT sequence as efficiently as it uses its own sequence in the plus strand priming reaction (Pullen and Champoux, 1990). Previously, a collection of M-MuLV PPT mutants was used to dissect the sequence requirements for M-MuLV plus strand priming (Rattray and Champoux, 1989). To use these same mutants to similarly analyze the sequence requirements for HIV-1 plus strand priming, we needed to determine whether the wildtype M-MuLV PPT could function as asuitable substrate for the HIV-1 RNase H. Thus, a single-stranded minus DNA fragment containing the M-MuLV PPT was annealed to the corresponding complementary plus RNA, and the resulting hybrid was used as a substrate for plus strand priming reactions by both M-MuLV and HIV-1 reverse transcriptases. The sites of initiation of plus DNA synthesis were mapped by an oligonucleotide extension assay and analyzed on a gel next to a sequence ladder generated using the same labeled oligonucleotide annealed to a template containing the M-MuLV PPT region in the plus polarity (Fig. 2 A ) . For comparison, parallel reactions were carried out using both enzymes with the HIV-1 PPT RNA-DNA hybrids as substrates (Fig. 2B). The spectrum of plus strand initiation sites observed when the HIV-1 reverse transcriptase was presented with the MMuLV PPT was very similar to what is observed with the homologous HIV-1 PPT (Huber andRichardson, 1990; Pullen and Champoux, 1990; also compare Fig. 2 A , lane 1 with Fig. 2B, lane 1 ) . The only reproducible difference was that slightly more product corresponding to initiation 1 nucleotide downstream from the true start was observed with the M-MuLV PPT (see below).

The Effect of Mutations in the M-MuLV PPT on Plus Strand Priming by HIV-1 Reverse Transcriptase--It has previously been shown that single-stranded plus RNA can direct the synthesis of a complementary minus strand DNA when incubated with M-MuLV reverse transcriptase, and that the resulting hybrid can serve asasubstrate for plus strand priming (Rattray and Champoux, 1989). Presumably, the 3‘ end of the RNA folds back and primes the synthesis of a minus DNA strand, generating a hybrid structure through the region of the PPT. Since this greatly simplifies the analysis of mutant PPTs, we used a similar strategy in the analysis of the specificity of the HIV-1 RNase H. For these experiments, we carried outa two-step reaction in which the singlestranded RNA was first converted to an RNA-DNA hybrid by RNase H- reverse transcriptase followedby incubation with the normal reverse transcriptase of interest. Using wildtype M-MuLV RNA and HIV-1 reverse transcriptase in such a two-step reaction yielded the same pattern of products as when the RNA-DNA hybrid was added to the reaction directly (compare Fig. 2 A , lane 1 and Fig. 3, W T ) .Control reactions

Plus Strand P r i m i n g by HIV-I RNase H

6224

-9

-4

+1

+5

"MULV HIV-1 FIG. 1. HIV- 1 and M-MuLV sequences near the PPTs.The regions of the M-MuLV and HIV-1 PPTs from which the plus strand primers are derived by RNase H cleavage are underlined. The boxed nucleotides indicate the left end of the integrated provirus that begins with the conserved TG dinucleotide. Sequences beyond the 20 bases shown here diverge completely for the two viruses. Bases that differ between HIV-1 and M-MuLV within the region shown are connected by vertical lines. Numbers above the sequences indicate the convention for numbering the bases in relation to the plus strand origin at the+1 position.

B

A Mol

PPI

HIV-1 PPT

C G A 1 2 3

RT

5' /

-

T C G A 1 2 3

Hiv Mol C

RT

I'/

A A A A

A A A

G G G

G G G G

-

T 3'

Hiv Mol C

A

-Y

FIG.2. Initiation of plus DNA synthesis in vitro by HIV-1 and M-MuLV reverse transcriptases.Panel A, plus strand priming at the M-MuLV PPT. An RNA-DNA hybrid containing the MMuLV PPT was constructed from the gradient-purified EcoRI fragment of ssB190(-) and RNA-synthesized using B190(-) DNA as a template. The hybrids were incubated with HIV-1 reverse transcriptase ( H i u ) (lane I ) and M-MuLV reverse transcriptase (Mol) (lane 2 ) , and the products were analyzed by oligonucleotide M7878(-) extension reactions, followed by electrophoresis in an 8% 8 M urea gel. For the control reaction ( C ) (lane 3), reverse transcriptase was omitted. Lanes marked A, C, C, and T show the products of dideoxy sequencing reactions using M7878(-) as a primer on M13mp7-Al5, a single-stranded DNA template containing the M-MuLV PPT in the plus polarity. The sequence of part of the M-MuLV PPT is drawn to the left, and thearrow marks the primary site of initiation of plus strands. Panel B, plus strand priming at the HIV-1 PPT. The same procedure was followedas for panel A, except the source of the minus DNA in the hybrid was M13mp7HXEV(-), the plus RNA was synthesized from pBHXS, and the oligonucleotide extension reactions were primed by H9119(-).Also, the single-stranded DNA sequencing template was M13mp7HXEV(+), which contains the HIV-1 PPT, and the oligonucleotideprimer was again H9119(-). The reactions contained HIV-1 reverse transcriptase (lane I ) , M-MuLV reverse transcriptase (lane 2); and no enzyme (lane 3). The sequence of part of the HIV-1 PPT is drawn to the left, and the arrow marks the primary site of initiation of plus strands.

in which RNase H- reverse transcriptase was added but the normal reverse transcriptase was omitted yielded no bands on the gel whenanalyzedby primerextension(datanot shown). Plus RNAwas transcribed invitro from each of the mutant used inthetwo-step M-MuLV PPT plasmidDNAsand reaction described aboveas a template for plus strand priming by HIV-1 reverse transcriptase. Point mutations a t positions from -9 to -5 and from +2 to +4 had little or no effect on the plus strand priming reaction (data not shown). Fig. 3 shows the results for some of the mutants that did have a significant effect. The +lAA-sG mutation caused essentially the same cleavage pattern as thewild-type, but the bands on the gel are shifted downward by 1 base. This result, which can be seen more clearly on a longer exposure of the gel (data

FIG. 3. The effects of mutations in the M-MuLV PPT on plus strand priming by HIV-1 reverse transcriptase. Plus RNA was synthesized from the M-MuLV wild-type and mutant dsB19O(-) clones and incubated with HIV-1 reverse transcriptase in two-step reactions as described in the text. The products were analyzed by oligonucleotide primer extension using oligonucleotide M7878(-) as described in the legend to Fig. 2. Designations for the RNAs are given above the corresponding lanes, in which WT indicates the wild-type and the other designations indicate the position of the mutation relative to thenormal plus strand origin a t +1 (see Fig. 1).The wildtype sequence is drawn to the left, and the normal site of RNase H cleavage is marked by an arrow.

notshown)isto beexpected, since deletion of the +lA positions the origin 1 base closer to the oligonucleotide used in the primer extension assay. Changing the G residues a t positions -4 and -2 dramatically affected the site of RNase H cleavage in the priming reaction. The -2A mutant shifted the major site of plus strand priming downstream by 1 base to the +1 position, with some priming also occurring at the proper site and at the-1 position. The -4A mutation resulted in extensive heterogeneity in the plus strand products, with themajority of thepriming occurring at the -1 and +1 positions, aswellfas at the normal site. The other two possible base changes at the-4 position, -4C and -4T, did not cause as drastic an effect as the -4A, but didcauseadefinite enhancement of priming at the-1 position (data notshown). Interestingly, for the-3A mutant, thecleavage at theproper site was enhanced relative to the spectrum of sites normally observed between +1 and +3. A double mutation, +1C+5C, caused extensive heterogeneity with significant cleavages occurring after basesa t positions -2 to +2. The Effect of Selected Mutations in theHZV-1 PPT on Plus Strand Primingby HZV-1 Reverse Transcriptase-To rule out any effect the Moloney context may have had on theexperiments with the HIV-1 reverse transcriptase and M-MuLV mutant PPTs, selected mutations were generated within the HIV-1 PPT and tested with the HIV-1 enzyme. One of the three differencesbetween the M-MuLV and HIV-1 PPT

Plus Strand Primingby HIV-1 RNase H sequences occurs at the +2 position (Shinnick et al., 1981; Ratner et al., 1985); for M-MuLV it is an A and for HIV-1 it is a C. We therefore constructed aplasmid containing a +2C to +2A change in the HIV-1 PPT. The results of the plus strand primingreactionwith this mutant RNA (Fig. 4A) showed that the spectrum of plus strand initiation products was identical with that of the wild-type, except for an enhancement of the band initiated at the+1 position. Four additional mutations,+1C, -2A, -3A, and -4A, were made in the HIV-1 PPT. It should be noted that due to a technical problem with the polymerase chain reaction mutagenesis procedure, the +IC, -3A, and -4A mutations were actually double mutations, being +1C+12A;-3A+9A; and -4A+8A, respectively. In the construction of each mutant plasmid, the oligonucleotide carrying the desired change was a 21-mer with the mutation at the central base. It appears that during step 1 of the polymerase chain reaction mutagenesis procedure, the DNA polymerase randomly added an extra base onto the 3‘ end of the product such that in step 2 a mutation was introduced (see “Experimental Procedures”). It is likely that this second, unexpected mutation is farenough away from the PPT that it had no effect on the priming results. The results of using RNA generated from these mutant plasmids as templates for plus strand priming reactions by HIV-1 reverse transcriptase were quite similar to the results obtained using the corresponding M-MuLV PPT mutants (compare Fig. 3 and Fig. 44). As seen previously, a G to A change inthe -2 to -4 region significantly altered the pattern of productsseenin the priming reaction. One difference, however, was that in these reactions using the HIV-1 PPT, the intensity of the band corresponding to initiation at the +1position was somewhat weaker than for the same mutants in the Moloney context. These results are consistent with the observation cited above that the +2A mutant in the HIV-1 PPT showed a slight enhancement inpriming at the +1 position. The Effect of Mutations in the HIV-1 PPT on the Specificity of M-MuLV RNase H-Previously, the sites of initiation of the plus DNA strands by M-MuLV RNase H were mapped for a series of double and single mutations in the M-MuLV PPT (Rattrayand Champoux, 1989). Using the series of mutationsinHIV-1context generated above, we asked whether the pattern of plus strand products would be the same when these mutant RNAs were used as substrates for plus strand priming by the M-MuLV reverse transcriptase.

A FIG. 4. The effects of mutations in the HIV- 1 PPT on plus strand priming by HIV- 1 and M-MuLV reverse transcriptase. Plus RNA was synthesized from the pBHXSclone containing the the HIV-1 wild-type PPT and pBHES clones containing themutant HIV-1 PPTs and incubated with HIV-1 reverse transcriptase (panel A ) or MMuLV reverse transcriptase (panel B ) . The products were analyzed by oligonucleotide primer extension using oligonucleotide H9119(-) as described in the legend to Fig. 3. Lanes are labeled as in Fig. 3.

+ o v a

6225

The results are shown in Fig. 4B. As observed previously for these mutants, the major site of initiation was not altered from that of the wild-type. For the +2A mutation, additional cleavage was also observed 1base downstream. In accordance with the earlier results (Rattray andChampoux, 1989) for the -2A, -3A, and -4A mutations, some cleavage and priming was also observed before the A mutation (Fig. 4B). DISCUSSION

RNase Hplays several critical roles in retroviral replication, one of which is its involvement in the plus strand priming reaction. A precise cleavage by RNaseH within the PPT generates the primer terminus for plus strand initiation, and a second cleavage removes the primer. The significance of these reactions is that they ultimately define the left end of the unintegrated linear retroviral DNA, and thesequences at the ends of this molecule are essential to the subsequent integration process. We have been interested in determining what featuresof the PPT direct the RNase Hto cleave within this region with such precision. A comparison of the PPT regions for retroviruses and retrovirus-like elements reveals a high degree of conservation over a sequence approximately 13 bases in length (Rattray and Champoux, 1989; Varmus and Brown, 1989). Moreover, secondary plus strand start sites have been identified upstream of the normal site in some retroviruses (Blum et al., 1985; Rattray andChampoux, 1987; Charneau and Clavel, 1991; Hungnes et al., 1991; TobalyTapiero et al., 1991; Charneau et al., 1992), and thesequences intheir immediate vicinity resemble the PPT sequences. These facts suggest that the PPT sequences are recognized by RNase H. We have further established the importance of the PPT to plus strand priming by our in vitro studies of MMuLV and HIV-1 reverse transcriptase. Although the PPT sequences for M-MuLV and HIV-1 are very similar (Fig. l), the sequences outside the 20 bases encompassing the PPT sites are quite different for the two viruses. We previously showed that the M-MuLV reverse transcriptase could recognize and correctly prime plus strand synthesis using the HIV1 PPT (Pullen and Champoux, 1990). Here we extend this observation by showing that the HIV-1 reverse transcriptase will similarly prime plus strand synthesis using the M-MuLV PPT. These results suggest that the PPT contains all of the determinants for proper cleavage by RNase H for the plus strand priming reaction. We have chosen to characterize these determinants by examining the effects of mutations in the PPT sequences on theRNase H cleavage reactions (Fig. 5).

Priming Strand Plus

6226

by HIV-1 RNase H

Previously we determined the effects of a series of mutations in the M-MuLV PPT on the Moloney plus strand priming reaction in vitro (Rattrayand Champoux, 1989). Although none of the mutations eliminatepriming within the PPT, a number of changes do influence the precision of the priming reaction (Fig. 5 C ) . Most important, insertion of 1 or 2 nucleotides at thejunction between the G residue at position -6 and the A residue at position -7 causes a corresponding upstream shift in the start site for plus strands (-7GV and -7AV-8TV). Thisresult, in conjunction with the results obtained with some of the other mutant PPTs (e.g. -7C), indicates that the M-MuLV RNase H is positioned for the cleavage reaction that generates the plus strand primer by measuring 6 residues from the end of the 5 contiguous A residues within the PPT. A less important, but nonetheless significant determinant of the precision of the reaction is the preference of the M-MuLV RNase H to cleave the RNA between the G andA residues found near the end of the PPT (positions -1 and +1) (Rattray and Champoux, 1989). The ability of the HIV-1reverse transcriptase to utilize the A vp

4 I A A'T'A

A A A A A G G G G G'G

+3C

A A A A A G G G C G'G

+2T +1C+5C

B

I4

A A A

E

+I

I4 I4 IIII I G

G A A A A G G G G G G C ' C T b G G A A

A A A

-3A

G A A A A G G G & G G A ' C T G G A A

C A T'G C A A

-IA

G A A A A C G A G G G A C T Q C ~ A ' A

A A A A A C G C G G A A A T ' C A A A

A A A

rAl

vp

141

-7A

A A A A A G G G G A G A A ' T G A A A

-2c

A A A A A G

-3A

A A A A A G G G A G G A ' A T G A A A

-4A

A A A A A G G I G G G A A ' T G A A A

-4c

A A A A A G

-4T

A A A A A

Ir

-7A

A A A A A G G G ~ G l ~ ~ JT AG ~A AA A

-3A

A A A A A G G G 1 b G G A ' A T G A A A

-4A

A A A A A G G1&'G'G'G

c

c

1,''

-7T

A A A A r c G c c G G l A ' A T G A A A

rl

-7GO

c G T G c

-1A-lAQ

II

G A

~

l

G G G'G

IA4A'T'G

A'A

T

C A A A

I I II

A A A

T A

& A'A T G A A A

I 1

C G G'G G C A'A

T G A

A A

A A A A A C G C G G G A A T C A A A

-5A

A A A A A G

A

-6T

A A A A A

c c

-6c-9~ A A

-,Av-~m

A A A A A G C G'G'G

A A~ A GA

L

I I

A A A A A G G G G C G G I A ' A T G A A ~

G G G A ' A ~ GA A A

-7A'J-BTP

-7Gv

I

A A A A A G G G G G C A ' A T G A A A

c c G C'G A A ' T ~ G A A A

I IC,

G A A

C

A A A

A'A'T'G

A C'T'G

II&b1

rlii

A A A A A C C G G G

rl I

G A A A A G G G G'I'G

-1A

-7T

II rl 1

G A A A A G G 0 G G'G A I'TlG G A A

-7A

A A A A A G G G G G'G T'A'T'G

-4C-6C

I

G A A A A G G G G G ' G A ' C T ' G G A A

A A A G

A T'G

A A A A A G G 0 G C'G A T'T'G A A A A A G G G G'G

+>A

A A A

A A'&G

A C G C C G'G

A A A

+1T

-1A-lAQ

WZ

A A A A A G G G G G ~ G A A ~ T ~ G A ~ A

+4A

+1M-0G

M-MuLV PPT as a substrate for plus strand priming has enabled us to use the setof M-MuLV PPT mutants generated in the study described above (Rattray and Champoux, 1989) to investigate the sequence determinants of the HIV-1 reaction. As in the case of M-MuLV, the results indicate that single- or double-base changes or even small insertions or deletions within the PPT do not abolish the plus strand priming reaction. However, the sequence features of the PPT that are necessary for the precise positioning of RNase H for the cleavage event that generates the plus strand primer are quite different for HIV-1, as compared with M-MuLV. In particular,thosemutations in the region from -5 to -9, including ones affecting the -7 position that is so critical for the Moloney enzyme, have no effect on the HIV-1 priming reaction, whereas mutations at the-2 and -4 positions, which have only subtle effects on the Moloney reaction, cause significant losses in precision by the HIV-1 reverse transcriptase. Most notably, the replacement of the -2G with an A causes the primary start site to be shifted 1 base downstream, whereas changes at the-4 position significantly increase the

c

A A

c

a

1

I

GaG A ~ A L T ~A GA A

G G G G'G I Ab A t T l G A A A

I I

A A A A

2 G G G c GAG A

~

A A~ A GA

A A A A

p

c c

~

A A~ A GA

A A A A A A

G t

G

A G

I

- 0 ~ A A A ~ A G G G G G ~ G A ' A T G A A A

A A A

c G

G'G

A

G G'G { Ab A i

Tb

G A A A

I

c c G G L G A ' A ~ GA A A

FIG.5. Summary of plus strand initiationsites for the PPT mutants. The wild-type and mutants are named as in Figs. 3 and 4. An underline indicates a mutated base. The size and location of the arrows indicate the strength and position of the plus strand initiation products, respectively. A, effects of mutations in the M-MuLV PPT on HIV-1 plus strand initiation. B, effects of selected mutations in the HIV-1 PPT on HIV-1 plus strand initiation. C, summary of previous results on the effects of selected mutations in the Moloney PPT on MMuLV plus strand initiation (Rattray and Champoux, 1989).

6227

Plus Strand Priming by HIV-1 RNase H priming observed 1 base upstream of the normal site. It is intriguing that the-2 and -4 positions, which are so sensitive to base changes in the HIV-1 system, are among the most highly conserved positions in the PPT sequences of retroelements (Rattray and Champoux, 1989). If they are conserved because of their importance to the plus strand priming reaction, then itis surprising that theM-MuLV reverse transcriptase is so unaffected by changes at these positions. It is also worth noting that by comparison with the M-MuLV reverse transcriptase, the HIV-1 enzyme appears to be intrinsically less precise for the plus strand priming reaction, with a significant amount of priming occurring 3 bases downstream of the normal site. Interestingly, the -3A mutation appears to increase the specificity of priming by HIV-1 reverse transcriptase at thenormal site. These results may be explained by hypothesizing that the HIV-1 RNase H is positioned for cleavage by forming close contacts with the -2G and -4G residues. Thus, substitution of an A for a G at the-2 position might cause the enzyme to adopt an alternate position on the substrateby forming close contacts with the G residues at -3 and -1, thereby forcing the cleavage to occur 1 base downstream of the normal site. When the -4G position is mutated to an A, it may similarly force the enzyme to position itself by forming contacts with the G residues at positions -5 and -3, forcing the cleavage to occur 1 base upstream of the usual origin. Perhaps the presence of an A between the 2 Gs at -4 and -2 is actually more favorable than the wild-type configuration containing the -3G. If this hypothesis is correct, it provides an explanation for the enhanced cleavage observed at theproper site for the -3A mutant, as well an explanation for the directionality of the shifts for the -2A and -4A mutants. Whereas mutations downstream of the origin had no effect on plus strand priming by M-MuLV reverse transcriptase, our results indicate that for the HIV-1 enzyme, some of the determinants for the specificity of the reaction must lie downstream of the origin site. First, we found that substitution of the M-MuLV PPT for the HIV-1 PPT resulted in a slight increase in priming 1base downstream from the normal origin by the HIV-1 reverse transcriptase. A comparison of the results shown in Fig. 5B (+2A versus W T ) with the pattern shown in Fig. 5A ( W T ) reveals that this effect can be accounted for by the difference in the two PPTs at the +2 position (it is an A for M-MuLV and a C for HIV-1). In fact, in comparing the results for the plus strand priming reactions by HIV-1 reverse transcriptase with the HIV-1 PPT mutants (summarized in Fig. 5B) with the corresponding results obtained using the M-MuLV mutants, it appears that most of the differences in the priming patterns can be attributed to this A to C change. Second, a loss of specificity was observed with the +1C+5c mutant in the M-MuLV context (Fig. 5A). The +lC mutation in the HIV-1 context (Fig. 5 B ) exhibits a less severe phenotype, but if one considers the effect of the A to C change at the +2 position on going from M-MuLV to HIV-1, it appears that most if not all of the defect in the +1C+5C mutant is caused by the C residue at the+1position. Given the fact that the PPT is so highly conserved among

retroviruses and related elements and that theHIV-1 and MMuLV reverse transcriptases can each properly initiate plus strands using the PPT derived from the other virus, it is surprising that thereis such a strikingdifference in how PPT mutations affect the RNase H cleavage patterns generated by these two enzymes (compare Fig. 4A with 4B and Fig. 5A with 5C). However, it is clear in both cases that a combination of two or more features of the PPT region determine the specificity of cleavage. Even the mutations that cause significant shifts in cleavage do not abolish it all together, and many other point mutations that do not have drastic effects still cause some alteration in the cleavage pattern. It is likely that the RNase H recognizes the whole PPT region, but that the way in which it binds causes it to have closer contacts with some of the bases than with others, and mutations in those particular bases cause more noticeable alterations in the precision of the cleavage reaction. Acknowledgments-We thank Sharon Castaneda and Andrew Ching for helpful comments during the preparation of the manuscript. REFERENCES Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A,, and Struhl, K. (1987) Current Protocols in MoleculurBiology, John Wiley & Sons, New York Baltimore. D.. and Smoler. D. F. (1972) J . Biol. Chem. .. .. 247.7282-7287 Been M. D and Cham oux, J. J:(1983) Methods Enzymol: 101,90-98 Blum, H. E.','Harris, J. Ventura, P., Walker, D., Staskus, K., Retzel, E., and Haaae, A. T. (1985) Virology 142,270-277 Champoux, J. J., Gilboa, E., and Baltimore, D. (1984) J. Virol. 4 9 , 686-691 Charneau. P.. and Clavel, F. (1991) J. Vtrol. 66.2415-2421 Charneau; P.. Alizon M.. and Clavel F. (1992) 2. Virol. 66,2814-2820 Colicelli, J., ind God S. k. (1985) ClU 42,573-580 Colicelli, J., and Goff: S. P. (1988) J. Mol. Biol. 1 9 9 , 47-59 Finston, W. I., and Champoux, J. J. (1984) J. Virol. 6 1 , 26-33 Furfine, E. S., and Reardon, J. E. (1991) Biochemistry 30! 7041-7046 Geisselsoder, J., Witney, F., and Yuckenberg, P. (1987) BwTechnques 6,7 s 791 Gilboa, E., Mitra, S., Goff, S., and Baltimore, D., (1980) Cell 1 8 , 93-100 Huber, H. E., and Richardson, C. C. (1990) J. Bwl. Chem. 2 6 6 , 10565-10573 Hungnes, O., Tjotta, E., and Grinde B. (1991) Arch. Virol. 1 1 6 , 133-141 Keller, W., and Crouch, R. (1972) Phc. Natl. Acad. Sci. U. S. A. 69,3360-3364 Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 1 6 4 , 367-382 Leis, J. P., Berkower, I., and Hurwitz, J. (1973) Proc. Natl. Acad. Sci. U. S. A. 70,466-470 Mitra, S. W.,Chow,M., Champoux, J. J., and Baltimore, D. (1982) J. Biol. Chem. 267.5983-5986 - - ~ Nelson, R. M.,and Long, G. L. (1989) A d . Biochem. 180, 147-151 Omer, C. A., and Faras, A. J. (1982) Cell 3 0 , 797-805 Panganiban, A. T., and Temin, H. M. (1983) Nature 3 0 6 , 155-160 Pullen, K. A., and Champoux J. J. (1990) J. Virol. 64, 6274-6277 Pullen, K. A., and Cham oux: J. J. (1992) J. Virol. 66, 367-373 h t n e r , L., Haseltine,, !V Patarca, R., Livak, K. J., Starcich, B., Josephs, S. F., Doran, E. R., Rafalski, J. A., Whitehorn, E. A., Baumeister, K., Ivanoff, L., Peteway, S. R., Jr., Pearson, M.L., Sautenberger, J. A., Papas, T. S., Ghrayeb, J., Chang, N. T., Gallo, R. C., and Wong-Staal, F. (1985) Nature 313,277-284 Rattray, A. J., and Champoux, J. J. (1987) J . Vzrol. 6 1 , 2843-2851 Rattray, A. J., and Champoux, J. J. (1989) J. Mol. Biol. (1989) 2 0 8 , 445-456 Resnick, R., Omer, C. A., and Faras A. J. (1984) J. Viml. 61,813-821 Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. ~

E.,

~I

~~~~

74.5462-5AfiX ","" l " _

Shinnick, T. M., Lerner, R. A,, and Sutcliffe, J. G. (1981) Nature 2 9 3 , 543548 Starnes, M. C., and Cheng., Y.-C. (1989) J. Biol. Chem. 2 6 4 , 7073-7077 Tobaly-Tapiero, J.,Kupiec, J. J., Santillana-Hayat, M., Canivet, M., Peries, J., and Emanoil-Ravier. R. (1991) J. Gen. Virol. 72.605-608 V ~ U H.S E. (1982) Scierice 2i6,812-820 ~'""Varmus' H. E. and Brown P. (1989) in Mobile DNA (Berg D. E., and Howe M.M:, eds i p 53 108 Ahencan Societ for Microbiology Washin Varmus H. a n d i w a k t r o m , R. (1982rin RNA Tumor viruses (%%s:'z; Teich: N., Vhrmus, H., and Coffin, J., eds), Vol. 1, pp. 369-512, Cold Spring Harbor Laboratow. Cold Surine Harbor. NY Varmus, H. E. and swanstrim, R. (1985)'in RNA Tumor Viruses (Weiss, R., Teich, N., qarmus, H., and Coffin, J. e&), Vol. 2, pp. 75-134, Cold Spring Harbor Laboratory, Cold S ring Harbor, NY Zoller, M. J., and Smith, M. (1984) DNA ( N . Y . )3,479-488 ~~

k.

8.