THE JOURNAL Q 1991 by The American Society for Biochemistry and Molecular Biology,

OF BIOLOGICAL CHEMISTRY Inc.

Vol. 266, No. 36, Issue of December 25, pp. 24295-24301,1991 Printed in U. S. A.

Human Immunodeficiency Virus Reverse Transcriptase Displays a Partially Processive 3‘ to 5‘ Endonuclease Activity*

(Received for publication, June 20, 1991)

Jeffrey J. DeStefanoS, Rodolfo G . Buiserzs, Lisa M. Mallaberz, Robert A. Bambarazll, and Philip J. Fay$JJ** From the Departments of $Biochemistry and IlMedicine and the TCancer Center, University of Rochester, Rochester, Niw York 14642

We have examined the ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV-RT) using a natural sequence 83- nucleotide-long RNA template to which was hybridized a DNA oligomer. This substrate configuration allowed for the simultaneous electrophoretic resolution of 5’-, 3‘-, and internally derived RNase H cleavage products. Assays performed in the presence of excess challenger RNA to sequester the RT permitted the analysis of products resulting from a single round of binding of RT to substrate. Substrate cleavage was highly sensi- tive to ionic strength, showing greatest activity at low KC1 concentrations. The increase in cleavage corre- lated with an increase in the half-life of the enzyme on the RNA-DNA hybrid from approximately 31 s to 6.2 min at 80 and 5 mM KCl, respectively. Internally de- rived cleavage products generated in challenged reac- tions were primarily 2-9 nucleotides in length. These lengths indicate that the products were generated by a n endo- rather than an exonuclease activity. The di- rectionality and processivity of the endonuclease were also determined by examination of cleavage products from challenged reactions. Although the lengths of 5’- derived products markedly decreased with time, no change in the size distribution of 3’-derived products was observed, indicating that cleavage proceeded pro- cessively in the 3‘ to 5’ direction. The 5’-derived pro- ducts were shortened more in reactions performed un- der conditions allowing multiple versus single enzyme- binding events, suggesting that the endonuclease ac- tion of a single enzyme is not processive enough to generate the maximum possible amount of cleavage on each substrate. Therefore, HIV-RT displays a partially processive 3’ to 5’ endonuclease activity.

Retroviruses are the etiological agents of several forms of cancer and AIDS. Viral replication requires conversion of a single-stranded RNA genome to double-stranded DNA, which is carried out by the multifunctional viral RT,’ an enzyme

* This work was supported by National Institutes of Health Grant AI 27054 and in part by Core Grant CA 11198 to the University of Rochester Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 Ford Foundation predoctoral fellow. ** Established investigator of the American Heart Association. To

whom correspondence should be addressed Hematology Unit, P. 0. BOX 610, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-6576; Fax: 716-473-4314.

The abbreviations used are: RT, reverse transcriptase; HIV, hu- man immunodeficiency virus; AMV, avian myeloblastosis virus.

possessing both RNA- and DNA-dependent DNA polymerase and RNase H activities (Varmus and Swanstrom, 1974; Han- sen et al., 1987; diMarzo Veronese et al., 1986; Starnes and Cheng, 1989). The latter activity can cleave the RNA moiety of an RNA-DNA hybrid. This activity is proposed to be required at several stages of viral genome replication, includ- ing removal of the RNA template after the synthesis of the first strand of DNA (Watson et al., 1979), removal of the host tRNA from which first-strand DNA synthesis has initiated (Omer and Faras, 1982), generation of a specific oligopurine ribonucleotide primer from which second-strand DNA syn- thesis will initiate, and subsequent removal of the oligopurine primer (Champoux et al., 1984; Finston and Champoux, 1984; Huber and Richardson, 1990; Mitra et al., 1982; Resnick et al., 1984; Smith et al., 1984a, 1984b).

HIV-RT is a heterodimer composed of 66- and 51-kDa subunits (p66 and p51) (diMarzo Veronese et al., 1986; Light- foote et al., 1986). Mutational analysis of p66 suggests that the RNase H domain is localized to the C-terminal portion of this polypeptide (Hansen et al., 1988; Prasad and Goff, 1989; Hizi et al., 1990). Cleavage of p66 by a viral protease results in the generation of the N-terminal-derived p51 and a C- terminal-derived 15-kDa polypeptide termed p15, with the proposed RNase H domain contained in the latter fragment. Hansen et al. (1988) have reported that p15 exhibits a non- processive RNase H activity. In contrast, Hostomsky et al. (1991) reported that p15 is enzymatically inactive unless reconstituted with p51, at which point RNase H activity of the complex is restored.

Using RNA-DNA hybrids derived from M13 mp8 DNA, Hansen et al. (1988) found that unlike p15, the HIV-RT heterodimer degrades the hybrid by a processive mode. That is, the enzyme remains bound to the hybrid through several hydrolytic events. Studies by Gerard (1981) with Moloney murine leukemia virus RT and AMV-RT, using oligo(dT). poly(rA) as substrate, indicate that the RNase H activity of these enzymes is partially processive. This is in contrast to studies by others with AMV-RT that indicate a highly pro- cessive mechanism of degradation (Verma, 1975; Leis et al., 1973; Grandgenett and Green, 1974; Moelling, 1976).

The specificity of the RNase H activity of RTs has been a subject of debate. The apparent inability of RTs to cleave certain circular RNA-DNA hybrids led researchers to suggest that these enzymes are exonucleases (Keller and Crouch, 1972; Leis et al., 1973). However, using a relaxed, closed circular plasmid with a 770-nucleotide RNA segment in one strand, Krug and Berger (1989) showed that the RNase H of HIV- and AMV-RTs are capable of authentic endonucleolytic cleavage. Schatz et al. (1990) have reported that HIV-RT displays both endonucleolytic and 3’ to 5’ exonucleolytic activity. The designation of exonuclease was based partly on

24295

24296 HIV-RT Possesses a 3‘ to 5’ Endonuclease Activity

studies by Starnes and Cheng (1989) in which cleavage of poly(dC) .poly(rG) by HIV-RT resulted primarily in mono- mers, dimers, and trimers as final products. However, Mizrahi (1989) found that the final cleavage products of HIV-RT digestion of a hybrid derived from the gag region of the HIV- 1 genome are oligonucleotides ranging from 4 to 15 nucleotides in length. Hence, the products generated from hybrid diges- tion may depend on the nature of the hybrid.

We have further examined the directional activity of HIV- R T using a natural sequence RNA substrate, which was internally labeled so tha t sizes of the products generated by the nuclease activity could be accurately determined. Our results indicate that this degradative activity is best described as a partially processive 3‘ to 5’ endonuclease.

EXPERIMENTAL PROCEDURES

Materials

Recombinant HIV-RT, having properties described by Huber et al. (1989), was graciously provided to us by the Genetics Institute (Cam- bridge, MA). T4 polynucleotide kinase, T 7 RNA polymerase, E. coli ribonuclease H, and PvuII were obtained from U. S. Biochemical Corp. Bovine pancreatic DNase (RNase-free), placental RNase inhib- itor, RNase A (DNase-free), snake venom phosphodiesterase, and rNTPs were obtained from Boehringer Mannheim; dNTPs were obtained from Pharmacia LKB Biotechnology Inc. The 20- and 30- mer deoxyoligonucleotides were synthesized by Genosys Inc. (for- merly Genetic Designs, Houston, TX). Oligo(dT) and poly(rA) were obtained from Midland Certified Reagent Co. (Midland, TX). All other chemicals were from Sigma. Radiolabeled compounds were from Amersham Corp.

Methods

RNase H Assays-HIV-RT (amounts indicated in the figure leg- ends), was preincubated with 2 nM substrate (see below) for 3 min in 10 pl of 50 mM Tris-HC1 (pH 8.0), 1 mM dithiothreitol, 2% glycerol (w/w), 0.1 mM EDTA (buffer A), supplemented with 5, 40, or 80 mM KC1 (as indicated in the figure legends). Assays were initiated by addition of MgCl, in 2.5 p1 of buffer A to give a final concentration of 6 mM MgC1,. Reactions were run for 5 min a t 37 “C, unless otherwise indicated.

Challenged RNase H Assays-In the challenged assay, reaction conditions were the same as for the standard RNase H assay, except that 0.5 pg of oligo(dT) .poly(rA) was included along with the divalent cation at the start of the reaction. This modification limits the RNase H activity to preformed complexes of RT and hybrid substrate. Oligo(dT).poly(rA) used in the reactions was prepared by mixing oligo(dT)16 with poly(rA) a t a 1:8 ratio (w/w) in 10 mM Tris-HC1 (pH 8.0), and 1 mM EDTA. The mixture was incubated for 30 min a t 37 “C and then cooled slowly to room temperature.

RNA-DNA Hybridization-Each 20- or 30-nucleotide-long deoxy- oligonucleotide was hybridized such that its 3”terminal nucleotide was 45 nucleotides from the 5’ end of the transcript (refer to Fig. 1). The 20 nucleotides at the 3’ side of the 30-mer were identical in sequence with the 20-mer. The RNA-DNA hybrid was prepared by mixing primer and transcript at a 5:l ratio of 3’ termini in 10 mM Tris-HC1 (pH 8.0), 1 mM EDTA, and 100 mM KCl. The mixture was heated to 65 “C for 10 min and then cooled slowly to room tempera- ture.

Gel Electrophoresis-Denaturing 10% polyacrylamide sequencing gels (19:1, acry1amide:bisacrylamide) containing 7 M urea were pre- pared and subjected to electrophoresis as described (Sambrook et aL, 1989).

Run-off Transcript-Run-off transcription was done as described in the Promega Protocols and Applications Guide (1989). Plasmid pBSM13+ DNA was purified by CsC12 equilibrium sedimentation from transformed Escherichia coli XL1 Blue cells. The plasmid DNA was cleaved with MseI for which there are 21 sites. The promoter for T7 RNA polymerase is present on a 160-base pair MseI restriction fragment. Using the polymerase, a run-off RNA transcript 83 nucle- otides in length was generated in the presence of 500 p~ each of ATP, GTP, CTP, and 18 p~ [a-’”PIUTP (approximately 44 Ci/ mmol). The DNA template was digested with bovine pancreatic RNase-free DNase I (1 unitlpg of DNA). The RNA transcript was

then extracted with 1 volume of pheno1:chloroform:isoamyl alcohol (24:24:1) and then ethanol-precipitated twice using 3 volumes of ethanol and a final concentration of 1.25 M ammonium acetate. The run-off transcript was resuspended and purified by electrophoresis on an 8% polyacrylamide gel containing 7 M urea. The full-length transcript was located by autoradiography, excised from the gel, and eluted overnight in a buffer containing 0.5 M ammonium acetate, 1 mM EDTA, 10 mM MgC12, 0.1% sodium dodecyl sulfate, and 10 mM ribonucleoside vanadyl complexes. The eluate was separated from the polyacrylamide by centrifugation in a microcentrifuge and subsequent filtration through a 0.45-p, 25-mm disposable syringe filter (Nalgene). The filtrate was extracted 2 to 3 times with phenol: ch1oroform:isoamyl alcohol (24:24:1) to remove the ribonucleoside vanadyl complexes and then ethanol-precipitated with 3 volumes of ethanol and 0.1 volume of 2 M NaCl.

Determination of Enzyme-Substrate Complex Half-lives-The bind- ing half-life of HIV-RT on substrate C (see Fig. 1) was determined using the conditions of the challenged reverse transcriptase assay and 5 or 80 mM KC1 (as indicated in Fig. 8) with the following modifica- tion. After preincubation, 0.5 pg of oligo(dT).poly(rA) in 1.5 p1 of buffer A was added (to sequester enzyme that had dissociated from the hybrid). At a specific time after the addition of oligo(dT) .poly(rA) (indicated in Fig. 8), reactions were initiated by the addition of MgCl, in 1.5 pl of buffer A to give a final concentration of 6 mM MgC1,. Cleavage products were resolved by gel electrophoresis. Autoradio- graphy was performed, and the films were scanned with a densitom- eter. Film exposure times were chosen such that the densitometer readings were approximately proportional to the quantity of radio- activity in each band. The percentage of transcript cleaved was determined by subtracting the remaining portion of absorbance rep- resenting intact transcript a t each time point from the absorbance of unreacted transcript (no enzyme added). The half-life of enzyme- hybrid complexes was estimated from the time course of transcript cleavage. The same approach was used to determine the percentage of transcript cleaved in other experiments. Additional films were appropriately exposed to a lesser degree for densitometry than those shown.

RESULTS

RNase HActivity under Conditions Allowing a Single Versus Multiple Enzyme-binding Events-A uniformly radiolabeled 83-nucleotide-long T 7 RNA polymerase transcript generated from a 160-base pair MseI restriction fragment of pBSM13+ was used as the substrate for RNase H-mediated degradation. A specific 20- or 30-nucleotide-long DNA oligomer was hy- bridized to the region between 45 and 64 (20-mer) or 45 and 74 nucleotides (30-mer) from the 5‘ end of the transcript (Fig. 1). Since the HIV-RT has no nuclease activity on single- stranded RNA, only cuts within the hybrid region of the substrate should be possible. Consequently, the shortest pos- sible 5”derived cleavage products (45 nucleotides) are longer than the longest possible 3’-derived products (38 nucleotides). The shortest possible 3’-derived products are 20 or 10 nucle- otides long for the hybrids derived from the 20 or 30 nucleotide oligomers, respectively. These substrates allowed separation of the 5’- and 3’-derived cleavage products from internally derived oligomers shorter than 20 or 10 nucleotides (20-mer or 30-mer hybrids, respectively) that result from multiple cleavage events within the hybrid region. Therefore, the con- figuration of the substrate allows a subsequent simultaneous electrophoretic resolution of 5’- and 3”derived RNase H cleavage products from each other and from internal products.

Figs. 2 and 3 show the products resulting from HIV-RT mediated degradation of the substrate with the 20- or 30- nucleotide-long oligomer, respectively, hybridized to the in- ternally labelled RNA. The concentration of KC1 in the reactions was either 5 mM (lanes 2 and 3), 40 mM (lane 4 ) , or 80 mM (lane 5 ) . A large excess of oligo(dT).poly(rA), com- pared with the substrate described above, was included in all the reactions, except the ones shown in lane 2. The oligo(dT). poly(rA) was added at the start of the reaction (see “Meth- ods”) to sequester any enzymes that dissociate from the

HIV-RT Possesses a 3‘ to 5 ’ Endonuclease Activity A.

pBSMl3+ (3204 bp)

24297

1 2 3 4 5 a::;: 77 Promalar

Transcriplion Slarl

J I.....) ,

3’

3’ 3044 bp i” ‘ 1 ’ 2 77 bp 83 bp

3’ RNA

- 70 r

L

3’ RNA

FIG. 1. Production of the substrates. Plasmid pBSM13+ was cut with the restriction enzyme Msrl, as described under “Methods.” The fragment containing the T7 promoter was used to generate an 83-nucleotide-long RNA transcript ( A ) . This transcript was hyhri- dized with a unique 20- (substrate R ) or 30- (substrate C) nucleotide- long DNA oligomer that hybridized from nucleotides 45 to 64 or 45 to 74, respectively, from the 5’ end of the transcript. bp. base pairs.

substrate (DeStefano et QL, 1991). This experimental design, designated a challenged reaction, confines the action of each R T t o a single round of processive cleavage during the course of an experiment. A 10-fold reduction in the amount of oligo(dT) .poly(rA) added to the reaction had no effect on the quantity or distribution of the resulting cleavage products (data not shown), indicating that oligo(dT)-poly(rA) was not influencing the processivity of cleavage events.

The relative quantities of RT and substrate in the chal- lenged reactions have been adjusted such that approximately 50% of the substrate molecules sustain no RNase H activity in the reactions containing 5 mM KCI. At higher salt concen- trations, less than 50% of the substrate was cleaved by this amount of enzyme. This ensures, assuming a Poisson distri- bution of enzyme on polymeric substrate, that in most cases the cleavage observed results from the action of one R T on one substrate molecule. When the concentration of the en- zyme was increased approximately 10-fold in the challenged reaction, nearly all of the substrate was cleaved, resulting in a n increase in the amount but not the distribution of cleavage products (Fig. 4). Evidently, even at this relatively high en- zyme concentration, the action of a single R T on one substrate molecule was still being observed. I t is likely that any excess enzyme not bound to the hybrid region of the substrate was rapidly sequestered when the oligo(dT) .poly(rA) was added at the start of the reaction. Furthermore, given the small size of the hybrid regions (20 or 30 nucleotides) and the proposed length of nucleotides (15-16) spanned from the polymerase to RNase H active sites of HIV-RT (Furfine and Reardon, 1991) it is unlikely that more than one RT would he efficiently bound to each hybridized oligomer.

The size distribution of the 3”derived cleavage products generated by HIV-RT in assays performed with 5 mM KC1 were slightly different depending on the substrate used. The 3”derived products generated a t 5 mM KC1 from cleavage of the RNA hybridized to the 20-nucleotide oligomer (the two predominant products of about 21 and 22 nucleotides; Fig. 2,

r

. -20

-10

Internal

L

FIG. 2. RT-mediated cleavage of substrate R (refer to Fig. 1). HIV-RT (lanr 2, 170 ng; lanes 9-5, 17 ng) was preincuhated with suhstrate R (2 nM). The concentration of KC1 in the assavs was 5 (lanes 2 and 3 ) . 40 (lane 4 ) . or 80 mM (lane 5 ). Reactions were started as described and stopped after 5 min. All assays were performed under challenged conditions (refer to “Methods”). except that shown in l a w 2, in which the poly(rA).oligo(dT) WAS omitted. Products were sepa- rated on a 10% polyacrylamide, ’7 M urea gel and visualized hv autoradiography. The positions of 5”. 3‘-, and internallv derived cleavage products are indicated by brackets. RSA size markers were produced hy alkaline hydrolysis of the “‘P-5’-end-Iaheled MseI-de- rived transcript. The undegaded suhstrate is shown in lane 1.

lane 3 ) were about 3 nucleotides longer than 3”derived prod- ucts generated from the 30 nucleotide oligomer-hybrid (pre- dominant products of about 18 and 19 nucleotides; Fig. 3, lane 3 ) . Since the position of the 3”terminal deoxyribonucleotide was the same in both hybrids, these results indicate that the length of the RNA-DNA hybrid region can influence the position of cleavages by HIV-RT.

The 5”derived cleavage products generated in the chal- lenged assay ranged in size from an upper limit of approxi- mately 65 down to about 53 nucleotides in length (Figs. 2 and 3, lane 3 ) , with a prominent product of about 57 nucleotides. Cleavage of the substrates also generated some products shorter than 10 nucleotides, presumed to result from multiple cleavages within the hybrid region of a single RNA molecule. Since these reactions were challenged, the production of a substantial quantity of products resulting from multiple cleav- ages indicates that HIV-RT is capahle of more than one cleavage per enzyme binding event. Therefore, cleavage is occurring via a processive mechanism.

The extent of cleavage was evaluated in reactions carried out in the absence of challenge template. In these reactions, multiple binding and dissociation events can occur since there is nothing to trap the RT after dissociation from the suhstrate. When HIV-RT was assayed (Figs. 2 and 3, lane 2 ) . the quantity hut not the distribution of 3”derived cleavage prod- ucts changed in comparison with the same assav performed in the presence of challenge template (Figs. 2 and 3 , lone 3 ) .

The quantity of products less than 10 nucleotides in length

24298 HIV-RT Possesses a 3‘ to Ei‘ Endonuclease Activity

1 2 3 4 5 1 2 3 4 5 6 7

r -

r Internal 1 i T

-70

-50

-30

-20

-10

FIG. 3. RT-mediated cleavage of substrate C. HIV-RT (lane 2, 110 ng; lanes L 5 , 1 1 ng) was preincuhated with suhstrate C (2 nM). Reaction conditions, product separation, and figure lahels were as descrihed in the legend to Fig. 2.

increased considerably in the unchallenged assay, and most of the larger (>55 nucleotides) 5”derived cleavage products decreased. There was an increase in 5”derived products be- tween 50 and 55. The likely explanation of these results is that the large 5”derived cleavage products are further hydro- lyzed, producing shorter 5’ products and internally derived small oligomers. The responsible activity would be best de- scribed as a 3’ to 5’ directional nuclease. Schatz et al. (1990) also came to this conclusion following analysis of end-labeled RNA substrates. Since only a portion of the potentially de- gradable 5”derived products were hydrolyzed in the chal- lenged reaction, the directional nuclease of HIV-RT is par- tially processive under the assay conditions.

The concentration of enzyme was increased 10-fold in the unchallenged reactions (lane 2) shown in Figs. 2 and 3 relative to the challenged reactions (lane 3 ) . This additional enzyme was necessary to allow substantial digestion of the 5”derived cleavage products in the time frame of the reactions. The differences in the lengths of 5’4erived products observed in the challenged versus unchallenged reactions was not due to the different quantity of enzyme used. When 10-fold more enzyme was used in the challenged reaction (Fig. 4, lane 2 ) , over 80% of the total suhstrate was cleaved, resulting in an approximately 2-fold increase in cleavage products, as com- pared with assays performed with less enzyme. However, the distribution of products did not change over this wide range of enzyme concentrations (Fig. 4). Shown in Fig. 5 is an experiment where the same low quantity of enzyme normally used in the challenged assay was used in both the challenged (lane 4 ) and unchallenged reactions (lanes 2 and 3, 10- and 5-min reactions, respectively). In the absence of the challenge template, a greater extent of substrate was cleaved, and there was a shift in the lengths of 5”derived cleavage products, with smaller products heing more prominent in the unchal- lenged reactions. These results indicate that even when the same low amount of enzyme is used in the challenged and

r 5’ L

3‘ r 1

Internal [

- 70

- 50

- 30

- 20

- 1 0

FIG. 4. Cleavage of substrate R (refer to Fig. 1) with differ- ent concentrations of HIV-RT. IIT (Innes 2-7, 170, 85. 42.5. 21.3. 10.6, or 5.3 ng, respectively) was preincuhated with suhstrate H in assays containing 5 mM KCI. All reactions were performed under challenged conditions (refer to “Methods”). Reactions were started as descrihed and stopped after 5 min. Product separation and figure lahels were as descrihed in the legend to Fig. 2. The undepaded suhstrate is shown in lane 1. The percentage of suhstrate cleaved (as determined hy densitometry scanning) in this particular assay. as compared with the undegraded suhstrate. was 82. 77, 71, 49. 37. and 26% for Lanes 2-7, respectively,

unchallenged reactions, enzyme-rebinding events in the un- challenged reaction result in a further decrease in the length of 5”derived cleavage products.

The Directional 3‘ to 5 ’ Activity of HIV-RT Is Endonuclm- lytic-The use of internally labeled RNA transcripts as sub- strates for RNase H activity allowed us to visualize products derived from multiple cleavage events within the RNA-DNA hybrid region of the suhstrate. We conclude from these anal- yses that the directional nuclease activity of HIV-RT is a 3‘ to 5’ endonuclease. The conclusion is based on the definition of an exonuclease as an enzyme that cleaves nucleotides one a t a time from the end of a polynucleotide chain, yielding mononucleotide products. A directional endonuclease would then be defined as an enzyme that traverses the suhstrate polymer in one direction, periodically exhibiting nuclease activity and generating oligonucleotide products. The inter- nally derived products generated by HIV-RT in the reactions shown in Fig. 3 were resolved on a 20% polyacrylamide gel (Fig. 6). As was the case with the 3’- and 5’derived products. the internally derived products generated in challenged reac- tions increased in quantity with decreasing salt concentration (lanes 4-6; 5 , 40, and 80 mM KCI, respectively). There was a further substantial increase in the unchallenged reaction (lane 3 ) . The distribution of internal products did not vary sub- stantially with varying salt or in the challenged uersu.7 un- challenged reactions. The internally derived products ranged in size from monomers up to decamers. However, the vast majority of these products were larger than monomers, indi- cating that they were produced hy an endonucleolytic activitv.

HIV-RT Possesses a 3 ’ to .5‘ Endonucleaqe Activity

1 2 3 4

c- - 0 -

m r ~

CL)

-70

- 50

- 30

3 f

- 20 I “ ..

Internal I -10

L

FIG. 5. Cleavage of substrate C with a fixed enzyme concen- tration in challenged or unchallenged reactions. HIV-RT (1 1 ng) was preincubatetl with substrate (‘2 nM) in reactions containing 5 mM KCI. Reactions were started as descrihed and stopped after 5 (lanes 3 and 4 ) or 10 min ( h e 2). React,ions were performed under unchallenged (lanrs 2 and 3 ) or challenged (/ana 4 ) conditions. Product separation and figure Iahels were as described in the legend 1.0 Fig. 2. The undegraded substrate is shown in lanr 1.

In Fig. 6, the position where a monomer would migrate on this gel is indicated by the position of [F;’-:”P]pU shown in lane 1 . In this reaction, the RNA portion of the substrate was digested to completion with snake venom phosphodiesterase to produce mononucleotides, and the labeled one is seen.

The Time Course of 3 ’ to 5’ Endonucleolytic Degradation in the Challenged R T Assay-Based on results presented in this report we conclude that HIV-RT possesses endonucleolytic activity that can cleave the RNA portion of an RNA-DNA hybrid producing 3’- and 5”derived cleavage products. Fur- thermore, after making this cleavage, the enzyme is capable of additional 3’ to 5’ endonucleolytic cleavages on the 5 ’ - derived portion of the RNA, generating 5”derived products shorter than the original cleavage products. Initially, the R T should produce large 5”derived cleavage products along with corresponding 3”derived products, the sum of which equal t h e full length of the substrate RNA (83 nucleotides). As the reaction proceeds, the 5”derived products would become pro- gressively shorter and small products resulting from this shortening would begin to accumulate. T o test these predic- tions, a time course of cleavage was performed in the presence of challenger template using the RNA hybridized to the 30- deoxynucleotide oligomer as substrate, with results shown in Fig. 7. After 2 s, most of the 5”derived cleavage products clustered around 65 nucleotides in length. As the reaction progressed, much of this RNA was shifted toward shorter products, and as predicted, the accumulation of very small products accompanied this shift. The distribution of the 3’- derived cleavage products remained constant during the time course. There was a small increase in the quantity of 3’- derived products between 2 and 10 s that remained constant

24299

I 2 3 4 5 6

” - 9

- 1

FIG. 6. Resolution of internally derived cleavage products produced by HIV-RT. HIV-KT Ilnnr, 3 , 1 I O ng: lnnr7.s .1-6, 1 1 ng) was preincuhated with substrate C ( 2 nM). Reaction ronditions for assays shown in lnnrs .?-6 correspond with those in lnncs 2Li. respec- tively, of Fig. 3. In the reactions shown in Inn,, 1, the R S A port ion o f the srlhstrate was digested to completion with snake venom phospho- diesterase producing .5’ mononucleotides. I’rodrtcts were analyzed o n a 20% polyacrylamide gel. Figure Iahels were as descrit)rd in Fig. ’2. The undegraded substrate is shown in lano 2.

thereafter. These results are consistent with the proposed model.

The Effect of Salt Concentration on thr RNase H Activity of HIV-RT-Some of the parameters that could potentiallv af- fect the endonuclease activity of HIV-RT were examined. Variations in the MgCI, concentration between 1 and 16 mM did not significantly affect the amount of substrate cleaved or the distribution of cleavage products in the challenge assay (data not shown). The addition of those deoxynucleotides that did not allow extension of the DNA portion of the hybrid (ATP, GTP, and CTP; addi t ion of TTP alone would allow extension by 1 nucleotide) also had no effect on the quantitv or distribution of cleavage products (data not shown). In contrast, the quantity of cleavage products was altered when the KC1 concentration was varied (Figs. 2 and 3 ) . When HIV- RT was assayed in challenged reactions containing 5 , 40, or 80 mM KC1 (lanes 3-5, respectively), the quantitv of RNA cleaved decreased considerably as the salt concentration in- creased. The distribution of the cleavage products did not change substantially with varying salt concentration.

Salt Dependence of the Half-life of HIV-RT Binding to H.ybrid Substrate-One possible explanation for the decrease in cleavage of the template at higher salt concentrations is that the enzyme affinity for the substrate is lower a t higher salt concentration. To test this, we measured the dissociation rate of preformed enzyme-subst,rate complexes at *5 or 80 mM KC1 (Fig. 8). In these assays, the concentration of enzvme was about 8-fold higher than that used in the normal chal- lenged assay. This concentration was necessary in order to observe substantial cleavage in the ass:~vs performed with 80 mM KCI. It was evident from this measurement that enzyme-

24300 HIV-RT Possesses a 3’ to 5‘ Endonuclease Activity

(Ime (rec) 0 2 10 20 60300

r

-70

-50

-30

-20

-10

Internal 1 FIG. 7. Time course of substrate degradation in the chal-

lenged reaction. RT (11 ng) was preincubated with substrate C (2 nM) in reactions containing 5 mM KCI. Reactions were started as described and stopped at, the t,ime indicated above each lanr. Product separation and figure labels were as described in the legend to Fig. 2.

100, 1

Time (min)

FIG. 8. Determination of the half-life of enzyme-substrate complexes in reactions with 5 and A 0 mM KCI. RT (1 10 ng) was preincubated with substrate C (2 nM) in reactions containing 5 (open circles) or 80 mM KC1 (closed circlrs). Reaction conditions were as described under “Methods.” The dashad lines labeled t l $ (initial phase half-life) represent a least squares fit of the first three data points (representing the initial decay phase) in each time course. The percent Mse transcript cleaved was determined as described.

substrate complexes were substantially stabilized by low salt. In both cases, the decay curves were biphasic with a portion of the enzyme dissociating rapidly and a portion dissociating more slowly. A similar phenomenon has been observed pre- viously with different substrates in our laboratory (DeStefano et al., 1991) and by others (Huber et al., 1989). The half-life for the initial decay phase for each reaction was estimated by least squares analysis, using those points that fell within the initial phase on the decay curves. Estimates of initial phase half-lives were approximately 31 s a t 80 mM KC1 and 6.2 min at 5 mM KCI. These results indicate that the preformed

enzyme-substrate complex is markedly more stahle under low salt conditions. However, the results also show that at high enzyme concentration, HIV-RT can cleave most of the suh- strate (approximately 80% at the concentration used), even when the reactions are performed with 80 mM KCI.

DISCUSSION

We have investigated the RNase H activity of HIV-RT on a specific natural sequence RNA-DNA hyhrid. Our results are in agreement with those reported by Schatz et al. (1990) in that HIV-RT exhibits a directional 3‘ to 5’ nuclease activitv. However, our results further indicate that this activity is endonucleolytic in nature. This assessment is hased on the length of the cleavage products generated by the directional endonuclease. Although low levels of mononucleotides were observed, most products were oligonucleotides between 2 and 9 nucleotides in length (Fig. 6). That a small proportion of mononucleotide products were generated indicates that an occasional exonucleolytic cleavage was performed, hut these events were rare. Schatz et al. (1990) speculated that the directional nuclease activity of HIV-RT is a 3’ to 5’ exonu- clease. However, the end-labeled RNA substrates employed in their experiments did not allow them to visualize the products of the directional nuclease.

The use of the challenged assay descrihed ahove allowed a determination of the cleavage that occurs during one inter- action between the RT and the RNA-DNA hyhrid. The results of this assay indicate that the 3’ to 5’ endonuclease of HIV- R T is partially processive (Figs. 2 and 3 ) . That is, the RT cleaves the substrate RNA and then proceeds 3’ to 5 ’ , cleaving the substrate several times, but often dissociates before gen- erating the 5’ products of maximum reaction. These products are hereby defined as 5’ segments of RNA that are no longer recognized as nuclease suhstrates by R T even though a region of potential RNA-DNA hybrid still remains. In the case of HIV-RT, this potential hybrid region is 6-11 nucleotides in length, based on the lengths of 5”derived cleavage products that accumulate in the unchallenged assay (Figs. 2 and 3, l n n ~ 2).

The results of the challenged reaction shown in Fig. 6 are also consistent with a partially processive cleavage mecha- nism. The RT initially cleaves the suhstrate at a preferred cleavage site, which is determined by the distance of a parti- cular preferred phosphodiester bond from the 3’ terminus of the DNA oligomer and may also be influenced hv the sequence of the RNA (Furfine and Reardon, 1991). The initial 5 ’ - derived cleavage products are then further shortened in a 3’ to 5’ direction, resulting in the accumulation of internallv derived oligomers. If the directional endonuclease responsihle for this shortening were highly processive, then the activity would have produced a large proportion of 5”derived products with sizes similar to those generated under unchallenged reaction conditions (Fig. 3, lune 2). An endonuclease that cleaved by a strictly distributive mode of action would have been unable to shorten the initial 5”derived products in the challenged reaction. The 5”derived products generated in the challenged reaction were on average considerably shorter than the initial 5’-derived cleavage product but longer than those produced in unchallenged reactions. Again, this is consistent with a partially processive mode of cleavage.

The predominant ,?’-derived cleavage products generated in our experiment,s varied in length depending on the length of the DNA oligomer portion of the byhrid suhstrate. This variation was not the result of differences in sequence since the last 20 nucleotides of the 30-mer (reading 5’ to 3’) were identical with t,he 20-mer. It has heen shown hv Furfine and

HIV-RT Possesses a 3’ to 5’ Endonuclease Activity 24301

Reardon (1991) that the major polymerase-dependent cleav- age site made by HIV-RT is between the 15th and 16th ribonucleotide from the 3’ terminus of the DNA primer. (Polymerase-dependent cleavages advance upon primer ex- tension and remain a fixed distance from the extended primer terminus). They propose that this distance may reflect the spacial arrangement of the polymerase and RNase H active sites, These authors also indicate that the position of clea- vages can be influenced by sequence preferences. The lengths of the predominant 3’-derived cleavage products in our assays were somewhat shorter than those expected from cleavages between the 15th and 16th ribonucleotides back from the 3‘ terminus of the DNA oligomer. This may be due to the presence of preferred cleavage sequences more distal from the DNA 3‘ terminus. The change in cleavage preference observed with the 30-nucleotide DNA oligomer compared with the 20- mer may reflect the presence of a new preferred cleavage site greater than 20 nucleotides from the 3’ terminus of the DNA oligomer.

Under the conditions of the challenged assay, RNase H- mediated cleavage by HIV-RT was highly salt-sensitive such that the amount of cleavage of substrate at a fixed time decreased with increasing salt concentration (Figs. 2 and 3). The decrease in cleavage correlated with a decrease in the stability of enzyme-substrate complexes with higher salt as was indicated by the substantially lower half-life of the com- plexes relative to the half-life with 5 mM KC1 (Fig. 8). The decrease did not necessarily result from a decrease in the catalytic capacity of the enzyme at the higher salt concentra- tion. This interpretation is consistent with the observation that nearly all the substrate was cleaved when the enzyme concentration was substantially increased even when the salt concentration was 80 mM (Fig. 8).

We note that the decay of preformed enzyme-substrate complexes was measured in the absence of MgClZ, whereas this cation was present during the RNase H assay. Ideally, divalent cation should have been included when measuring complex decay. However, since in its presence, the RT can cleave and potentially destabilize the hybrid substrate, it was not possible to measure the dissociation of enzyme from intact substrate under these conditions.

Using poly(rA) .poly(dT) as substrate, Gerard (1981) found that Moloney murine leukemia virus RT was capable of both 3’ to 5’ and 5’ to 3’ RNase H-mediated cleavage of the substrate. A similar conclusion was drawn by others for AMV- RT (Grandgenett and Green, 1974; Leis et al., 1973). We observed no 5‘ to 3’ nuclease activity in our assays. However, the substrates employed in our experiments were different from those used in the above studies in that the RNA portion of our substrates was not homopolymeric and the RNA-DNA hybrid region of our substrates was relatively small. Also, the substrates employed in our experiments were designed such that the 3’-terminal deoxynucleotide of the DNA oligomer hybridized 45 nucleotides from the 5‘ end of the RNA portion of the substrate. This created an RT polymerization initiation site on these substrates. RTs are known to have a high affinity for 3’ termini of primers recessed on long templates (Matson and Bambara, 1981). It is possible that the construction of our substrates permitted only 3‘ to 5’ endonuclease activity. For example, the RT may cleave the substrate at a position distal from the DNA 3‘ terminus, and subsequent cleavages may be influenced by the affinity of the polymerase active site for the 3’ terminus. If the enzyme proceeded toward the 3‘ terminus of the DNA oligomer after the initial cleavage

event, subsequent cleavages would decrease the length of the initial 5’-derived cleavage product, as was observed in our assays. The small size of the hybrid region (20 or 30 base pairs) of the substrates used in our experiments may constrain the RT such that it is always in close proximity to the 3’ terminus of the DNA oligomer, a condition that may strongly influence its directionality. Further experimentation will be necessary to test this hypothesis.

Acknowledgment-We wish to thank Drs. John McCoy and Jasbir Seehra representing the Genetics Institute for the generous gift of HIV-RT, without which this work would not have been possible.

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