An Active Recombinant p15 RNase H Domain Is Functionally

THE JOURNAL OF BIOIOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 34, Issue of August 26, pp. 21741-21747, 1994 Printed in U S A .

An Active Recombinant p15 RNase H Domain Is Functionally Distinct from the RNase H Domain Associated with Human Immunodeficiency Virus Type 1 Reverse Transcriptase*

(Received for publication, January 5, 1994, and in revised form, June 2, 1994)

David B. Evans, Naisheng Fan, Steven M. SwaneyS, W. Gary TarpleyS, and Satish K. Sharmas From Biochemistry and $Cancer and Infectious Diseases Research, Upjohn Laboratories, Kalamazoo, Michigan 49001

An active p16 RNase H domain, consisting of amino acids 427460 of human immunodeficiency v i rus type 1 (HIV-1) reverse transcriptase (RT) and a genetically engineered penta-histidine N-terminal affinity tag, was expressed in Escherichia coli and purified to apparent homogeneity by immobilized metal affinity chromatog raphy. The purified pl6 RNase H domain exhibited no substrate preference for [SHlpoly(rG)*poly(dC) compared to [sH]poly(rA).poly(dT), in contrast with the HIV-1 RT-associated RNase H, which showed a 30-fold preference for the former substrate. Unlike the HIV-1 RT-associated RNase H, when challenged with unlabeled substrate, the recombinant pl6 RNase H domain was relatively nonprocessive in RNA degradative activity of the [sH]poly(rA).poly(dT) duplex. Kinetic studies using pl6 RNase H showed substrate inhibition with an apparent K, value of 0.12 p~ for the [SHlpoly(rA). poly(dT) hybrid. Substrate inhibition was not observed for the HIV-1 RT-associated RNase H. The results show that the isolated pl6 HIV-1 RNase H domain is functionally distinct from the recombinant HIV-1 RT-associated RNase H.

Human immunodeficiency virus type 1 (HIV-1)' reverse transcriptase (RT) exhibits RNA- and DNA-dependent DNA polymerase and RNase H activities essential for the replication ofviral genomic RNA (1, 2). The role of RNase H activity in viral replication is evidenced by mutagenesis studies that demonstrate that a mutant provirus defective in RNase H function cannot produce infective virus particles (3). The HIV-1 RT-associated RNase H activity, a target for development of antiviral drugs, is confined to the 15-kDa C-terminal portion of the p66 subunit (4-6). This portion of reverse transcriptase from HIV-1 and other retroviruses shows a significant degree of structural simi- larities to the Escherichia coli RNase H (7, 8). However, there are reported biochemical differences between the HIV-1 RT-associated RNase H and bacterial RNase H (9,lO). Recently, it has been shown that, despite some minor differences, the structure of the inactive RNase H domain (8) conforms well to the structure of the p15 RNase H domain associated with heterodimeric HIV-1 RT (11,121. However, active p15 HIV-1 RNase H domains of HIV-1 RT have also been documented (13, 14). To obtain in-

* This work was supported in part by NCDDG-Human Immunodefi- ciency Virus Grant UO1 AI25696-7. 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.

Biochemistry, 7240-267-117, Kalamazoo, MI 49001. Tel.: 616-384-9413; 8 To whom correspondence should be addressed: The Upjohn Co.,

' The abbreviations used are: HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase, IMAC, immobilized metal affinity chromatography; MES, 2-(N-morpholino)ethanesulfonic acid.

Fax: 616-385-5488.

formation on the detailed catalytic mechanism of the active p15 HIV-1 RNase H domain and its interaction with specific RNase H inhibitors, it is necessary to understand its biochemical prop- erties in relation to the RNase H domain associated with the heterodimeric HW-1 RT engineered with a polyhistidine tag (15). In this work, we sought to re-engineer the p15 RNase H domain (14) to introduce strong binding, metal-chelating sites utilized to obtain pure heterodimeric HIV-1 RT from crude E. coli extracts in a single step by IMAC (15). Here we present data that show functional differences between the isolated p15 HIV-1 RNase H domain and the C-terminal RNase H domain associated with heterodimeric HIV-1 RT.

MATERIALS AND METHODS

Chemicals General laboratory chemicals were purchased from Sigma. Poly(dT),

(rA)ls18 oligonucleotide, pM-223-3 plasmid vector, and NAP-5 columns were obtained from Pharmacia Biotech Inc. GF/C papers were purchased from Whatman. Tritiated poly(rA) was obtained from Amersham Corp., and PPIATP and 13HlGTP were purchased from DuPont NEN. The scintillation fluid Ultima Gold was from Packard Instruments. The p66/p51 heterodimeric HIV-1 RT was prepared as described earlier (15). The E. coli RNase H preparation was obtained from Boehringer Mann- heim. DNA sequencing was done using the Version 2 Sequenase kit from United States Biochemical Corp.

Construction ofpl6 HN-1 RNase H Domain The DNA sequence coding for the HIV-1 RT-associated RNase H

domain was cloned in the pKK-223-3 plasmid vector, with an affinity tag fused on the N-terminal region to facilitate its purification by IMAC. The original tag (14), which had the amino acid sequence MPIHDHDH- PFHGY, was changed to MPIHHHHHPFHGY by site-directed mutagenesis using polymerase chain reaction-based amplification. Two oligonucleotides were chemically synthesized and used as primers for polymerase chain reaction. The first oligonucleotide (5"AAACAGMT- TCATGCCCATTCACGATCACCATCACCC-3') containing the desired changes (underlined) represents the nucleotide sequence around the His polylinker of the DE10.3 plasmid (14) containing the EcoRI site of DE10.3. The second primer (5'-CAC""I"I'ATCTGGTTGTGC-3') spans the unique NsiI site of DE10.3 and is complementary to the positive strand. Plasmid DE10.3 was linearized by digestion with PuuII, and 20 cycles of polymerase chain reaction were performed using the above two primers. The amplified DNA (-300 base pairs long) was digested with EcoRI and NsiI and inserted into the EcoRI and NsiI sites of DE10.3. The resulting plasmid (AB 1) was used to transform E. coli (strain JM109), and the clone AB 1.6 was found to express the p15 RNase H domain of HIV-1 RT at high levels. DNA sequencing of the 5'-region of AB 1.6 confirmed that it had the desired changes.

Expression and Purification ofp l6 HN-1 RNase H Domain Expression and disruption of the cells were done exactly as described

earlier (14). The enzyme was purified by IMAC on a Ni'+-iminodiacetic acid-Sepharose column as described by Evans et al. (14) with the following modifications. The crude extract was loaded on a Ni'+-iminodiacetic acid-Sepharose column pre-equilibrated with 20 mM Tris-HC1, pH 8.0 (buffer A), and washed sequentially with 10 column volumes of buffer A containing 1 M NaCl, buffer A alone, and buffer A containing 60 mM imidazole. The protein was eluted with a stepwise imidazole gradi-

21741

21742 Functional Differences

ent (100 and 200 mM) in buffer A. Fractions were analyzed for purity on 15% SDS-polyacrylamide gels (16). Fractions containing the RNase H domain were pooled, concentrated, and dialyzed against 50 mM Tris- HCl, pH 8.0, containing 100 mM NaCl.

RNase H Substrates The RNase H substrates used in this work were prepared as follows. fHIPoly(rA).PolyfdTj-To 50 pCi of [3Hlpoly(rA) in 2.5 ml of water

were added 2.65 ml of (dT),,, containing 0.5 A,,, unitlml. The mixture was incubated at 70 "C for 5 min, cooled on ice, and stored in 200-pl aliquots at -20 "C until use. PHIPoly(rGj.PolyfdCj-This substrate was prepared by polymeriz-

ing C3H1GTP to a poly(dC) template as follows. 0.232 mg/ml poly(dC), 0.582 mM unlabeled GTP, and 200 pCi (20 nmol) of t3H]GTP were incubated with 550 units of DNase-free RNA polymerase (Pharmacia Bio- tech Inc.) in a total of 860 p1 for 1 h at 37 "C in buffer containing 25 mM Tris, pH 7.6, 50 mM KCl, 2.5 mM MgCI,, 2.5 mM dithiothreitol, and 2.5% glycerol. The incubation was terminated by the addition of 140 pl of 10 mM Tris, 1 m~ EDTA. Fractions (0.5 ml) of the polymerized material were passed over a NAP-5 column and eluted with six 500-pl aliquots of 10 mM Tris, 1 mM EDTA. Samples (10 pl) of each eluted fraction were counted, and peak fractions were pooled, aliquoted into 200-pl fractions, and stored at -20 "C until use. The stock substrate was calculated to be 132 p~ with respect to GTP. 5'-P3P1PolyfrA).Poly(dT)-The (rAIl5 oligonucleotide (250 pmol) was

end-labeled with 100 pCi of [33P]ATP by incubation a t 37 "C for 30 min. This was followed by the addition of unlabeled ATP to a final concentration of 4 p ~ , and the incubation was continued for another 30 min. The sample was then placed at 65 "C for 10 min to inactivate the kinase. Following inactivation, 15.25 pg of poly(dT), 50 mM NaCl, and water were added in a final volume of 1 ml, which corresponded to a 250 nM solution with regard to oligonucleotide content. The mixture was incubated at 65 "C for 10 min, allowed to cool at room temperature for 10 min, and stored in 100-pl aliquots a t -20 "C until use.

HN-1 gag-based PHIRNAIDNA Hybrid-Plasmid pSMP-15 is a Bluescript KS' (Stratagene) derivative that carries the HIV-1 gag gene. This vector was prepared by cloning the 1.4-kilobase pair SacIiBglII DNA fragment from the HIV-1 variant HXB2 (GeneBankm accession number K03455, nucleotides 679-2095) into the SacIIBamHI site of pBluescript KS'. In this vector, the HIV-1 DNA is under the transcrip- tional control of the T7 RNA polymerase promoter. Purified plasmid DNA was used for in vitro transcription, which was accomplished using a commercially available transcription kit (Stratagene) containing r3H]ATP and L3H]UTP (DuPont NEN). The DNA template was digested with 1 unit of RNase-free DNase (Promega) per pg of DNA for 20 min a t 37 "C. The RNAtranscripts, predicted to be 1463 bases, were purified by standard protocols and monitored at 260 nM.

'Ib prepare a complementary HIV-1 gag DNA strand, pSMP-15 was used for polymerase chain reaction with two primers: 5"biotin- CTCTCTCGACGCAGGACTCG-3' and 5'-TTCCACATI'TCCAACAGC- CC-3' (Research Genetics), After 35 cycles, the biotin-labeled DNA was separated from the complementary strand with streptavidin-Dyna- beads (Dynal, Inc.). The non-biotinylated DNA strand was recovered and analyzed at 260 nM. The radiolabeled HIV-1 RNA and nonradioac- tive complementary DNA strands were hybridized using equimolar concentrations in 0.15 M NaCl, 0.01 M Na,HPO,, pH 7.4, 1 mM EDTA, and 1 x Denhardt's solution (United States Biochemical Corp.). The r3H]RNA/DNA hybrid was heated a t 85 "C for 5 min, followed by incubation a t 20 "C for 1.5 h in a thermocycler. The annealed HIV-1 gag- based [3HIRNAiDNA substrate was diluted to 50,000 d p d l O pl and stored at -20 "C.

RNase H Activity Assays RNase H activity assays using various substrates were performed as

follows. fH]Poly(rA).Poly(dTj-Routine RNase H activity assays were done

in buffer containing 25 mM Tris, pH 8.5, 5 mM MgC1, or 8 mM MnCI,, 1.5% glycerol, 50 pg/ml bovine serum albumin, 0.01% Nonidet P-40, and 6.5 p~ [3Hlpoly(rA).poly(dT) (15). The substrate concentration is based on the total content of AMP residues. Assays were performed in a total volume of 50 pl containing 30 pl of buffer, 10 p1 of substrate, and 10 1.11 of enzyme. Reactions were terminated by the addition of 150 pl of cold 10% trichloroacetic acid and 10 p1 of 0.5 mg/ml salmon sperm DNA (17). Samples were kept on ice for 10 min, followed by centrifugation for 10 min at 12,000 rpm. Each supernatant (75 pl) was added in duplicate to a 4-ml Omni vial (Wheaton) containing 3 ml of Ultima Gold scintillation fluid and counted in a Packard 1900 TR liquid scintillation counter. One unit of RNase H activity is defined as 1 nmol of I3H]adenylate produced

in RNase H Domains in 1 h at 37 "C. For comparison of the relative specific activities of the p15 RNase H domain and the heterodimeric RT-associated RNase H, activity was determined under identical experimental conditions. The Bradford protein assay (39) was used to determine protein concentration with bovine serum albumin as a standard. 5'-~3P]Poly(rA)~Poly(dTJ-RNase H assays using 33P-labeled sub-

strate were carried out in the RNase H buffer described above for the [3Hlpoly(rA).poly(dT) substrate. The substrate (9 nM) was incubated with a 500 nM concentration of the p15 RNase H domain in a total volume of 75 pl. Two 15-pl samples were spotted on GF/C papers at t = 0 and dropped into ice-cold 5% trichloroacetic acid. Samples were then incubated a t 37 "C for 4 min. Following incubations, another duplicate set of 15-1.11 aliquots were spotted on papers and placed into the trichloroacetic acid. Samples were then washed, dried, and counted as described (15). RNase H activity was measured in terms of loss of trichloroacetic acid-precipitable counts as a function of time. PHIPoly(rGj.Poly(dCj-RNase H assays using the [3Hlpoly-

(rG).poly(dC) substrate were done following the same procedure out- lined above for the [3H1poly(rA).poly(dT) substrate. In this case, 26.4 p~ 13HlGTP substrate and 1.2 p~ RNase H enzyme were used.

HN-1 gag-based PHIRNAIDNA Hybrid-The RNase H activity determinations were carried out as described above for the [3Hlpoly(rA).poly(dT) substrate. The substrate concentration was 0.25 p~ based on the total nucleotide content. The RNase H activity of heterodimeric HN-1 RT or p15 RNase H was determined both at pH 8.5 (5 mM Mg", 0.17 pmol of enzyme) and pH 6.0 (8 mM Mn"', 3.4 pmol of enzyme) in the presence of 25 mM Tris and 20 mM MES buffers, respectively. Samples incubated at 37 "C were withdrawn at an interval of 2 min to determine RNase H activity.

Effect of Challenger Substrate on RNA Hydrolysis To assess whether p15 RNase H degrades RNA in a relatively pro-

cessive or nonprocessive manner, the RNase H activity was monitored following the addition of excess unlabeled substrate after the onset of the reaction (4). The conditions of the assay are described below.

Purified p15 HIV-1 RNase H (1.25 p ~ ) or heterodimeric HIV-1 RT (60 nM) was incubated at 37 "C with 0.19 pg of [3Hlpoly(rA).poly(dT) in a total volume of 90 pl containing the assay buffer described above. One min after the onset of the reaction, 10 pl of unlabeled poly(rA).poly(dT) (0.2 mg/ml) were added to give a 10-fold excess over labeled substrate. Aliquots (30 pl) were removed at 3,6, and 15 min and placed into a tube containing 160 pl of cold 10% trichloroacetic acid and 10 p1 of 0.5 mdml salmon sperm DNA. All samples were processed and counted for RNase H activity as described above for [3H]poly(rA).poly(dT).

Enzyme Kinetics The effect of substrate concentration on the enzymatic activity of the

p15 HIV-1 RNase H domain, E. coli RNase H activity, and HIV-1 RT- associated RNase H activity was determined using a modified procedure of the one described above for poly(rA).poly(dT). Samples (300 pl) were incubated at 37 "C at various substrate concentrations (6.5, 5.4, 4.3, 3.2, 2.1, and 1.6 w [3Hlpoly(rA).poly(dT)). A 50-pl aliquot was

cold 10% trichloroacetic acid and 10 pl of 0.5 mg/ml salmon sperm DNA, removed at various times points, added to a tube containing 150 pl of

and processed as described above for [3Hlpoly(rA).poly(dT). Linear release of 3H as a function of time was used to ensure that the experiments were performed at saturation conditions for all the substrate concentrations. Enzyme activity was calculated as nmol of E3H1AMP released per mVh at the various substrate concentrations. For kinetic evaluation, various substrate concentrations, in the 6.5 to 1.6 w [3H]poly(rA).poly(dT) range, were incubated with 2 p~ p15 RNase H, 16 nM E. coli RNase H, or 100 nM p66/p51 heterodimeric RT-associated RNase H for 10 min at 37 "C. The velocity (nmol of L3H1AMP released per mVh) of the reaction was determined as a function of both time and substrate concentration.

Enzyme Kinetics with Substrate Znhibition The simplest enzymatic reaction with substrate inhibition is repre-

sented by Equation 1,

E . S K, = - E = E S X - K,

ES S

(Eq. 1)

(Eq. 2)

Functional Differences in RNase H Domains 21743

w x 103

loo - 71 - 43- 28-

18 - 15 -

I -

MW 1 2 3 4 5 6 7 8 9 MW 10 11 12 13 14 15 16 17 10 Fro. 1. SDS-polyacrylamide gel electrophoresis (151) of recombinant p15 HIV-1 RNase H domain purified from crude E. coli

extract by IMAC. Lane MIV, molecular weight markers; lane 1, crude E. coli lysate; lane 2, flow-through fraction from Ni"4minodiacetic acid-Sepharose column; lanes 3-5. buffer A with 1 x! NaCI, buffer A alone, and buffer A with 30 mal imidazole, respectively; lanes 6-9, 60 mxr imidazole fractions; lanes lO-ZFi, 100 mxr imidazole fractions; lanes 16-18, 200 mxr imidazole fractions.

and

(Eq. 3 )

Under conditions where E,, << S,, and for the initial phase of the reaction, ;.e. S = S,,, the rate of the reaction, u,,, is given by Equation 4.

(Eq. 4)

Substituting the values ofE and ES, into the equation E,, = E + ES + ES? and solving for ES, we obtain Equation 5.

Thus, the initial rate of the reaction is given by Equation 6.

(Eq. 5 )

(Eq. 6)

Equation 6 was used with a nonlinear least-squares algorithm to ana- lyze the experimental u, , wrsus S data and to calculate the best fit parameter K,.

RESULTS AND DISCUSSION

IMAC-purified p15 HN-1 RNase H Dom.ain-Fig. 1 shows a typical example of the IMAC-purified material on SDS-polyacrylamide gel electrophoresis. As expected, a single band around 15 kDa was observed. The penta-histidine-containing p15 RNase H was strongly retarded on immobilized Ni" and was eluted at imidazole concentrations above 100 mh.1. Although a significant portion of the bound p15 RNase H also eluted in the 60 mM imidazole (Fig. 1, lanes 6-9) and 100 mnf imidazole (lanes 10-15) fractions, the 200 mu imidazole fraction (lanes 16-18) was preferred for subsequent characterization. This was done to ensure that the purity of the p15 RNase H preparation was comparable to that of heterodimeric HIV-1 RT (15).

RNase H Activity and Substrate Preference-The RNase H activity of the purified protein was initially determined using the ['Hlpoly(rA).poly(dT) substrate under conditions found to be optimal for the HIV-1 RT-associated RNase H (18, 19). Table I shows the specific activity of the purified p15 RNase H domain against a number of synthetic RNA/DNA substrates. The p66/p51 heterodimeric HIV-1 RT-associated RNase H was in- cluded for comparison purposes. In agreement with the results of other investigators (181, the HIV-1 RT-associated RNase H showed a marked preference for poly(rG).poly(dC) with a specific activity at least 30-fold higher than those of the other two substrates (Table I). In contrast, p15 RNase H showed no substrate preference, and its specific activity did not vary more than 2-fold between the other two substrates (Table I). Stated

TAI~I.: I

Aeterodimeric HIV-I RT and the ,111.5 HIV-I RNase H domain Substrate preferencr of RNase H activity associated with

Specific RNase H activity Substrate

HIV-I RT p l 5 RNasr H ~~

rrnils/nlg"

I:'HIPoly(rA).poly(dT) 718 -c 71 410 f 10 IRHIPoly(rG).poly(dC) 19,720 5 2930 659 t 18 5'-1"'P1Poly(rA)~poly(dT) 190 t 15 357 -c 59

" One unit is defined as 1 pmol o f trichloroacetic acid-soluble radiolabeled adenylate released in 1 h a t 37 "C. The data represent means t S.D. ( 1 1 = 3).

differently, the relative specific activity of the p15 RNase H domain is at least 30-fold lower than that of the heterodimeric H N - 1 RT-associated RNase H using the poly(rG).poly(dC) substrate.

Divalent Cation Requirement-The RNase H activity of the purified p15 RNase H was initially determined with I"H Ipoly(rA).poly(dT) or a circular RNA/DNA hybrid substrate under buffer conditions previously found (14) to be optimal for the RNase H associated with heterodimeric HIV-1 RT (50 mhl Tris-HCI, 5 mM MgCI,, pH 8.5). With these substrates, subsequent studies showed Mn'+-dependent RNase H activity of the p15 RNase H domain at pH 6.8 (data not shown). Here we characterize the effect of divalent metal ions on the relative RNase H activity of p15 RNase H and heterodimeric HIV-1 RT by using a HIV-1 gag-based heteropolymeric RNA/DNA substrate.

Fig. 2 shows Mn"-dependent RNase H activity at pH 6.0 of the heterodimeric HIV-1 RT-associated RNase H and p15 RNase H by using this heteropolymeric RNA/DNA substrate. The relative RNase H activity of p15 RNase H was -66% that of the RNase H associated with heterodimeric HIV-1 RT. As shown, the RNase H of heterodimeric HIV-1 RT was also active at pH 8.5 in the presence of Mg'"', while the 15 RNase H domain at the same enzyme concentration showed no detectable activity. However, p15 RNase H was active under these conditions, but required 10-fold more enzyme (data not shown), suggesting very high affinity of the heterodimeric HIV-1 RT-associated RNase H for this HIV-1 gag-based substrate in the presence of M e . These results show that p15 RNase H differs from the heterodimeric HIV-1 RT-associated RNase H with regard to divalent metal ion requirements.

Effect of Added Unlabeled Substrate on RNA Hydrol.ysis- Hansen et al. (4) suggested that the p15 RNase H fragment isolated from virus particles exhibited a random degradation of RNA from the RNA/DNA substrate. Based on this assay, where RNase H activity is measured following the addition of excess unlabeled substrate 1 min after the onset of the reaction, we observed that our recombinant preparation of the p15 HIV-1

21744 Functional Differences in RNase H Domains RNase H also cleaved RNA from poly(rA).poly(dT) in a similar nonprocessive manner (Fig. 3). As shown, the HIV-1 RT-associated RNase H was much more processive under similar experimental conditions. Recent elucidation of the three-dimen- sional structure of p66/p51 HIV-1 RT has revealed that most of the template-primer-binding site is provided by the polymerase domain (p51) of the p66 subunit and portions of the p51 subunit of the p66/p51 heterodimer (11, 12). This is supported by our results and by earlier studies (20) that showed that the addition of the polymerase domain (p51 subunit) stimulated the RNase H activity of the p15 RNase H domain. We conclude that p15 RNase H, without the supportive role of the p51 polymerase domain, cannot remain bound to the substrate to carry out cleavage in a processive manner.

Substrate Inhibition of RNase H Activity by Poly(rA). Poly(dT)-Our previous kinetic studies of the HIV-1 RT-asso-

l'O 1

0 1 I I I , I 0 2 4 0 8 10

TIME (mid

RNase H domain and RNase H activity of heterodimeric HlV-1 FIG. 2. Effect of divalent metal ions on RNase H activity of p15

RT using radiolabeled HlV-1 gag-based RNMDNA substrate. B, heterodimeric HIV-1 RT (5 rn M P and pH 8.5); 0, p15 RNase H (5 rn M e and pH 8.5); 0, heterodimeric HIV-1 RT (8 rn Mn2+ and pH 6.0); 0, p15 RNase H (8 mM Mn2+ and pH 6.0). All the data points were plotted after subtracting the background counts from controls run without the enzymes.

strate on RNA degradative activity of FIG. 3. Effect of challenger sub-

pl6 HlV-1 RNase H domain versus pl6 RNase H associated with heterodimeric HlV-1 RT. After 1 min at 37 "C, 10 pl of water or 2 pg of the challenger poly(rA).poly(dT) in 10 pl of water were added. At the times indicated, 30 pl of the reaction were quenched and as- sayed for RNase H activity expressed as cpm released from [3Hlpoly(rA).poly(dT)

which the enzymes were added after the and substracted from the controls, in

trap (see "Materials and Methods"). H, heterodimer without challenger; 0, heterodimer in presence of challenger; 0, p15 RNase H domain without challenger; 0, p15 RNase H with challenger.

11000-

10000-

9000-

8000-

U g 7000-

2 eooo- a

Q

0

v) 6000-

0" 4000-

3000 -

'E

2000-

ciated RNase H, carried out with the [3Hlpoly(rA).poly(dT) substrate, have exhibited linear Lineweaver-Burk plots and follow the Michaelis-Menten equation for a unisubstrate condition (15). However, as shown in Fig. 4, in the presence of p15 RNase H, the rate of reaction decreased with increasing [3H]poly- (rA).poly(dT) concentrations under reaction conditions of pH 8.5 and 5 nm Mg2' (15). In contrast, under the same assay conditions, the RNase H associated with heterodimeric HIV-1 RT (Fig. 4, inset) showed no apparent inhibition of RNase H activity with increasing substrate concentrations, consistent with our earlier results on the heterodimeric HIV-1 RT-associated RNase H (15). Likewise, substrate inhibition was not de- tected with a commercial preparation of E. coli RNase H (data not shown).

To ensure that substrate inhibition was not an artifact of assay conditions, identical experiments were performed under conditions different from 5 nm Mg2' and pH 8.5 (Fig. 4). This was based on our other studies with p15 RNase H, where we found that our preparation of p15 RNase H showed a pH opti- mum between 6 and 8 in the presence of 8 nm Mn2+. Notably, in the presence of 8 mM Mn2+, the RNase H activity dropped to zero at pH 8.5 and above. Therefore, we investigated the effect of [3H]poly(rA).poly(dT) concentration on the RNase H activity of p15 RNase H under another set of conditions for optimal activity (8 mM Mn2+ and pH 7.0). As shown in Fig. 5, inhibition of RNase H activity at high substrate concentration was again observed, confirming that the substrate inhibition also occurs when both pH and metal ion are altered.

The data shown in Fig. 5 were analyzed in terms of an equation derived for substrate inhibition (see "Materials and Meth- ods"). As shown in Fig. 6, the agreement between the theoretical curve and experimental data points validates the use of this equation. The downward deflection in the curve at high substrate concentration is typical of substrate inhibition, indi- cating that poly(rA).poly(dT) inhibits the RNase H activity of the p15 RNase H domain with an apparent Ki of 0.12 f 0.01 p ~ .

To determine specificity of substrate inhibition, we examined the effect of increasing concentrations of the two other substrates shown in Table I: [3Hlpoly(rG).poly(dC) (319-mer) and 5'-[33P]poly(rA).poly(dT). The 5'-33P-labeled substrate was in- cluded to see if the observed inhibition would still occur when

Time (mid

l0O0 1 Functional Differences in

,000 r

RNase H Domains 21745

TIME (rnin) FIG. 4. Time course of RNA hydrolysis from [8Hlpoly(rA).poly(dT), determined as release of radioactive rA, at various substrate

concentrations. The assays were performed at pH 8.5 in the presence of 5 m~ M e . m, 1.6 p ~ ; 0, 3.2 p; 0 , 4 . 3 p ~ ; 0, 6.5 p ~ . Inset, similar plot generated under identical conditions using the heterodimeric HIV-1 RT-associated RNase H.

7000

6000

6000

0 0) rn 2 4000 0)

rn

- a *E 3000

0 a

2000

1

A, 2.2 PM; 0, 3.3 p ~ ; 0, 4.3 p ~ ; 0, 5.4 p; m, 6.5 PM. For other details, refer to “Materials and Methods.” FIG. 5. Time course of RNAhydrolysis from [3H]poly(rA).poly(dT), determined as release of radioactive rA, at pH 7.0 and 8 m~ Mn2+.

21746

2.75 7 2.5 -

E 2.25- E 2.0- h 5 1.75-

1.5-

1.25-

- -

.- ; 1.0-

8 0.75 - 0.5-

-

0

Functional Differences in RNase H Domains

0.25 0.0 i 0 1 2 3 4 5 6 7 0

[Substrate], pM

FIG. 6. Effect of [sHlpoly(rA)~poly(dT) concentration on RNase H activity of pl6 RNase H. The theoretical curve was calculated based on an equation derived for substrate inhibition, and experimental data points were taken from Fig. 5. The kinetic equation is given under “Materials and Methods.”

loss of the 5”labeled products is quantitated, irrespective of their size and mechanism of release from the RNA/DNA hybrid. The inhibition phenomenon was also observed in the presence of these substrates (data not shown), suggesting that the observed substrate inhibition is not only confined to the [3H]poly(rA).poly(dT) substrate. However, from studies with these three substrates, it cannot be said that this phenomenon would be characteristic for all other substrates. These results (Figs. 4 and 5 ) , in addition to the above findings (Figs. 2 and 3 and Table I), have led us to conclude that there are functional differences between the recombinant p15 RNase H domain and that of the heterodimeric HIV-1 RT-associated RNase H under defined conditions.

Smith and Roth (13) have reported the purification and characterization of an active HIV-1 RNase H domain. Their p15 RNase H construct contains the 427-560 residues from HIV-1 RT, but is significantly different from our penta-histidine construct with regard to the N-terminal extension. The construct published by Smith and Roth (13) is represented by the sequence MGSS(H),SSGLVF’RGSHM, containing a thrombin cleavage site. Another notable difference is related to the soluble p15 RNase H domain (Ref. 14 and this study) versus complete insolubility of the expressed p15 RNase H polypep- tide, which requires in vitro manipulations to acquire enzymatic activity (13). Likewise, Cirino et al. (21) have obtained a hexa-histidine-containing p15 HIV-1 RNase H that required denaturatiodrefolding from inclusion bodies. These differences (the N-terminal extension, native versus denatured, and in vitro enzymatic cleavage) could explain the contradictory results concerning catalytic activity and metal ion requirements among various preparations of the recombinant p15 RNase H domain (8, 13, 14, 20, 21).

It is unknown why our previous (14) and present constructs are expressed in a soluble native form compared with similar constructs reported by others (13, 21). However, inconsistent results have also been reported in regard to the activity of the p51 domain of HIV-1 RT (22-26). It has been shown recently that temperature during growth and induction of bacterial cul- ture are critical for the preparation of the enzymatically active p51 domain of HIV-1 RT (27). Thus, in addition to the above- mentioned factors, expression conditions may play an impor- tant role in determining ratios of active uersus inactive p15 proteins.

The exact binding sites of the various enzymes for the RNA/ DNA substrate are largely unknown. There is general agree-

ment that as little as 4-5 base pairs of the duplex might be in contact with the RNase H domain. Therefore, there may exist a series of subsites across the enzyme surface for interaction with a substrate. As noted previously (28, 29), in E. coli RNase H, basic amino acids are clustered in two short sequences from positions 27 to 33 and 85 to 89. Clustering of these positively charged residues, which may be involved in the substrate-binding site, is absent in the p15 RNase H domain. This and other structural differences (30) between the two enzymes may ac- count, at least partially, for higher substrate affinity of the E. coli enzyme and hence provide it with an overall catalytic ad- vantage over the retroviral p15 RNase H. Likewise, the interaction of p15 RNase H with the p51 polymerase domain in heterodimeric HIV-1 RT may be responsible for providing higher substrate afiinity toward [3H]poly(rG).poly(dC) compared with the isolated p15 RNase H domain (Table I). This is consistent with known functional interdependence of the polymerase and RNase H domains as linker insertion mutations, point mutations, or chemical modifications in the polymerase domain can affect the RNase H activity, while such modifications in the RNase H domain can disrupt polymerase activity

Reverse transcriptase activity is crucial for the life cycle of retroviruses; it is therefore a favorite target for development of antiviral drugs. Recently, however, there is some trend toward targeting the RNase H activity of HIV-1 RT (8, 13, 14, 20, 21, 35). In fact, recent reports show inhibition of the retroviral RNase H in U&O by agents such as heparin (36), illimaquinone (9), vanadyl complexes (lo), and HP 0.35, a cephalosporin degradation product (37).

Our findings demonstrate for the first time that the recombinant p15 HIV-1 RNase H domain is kinetically distinct from the HIV-1 RT-associated RNase H under defined conditions. Recently, Smith and Roth (13) have reported that the isolated RNase H domain is more susceptible to RNase H inhibitors than the HIV-1 RT-associated RNase H. This is consistent with a recent paper that describes conformational differences, based on characterization with antibodies, between the RNase H domains of p66 and p15 (38). These studies, together with our kinetic studies with p15 HIV-1 RNase H, point to the need for caution in the application of this p15 RNase H domain in the evaluation of RNase H inhibitors targeted for the RNase H domain-associated HIV-1 RT-associated RNase H. However, inhibition of the RNase H activity of the p15 RNase H domain, in theory, should still serve as a useful indicator of potential HIV-1 RNase H inhibitors acting on this domain of heterodimeric

(31-34).

HIV-1 RT.

Acknowledgments-We thank Dr. F. J. Kezdy for help with data anal- ysis. We also thank Dr. R. C. Thomas for helpful discussions and Dr. A. Basu for technical contributions.

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An Active Recombinant p15 RNase H Domain Is Functionally