fluorimetric analysis of recombinant p15 hiv- 1 ribonuclease h*

THE JOURNAL OF B~OLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 20, Issue of July 15, pp. 14743-14749, 1993 Printed in U. S. A.

Fluorimetric Analysis of Recombinant p15 HIV- 1 Ribonuclease H* (Received for publication, January 27, 1993)

Nick M. Cirinot, Robert C. Kalayjiane, Joyce E. Jentoftt, and Stuart F. J. Le Griceen From the $Department of Biochemistry and the $Division of Znfectious Diseases, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

We have exploited the sole tryptophan residue (Trp6SB) in the ribonuclease H (RNase H) domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) to study features of the isolated polypeptide (pl5 RNase H) by fluorescence spectroscopy. Incubation of purified p l 5 RNase H with a synthetic RNA/DNA hybrid was accompanied by an alteration in TrpbS6 fluorescence intensity. This property was used to determine an apparent binding constant (Kapp) of 3.5 x lo6 M” for p15 RNase H complexed with poly(rA)/oligo(dT)12-le and an occluded site size of 4 nucleotides. A cooperativity coefficient (w) of 910 was also determined which indicated that nearly three logs of the Kapp were due to cooperativity effects. Re- combinant p15 RNase H preparations containing mutations at position 478 ( G ~ u ~ ~ ’ + G ~ I I ~ ~ ’ ) or 539 (HisBSe + Phe6”), which are highly conserved between bacterial and retroviral RNases H, were also analyzed. Un- der the same conditions, these mutants failed to bind the RNA/DNA hybrid, although they were structurally similar to the wild type polypeptide. Fluorescence spectroscopy thus appears to be an alternative and sensitive means of analyzing functional properties of the purified RNase H domain of HIV-1 RT under a variety of conditions.

Conversion of the single-stranded retroviral RNA genome into double-stranded DNA, uninterrupted by ribonucleotides, requires a contribution from the C-terminal, ribonuclease H (RNase H)’ domain of reverse transcriptase (RT) (1-3). Mu- tagenesis studies with HIV-1 RT (4-6) have indicated that RNase H function is indispensable for viral infectivity, suggesting this as a novel target for the development of antiviral drugs to combat the continuing spread of AIDS. Preliminary investigations have been encouraging, demonstrating that agents such as heparin (7), illimaquinone (8), and H P 0.35, a cephalosporin breakdown product (9), selectively impair RNase H function in uitro. Future antiviral strategies would be facilitated by more detailed knowledge of the mechanism of RNase H action, and possibly a comparison between retroviral and bacterial RNase H, for which considerable mutagenesis (10-12) and x-ray crystallographic data (13-15) are now available.

* This work was funded by National Institutes of Health Grants GM 46623 (to S . F. J. Le G.) and AI 07381 (to R. C. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

dressed. 7l TO whom correspondence and reprint requests should be ad-

’ The abbreviations used are: RNase H, ribonuclease H; HIV, human immunodeficiency virus; NTA, nitrilotriacetic acid; RT, reverse transcriptase; bp, base pair(s).

Recently, Davies et al. (16) reported the crystal structure of a recombinant polypeptide representing the RNase H domain of HIV-1 RT. While remarkably similar to the RNase H domain deduced from the crystal structure of p66/p51 HIV- 1 RT (17, la), as well as the 16-kDa Escherichia coli enzyme (13-15), both Davies et al. (16) and others (19,ZO) have failed to detect activity in their purified polypeptides. Hostomsky et al. (21) have indicated that, while purified p15 RNase H itself was inactive, recovery of enzymatic function could be achieved by reconstitution with the 51-kDa RT polypeptide. In contrast, Evans et al. (22) claim to have prepared a recombinant HIV-1 RNase H capable of hydrolyzing an RNA/DNA hybrid. In preliminary investigations, we have been able to detect minimal activity in our recombinant RNase H, but could not completely rule out the possibility of trace contamination by E. coli RNase H. In light of this, we have sought to develop an alternative strategy whereby the interaction between recombinant HIV RNase H and its RNA/DNA substrate could be monitored independent of hydrolysis and potential contamination by the bacterial enzyme.

Maturation of p66 HIV-1 and HIV-2 RT by the pol-coded protease (PR) via cleavage between Phe440 and Tyr441 yields a 120-residue polypeptide representing the RNase H domain (23, 24). This domain contains a single tryptophan residue at position 535, which suggested to us that fluorescence spectroscopy might be a useful tool to study the recombinant enzyme and genetically engineered mutants. We have therefore prepared and analyzed recombinant HIV-1 RNase H and derivatives containing point mutations at position 478 ( G ~ u ~ ~ ’ + Gln) or 539 (His539 + Phe). Each of these polypeptides was designed to incorporate residues 436-560, thereby re- creating @-strand 1, elucidated from crystallographic studies of Davies et at. (16). This structure would be either absent or disordered in the RNase H domain released from homodimer p66 RT during maturation into heterodimer (25) and may contribute to RNase H function. Addition of a small polyhis- tidine extension at the N terminus permitted purification of the polypeptides by metal chelate affinity chromatography (26,27). Following renaturation, both spectropolarimetry and spectrofluorimetry indicated each RNase H domain was folded similarly. The interaction of both wild type and mutant polypeptides with a synthetic homopolymer RNA/DNA hybrid was thereafter investigated by following the intrinsic fluorescence of Trp535.

EXPERIMENTAL PROCEDURES

Cloning and Expression of RNase H Polypeptides-The DNA seg- ment encoding a 125-residue HIV-1 RNase H domain, ( G l ~ ‘ ~ ~ - L e u ~ ) was amplified from plasmids p6H RT (wt RNase H), p6HRTE - ( G ~ u “ ~ --* Gln mutant), and p6HRTH - (Hiss3’ + Phe mutant) by the polymerase chain reaction (28) and inserted into pDS56, RBSII, 6 X His, creating plasmids pGHRNaseH, p6HRNaseHE + and p6HRNaseHH -. ’, respectively. Based on the crystal structure of the HIV-1 polypeptide (16), we extended the N terminus to residue 436 to restore &strand 1, otherwise incomplete in RNase H released from

14743

14744 HIV-1 RNase H / S ~ ~ s t r a ~ e Interactions

p66 RT by the pol-coded protease (23,24). Expression of recombinant RNase H was placed under control of lac regulatory elements. Recom- binant plasmids were selected in the E. coli strain M15::pDMI.l (29) in the presence of 100 pg/ml ampicillin and 25 pg/ml kanamycin,

For induction of gene expression, recombinant bacteria were grown in antibiotic-supplemented L-broth until midlogarithmic phase, after which isopropyl P-D-thiogalactopyranoside was added to a final concentration of 200 pg/ml. Cultures were incubated a further 3-4 h, after which cells were harvested and stored at -20 "C until required for purification.

RNase H Purification-Both wild type and mutant RNase H were purified by metal chelate affinity chromatography (26, 27) under denaturing conditions. Gel1 pellets were resuspended in 5 ml of 0.1 M Na2HP04/NaH2P04 buffer, pH 8.0, 6 M guanidinium hydrochloride per g wet weight and lysed by overnight incubation at room temperature. The lysate was clarified by centrifugation at 15,000 rpm for 30 min, and the supernatant was loaded directly onto a 2-ml column of Niz+-nitrilotriacetic acid-Sepharose (NTA-Sepharose), equilibrated in the same buffer, at a flow rate of 3 ml/h. After washing to remove unbound material, weakly adsorbed proteins were eluted with a buffer of 0.2 M NH,OAc buffer, pH 6.0, 6 M guanidinium hydrochloride. Finally, RNase H was eluted with a buffer of 0.2 M NH,OAc buffer, pH 4.0,6 M guanidinium hydrochloride. Aliquots of selected fractions were treated with cold 10% trichloroacetic acid, after which precipi- tated material was collected by centrifugation, washed with ice-cold 100% ethanol, dried, and resuspended in S D S / p o l y a c ~ l ~ i d e gel sample buffer. Following SDS/polyacrylamide gel electrophoresis (15% gels containing a 3.3% stacking gel), fractions containing pure RNase H were pooled and dialyzed into a final buffer of 20 mM Tris/ HCI, pH 7,2 mM MgC12, and 1.5 M guanidinium hydrochloride. Under these conditions, concentrated solutions of RNase H (>4.0 mg/ml) could be stably maintained and later diluted as required.

Circular Dichroism Memurements-Spectropolarimetric analysis of purified pl5RNase H, ~ l 5 R N a s e H ~ ' ~ , and p l 5 R N a ~ e H ~ ' ~ was performed with a Jasco-600 spectropolarimeter over the wavelength range 200-260 nm. Each sample was dialyzed to a final concentration of 0.325 mg/ml in a buffer of 60 mM Hepes, pH 6.0, 20 mM M&h, and 25 mM guanidinium hydrochloride. Spectra were obtained with a 1-mm pathlength cell and compiled from 5~ oversampling, with background subtraction of buffer.

Fluorescence Measurements-Fluorescence spectroscopy was performed with a Perkin-Elmer LS-5B luminescence spectrometer equipped with a xenon lamp, variable slits, and microprocessor- controlled photomultiplier gain. A 0.5 X 0.5-em quartz cuvette with a sample size of -250 pl was used for all experiments, using excitation and emission wavelengths of 280 and 340 nm, respectively. Excitation and emission slit widths of 3 and 10 nm, respectively, were used for all experiments. A jacketed cuvette holder was used to maintain the temperature of samples at 27 "C. The concentration of RNase H ranged from 0.5 to 2 p ~ . Fluorescence was stable with time under d l conditions. Under most conditions, the inner filter effect was negli- gible since theAzsa was generally less than 0.05. For those experiments where the Anso was greater than 0.05 due to high concentrations of RNase H in solution, a corrected fluorescence, F,, was determined using the following equation: F, = Fob X antilog (Amf2) (30). T W O types of titrations were performed to examine the binding parameters of the RNase H to a poly(rA)/oligo(dT) hybrid substrate. TO determine the number of nucleotides occluded upon RNase H binding, poly(rA) saturated with oligo(dT)12-18 (Pharmacia LKB Biotechnol- ogy Inc.) was titrated into a fixed concentration of RNase H under tight binding conditions, Le. 20 mM TrislHCl, pH 7, 2 mM MgClp, 0.2 M guanidinium hydrochloride. In order to determine the binding affinity and the degree of cooperativity, RNase H was titrated into a fixed concentration of poly(rA)/oligo(dT) under equilibrium binding conditions, i.e. 7 mM Tris/HCl, pH 7, 2 mM MgC12, 0.01% Tween 20, 1.5% glycerol, 0.5 M guanidinium hydrochloride, and the results were assessed as described by McGhee and von Hippel (31). By this method, any lattice effect of RNase H on the homopolymer substrate was included in the calculation of binding parameters.

Materiak-Poly(rA), fully saturated with oligo(dT)12-18, was pur- chased from Pharmacia. According to manufacturer's specifications, a concentration of 50 pg/ml = 1 AZm, and an average molecular weight of 334.7 per residue was used to determine the concentration of substrate.

RESULTS

Purification and Characterization of Recombinant HIV-1 RNase H-In Fig. 1, we have aligned the amino acid sequence of our recombinant HIV-1 RNase H with that of the E. coli enzyme, indicating residues most conserved between the bacterial and retroviral enzymes. Previous reports had suggested that the RNase H domain of HIV-1 RT results as a consequence of protease-mediated cleavage between PheP4' and T y P ' (23, 24). However, the x-ray structure presented by Davies e t al. (16) indicates that this would contain an incomplete copy of @ strand of the 66-kDa RT polypeptide (residues 437-447). Consequently, the RNase H domains studied in the present communication were extended at their N termini to include these residues in the hope of promoting more stable folding. The same figure indicates point mutations previously introduced into the RNase H domain of p66 RT and shown to influence specifically RNase H function of the parental heterodimer (4,32).

Expression of this extended HIV-1 pol~ept ide in E. coli gave rise to a protein of molecular mass slightly larger than 15 kDa, due to the addition of a small N-terminal polyhisti- dine extension (Hiss) to facilitate purification (Fig. 2a). An equivalent approach was taken by Evans et al. (22) in their studies with HIV-1 RNase H; however, in contrast to their studies, we observed that our recombinant polypeptide was almost completely insoluble. Accordingly, p15 RNase H was purified to homogeneity by metal chelate chromatography under denaturing conditions in the presence of 6 M guanidinium hydrochloride (Fig. 2a), followed by controlled dialysis to allow correct refolding. Analysis of the CD spectrum of the purified protein (Fig. 2b) indicated that it contained 18% LY helix, 45% p strand, 13% @ turn, and 24% random coil, using the program SSEAX provided by the manufacturer. These values are in reasonable agreement with previous CD studies by Becerra et al. (20) who have determined 15-20% a helix and -60% 0 strand for their purified RNase H. Later NMR studies by the same group with recombinant RNase have indicated that the solution structure is consistent with that found in the crystal structure (33, 34).

An interesting feature of the RNase H domain of HIV-1 RT is the presence of a single tryptophan residue, Trp535, situated at the extremity of @ strand 5 (Fig. 1). From tertiary structural data of E. coli and HIV-1 RNase H, it appeared that this residue might be surface-exposed, allowing us to consider altered ~uorescence emission at 340 nm as a means of characterizing the environment near the active site. This indeed turned out to be the case, as illustrated in Fig. 2c. An emission maximum at 340 nm for the RNase H domain was observed, consistent with a polar environment for Trp535 (331, Based on this observation, we attempted to determined whether the fluorescence spectrum derived from Trp535 could be modulated in the presence of a synthetic RNAPNA hybrid.

Interaction of Wild Type HIV-1 RNase H with a Synthetic RNAIDNA Hybrid-Although CD analysis suggested that our recombinant RNase H was most likely correctly folded, several enzymatic analyses indicated that it possessed little or no catalytic activity (data not shown). Despite this, we pro- posed that the polypeptide might still be capable of interacting with its RNA/DNA substrate. Consequently, a fluorescence study was initiated using the synthetic hybrid polyfrA)/ oligo(dT) as a representative substrate, the results of which are indicated in Fig. 3a. This analysis indicated that fluorescence of Trp5" was s i ~ i f i c ~ t l y quenched upon addition of the synthetic RNA/DNA hybrid suggesting that the environment in the vicinity of this residue had been altered. Reduc-

HIV-1 RNase HISubstrate Interactions 14745 - P1 - f" P 2 + "3-

440 450 460 47 0

P $ t--.-"----aE-

HIV p A H K G I I;G:./G : ~ : ~ ~ ~ . ~ . : , ~ : ~ v : : . : ~ : : : : ~ . ~ ~ . ~ L . : ~ : . ~ ~ : ~ $ ~ A G I R K I L

E .c . K G H A G ' ~ ~ , ~ ~ : ~ : ~ ~ : ~ N : ~ ~ : ~ ~ ~ ~ ~ ~ = ' : ~ : ' ~ , . ~ . ~ : ~ : ~ . ~ . A .:,R:i:?{,TA,! 'A :;H[ N p T L E D T G y Q V E V . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

5 4 0 550 560

FIG. 1. Amino acid sequence alignment of E. coli RNase H and the l25-residue, C-terminal domain of p66 HIV-1 RT used in the present study. Although several reports suggest Phe440/Tyr441 as the HIV-1 protease cleavage site delineating p51 and p66 RT, the amino terminus of our recombinant RNase H was extended to Gly436 to complete the sequence of @-strand 1 deduced from the crystal structure (16). Secondary structure elements of both polypeptides are enclosed in shaded boxes. Residues conserved between bacterial and retroviral RNases H are indicated by arrows. Double arrows above the aligned sequences at positions 478 (E478 c, Q) and 539 (H639 o F) indicate RNase H mutants used in the present study .

tion in Trp635 fluorescence intensity in the presence of substrate was subsequently used to determine the occluded site size for RNase H on poly(rA)/oligo(dT). This analysis is presented in Fig. 36, where an occluded site size of 4 nucleotides was determined for the recombinant polypeptide. The same figure indicates the results of a parallel experiment where recombinant RNase H was thermally denatured at 95 "C for 15 min prior to analysis. Although a similar level of Trp636 fluorescence could still be detected with the denatured protein, there was no quenching observed upon poly(rA)/ oligo(dT) addition, indicating loss of interaction with the substrate. The occluded site size of 4 nucleotides for the HIV- 1 RNase H binding site is in good agreement with x-ray crystallography of p66/p51 RT in the presence of duplex DNA, suggesting that 4-5 bp would contact the RNase H domain (IS).' Although the presence of trace amounts of contaminating E. coli RNase H (whose specific activity is substantially greater than that reported for the retroviral RNase H domain) might have contributed to an enzymatic analysis, equivalent levels of contamination would not affect determination of the occluded site size.

Binding data of RNase H with the synthetic hybrid substrate was determined under conditions indicated in the text and transformed as described by McGhee and von Hippel (31)

* E. A. Arnold, personal communication. ~ ~~

and Schwarz and Watanabe (35). Using these combined meth- ods, an apparent binding constant (Kapp) was determined which included a cooperativity parameter ( w ) multiplied by an intrinsic binding constant ( K ) . The line of Fig. 4a originating at zero represents the intrinsic fluorescence of RNase H in the absence of substrate. The line intersecting the abscissa at an RNase H concentration of -0.25 ~ L M represents the intrinsic RNase H fluorescence increase after 100% of the available substrate has been bound. These two lines are parallel, indicating a linear increase in fluorescence with added RNase H, independent of hybrid binding. These data could then be used to determine the binding density (u ) of RNase H according to Schwartz and Watanabe (35). The calculated binding density was subsequently used to generate a modified Scatchard plot (Fig. 46, closed circles). When these data from Fig. 46 are incorporated into Equation 15 of McGhee and von Hippel (31), a Kepp of 3.5 X lo6 M" was determined. Surpris- ingly, there was a very large cooperativity contribution (w = 910) which would indicate that the intrinsic binding constant was only 3.9 X lo3 M-'. These parameters were then reincor- porated into the McGhee and von Hippel equation (31) in order to determine a theoretical Scatchard curve (Fig. 46, solid line). As evidenced from the combined data of Fig. 4b, our observed binding fits very well with the theoretical curve.

Interaction of HIV-1 RNase Mutants with Their RNAIDNA

14746

(a)

kDa

200 - 98 - 66 - 45 - 28 -

18 - 14 -

HIV-1 RNase HISubstrate Interactions

"D

- RNaseH

-20' ' ' ' ' ' ' ' 195 215 235 255

Wavelength (nm)

40

k k 4 Buffer

0 300 320 340 360 380 400

Wavelength (nm) FIG. 2. a, analysis of purified p15 HIV-1 RNase H by SDS-poly-

acrylamide gel electrophoresis. +ZPW represents bacterial lysates that were analyzed prior to and 3 h postinduction, respectively. NTA- Seph, RNase H purified via Ni'+-NTA-Sepharose under denaturing conditions in the presence of 6 M guanidinium hydrochloride. M, protein M, markers. Material migrating at M, = 30,000 (D) was positive in an immunological assay with antibodies directed against p66 RT (data not shown) and may represent a small proportion of aggregated RNase H. b, spectropolarimetric analysis of renatured, wild type p15 HIV-1 RNase H. Purified protein (0.325 mg/ml) was analyzed in a buffer of 60 mM Hepes, pH 6.0, 20 mM MgC12, 25 mM guanidinium hydrochloride. The spectrum was obtained after buffer subtraction and oversampling to reduce background noise. c, fluorescence spectrum of purified RNase H. Protein was analyzed in a

.- v c k

90 - 0 RNaseH - (rA)/(dT]

80-

70 - 60-

RNaseH 0

40-

30 300 320 340 360 3

Wavelength (nm)

6

(rA)/(dT) : RNaseH ratio (uM)

HIV-1 p15 RNase H with the synthetic RNA/DNA homopolymer FIG. 3. a, quenching of Trp535 fluorescence following incubation of

poly(rA)/oligo(dT). 1 p~ purified RNase H was assayed in either the absence or presence of 1 p~ polynucleotide. Buffer conditions were similar to those described in the legend to Fig. 2c. b, determination of subsite size for p15 RNase H on poly(rA)/oligo(dT) by fluorescence spectroscopy. A solution of 1 p~ RNase H was titrated with increasing concentrations of RNA/DNA hybrid (closed circles); the intersection between the linearly increasing portion and the plateau occurs at saturation. As indicated in the figure, HIV-1 RNase H occupies 4 nucleotides of substrate (indicated by arrow). As control, RNase H was thermally denatured at 95 "C for 15 min prior to addition of substrate (open triangles). Buffer conditions were identical with those in the legend to Fig. 2c. Data presented are the average of four independent analyses.

Substrate-In recent publications, we indicated that point mutations introduced into the RNase H domain of p66 HIV- 1 RT (Glu4" + Gln and His639 + Phe) resulted in specific inhibition of RNase H activity (12), and in one case (Glu4" + Gln), loss of viral infectivity (4). To better understand the

buffer of 20 mM Tris/HCl, pH 7.0, 2 mM MgC12, 0.2 M guanidinium hydrochloride. Excitation wavelength was 280 nm, and slit widths of 3 and 10 were used for excitation and emission, respectively. The final concentration of RNase H was 1 pM.

HIV-1 RNase HISubstrate Interactions 14747

(b)

i >

0.00 0.20 0.40 0.60

pM RNaseH

0.75

0.50

0.25

0.00 - 0.00 0.10 0.20 0.30

v FIG. 4. a, equilibrium binding of p15 HIV-1 RNase H to poly(rA)/

oligo(dT). Equilibrium binding was analyzed by titrating a 1 FM solution of homopolymer with increasing concentrations of recombinant RNase H in 7 mM Tris/HCl, pH 6.0,2 mM MgC12, 1.5% glycerol, 0.01% Tween 20, and 0.5 M guanidinium hydrochloride. Lines representing the intrinsic fluorescence intensity prior to binding and following saturation have been indicated. The fractional change in fluorescence between these two lines was used to calculate the proportion of added RNase H bound to substrate, according to the method of Schwarz and Watanabe (35). b, modified Scatchard plot for bindingof p15 RNase H topoly(rA)/oligo(dT). The line represents the calculated curve obtained from an iterative fit of Kspp and w to the McGhee and von Hippel equation 15 (34) for Kint = 3.9 X lo3 M-', w , the cooperativity parameter, = 910, and n = 4. Closed circles represent the experimental values derived from a. u, nanomoles of RNase H bound/total nmol of poly(rA)/oligo(dT). L, concentration of free RNase H (PM). The intercept on the abscissa is fixed as l/n.

consequences of these alterations, the respective mutant RNase H domains were purified in an analogous manner (Fig. 5 a ) and assayed for binding to the synthetic RNA/DNA hybrid. The results of these studies are presented in Fig. 5 , b and c. In a preliminary analysis, spectropolarimetry indicated that the overall structure of both RNase H mutants had not been compromised (Fig. 5b) . Similarly, fluorescence emission from Trp636 was not affected by these mutations (data not shown). However, Fig. 5c further indicates that fluorescence quenching was not observed with either mutant in the presence of poly(rA)/oligo(dT). Such an observation suggests that, while structurally similar to the wild type polypeptide, the affinity of both mutants for their substrate was impaired.

200 - 98 - 66 - 45 - 28 -

18 - 14 - - RNaseH

+10 I h

v E o i + i - - - 7 "

Wavelength (nm)

( a

WT

25-

20-

15-

10-

5-

0 -

c 4 9 c 4 9 c 4 9

(rA)/(dT) : RNaseH ratio (PM)

FIG. 5. a, analysis of purified HIV-1 RNase H mutants ~ 1 5 ~ 4F and ~ 1 5 ~ -. by SDS/polyacrylamide gel electrophoresis. M, protein M, markers. b, CD spectra of purified RNase H mutants ~ 1 5 ' ~ ~ and p15" -F, compared to that of the wild type polypeptide. Conditions for spectropolarimetry were similar to those described in the legend to Fig. 2b. c, comparison of fluorescence quenching from wild type RNase H ( WT) and mutants ~ 1 5 ~ ' ~ (EQ) and ~ 1 5 ~ ' ~ (HF) in the presence of poly(rA)/oligo(dT). Histograms represent the reduction in fluorescence emission from Trp535 at nucleic acidprotein ratios of 4 (hatched bars) and 9 (stippled bars). C, change in fluorescence intensity in the absence of substrate. Data presented are the average of three experiments. Conditions for fluorimetric analyses were similar to those described in the legend to Fig. 36.

14748 HIV-1 RNase HISubstrate Interactions

These observations were particularly important for the RNase H mutant containing a substitution at which is in the immediate vicinity of Trp635 exploited in the present fluorescence studies.

Mutagenesis data with E. coli RNase H have indicated that an enzyme containing a substitution at i.e. the equivalent of His639 in the HIV-1 enzyme, leads to a 4-fold reduction in binding affinity, coupled with a 20-fold loss of catalytic function (10). His"' of E. coli RNase H has been shown to constitute part of a well-defined protruding loop which has been implicated in substrate binding (14). By analogy, it is therefore not unreasonable to assume that alteration of the equivalent residue, conserved among RNase H of retroviral enzymes, might have similar consequences. Furthermore, while it is clear that G I U ~ ~ ~ , and its equivalent in the E. coli enzyme ( G I U ~ ~ ) are absolutely required for catalysis, a role for this residue in substrate binding has not been ruled out; from the data presented here, this appears to be the case. While providing further information on the consequences of the point mutations, the data of Fig. 5c are a clear indication that E. coli RNase H contamination does not account for the binding data obtained with the wild type RNase H polypeptide. In summary, the combined data of Fig. 5 illustrate that fluorescence spectroscopy is a sensitive and useful tool for examining interactions between a purified domain of HIV reverse transcriptase and its substrate.

DISCUSSION

It remains unclear whether the isolated C-terminal domain of HIV RT retains sufficient RNase H activity relative to the parent enzyme (p66/p51) to justify its role in the retroviral replication cycle. X-ray crystallography data from Davies et al. (16) strongly suggest that the position at which HIV protease cleaves the 66-kDa RT polypeptide to yield a heterodimer (Phe'" c, Tyr") would result in an incompletely- folded 15 kDa RNase H domain. This suggestion has been substantiated by a series of communications indicating that recombinant polypeptides originating at this cleavage site are devoid of activity (19-21). However, Evans et al. (22) suggest that it is possible to prepare a recombinant RNase H domain whose catalytic function is preserved. Since the possibility of trace contamination with highly active E. coli RNase H could not be completely ruled out in these latter studies, we sought to develop an alternative strategy through which the RNase H domain of HIV-1 RT might be studied without interference from contaminating bacterial enzymes. Furthermore, loss of RNase H activity does not necessarily imply lack of interaction with the RNA/DNA substrate. Using this hypothesis, our intention was to determine if binding to an RNA/DNA duplex might be measured independently of its hydrolysis. In agreement with previous work from Becerra et al. (20), a spectropolarimetric analysis indicated that the secondary structure of our recombinant polypeptide was preserved. By taking advantage of a single tryptophan residue (Trp535) within the purified RNase H domain, we have been able to conduct a fluorimetric analysis of substrate binding. Loss of binding activity of recombinant RNase H following thermal denaturation further established that this was a function of the folded domain. Thus, although our preparation of wild type p15 RNase H did not exhibit catalytic activity, the 125- amino acid domain excised from p66 RT appears to have retained sufficient structural integrity for binding of its RNA/ DNA substrate.

Data presented here with wild-type p15 RNase H indicates an occluded site size of 4 nucleotides, usingpoly(rA)/oligo(dT) as a representative RNA/DNA duplex. The value is consistent

with related studies on this domain of p66/p51 RT, which have employed either chemical footprinting or x-ray crystallography. Hydroxyl radical footprinting of polymerization complexes containing p66/p51 RT and duplex DNA suggests that 18 bp (from positions +3 to -15) are protected from chemical modification. Hyperaccessibility to hydroxyl radi- cals was observed between base positions -12 and -15 in a complex containing wild type RT, while absent from one containing the RNase H-deficient enzyme ~ 6 6 ~ + Q/p51. Al- though duplex DNA was used in the experiments, these observations make a strong argument for 4 bp of nucleic acid in contact with the RNase H domain of heterodimer RT.3 In addition, examination of x-ray crystallography data from Ar- nold et al. (18), who have prepared a co-crystal of p66/p51 HIV-1 RT and duplex DNA, indicates that as little as 5 bp of the duplex might be in contact with the RNase H domain.' Finally, in a study with the E. coli enzyme, Wang et al. (14) have attempted to dock a decameric RNA/DNA duplex into the active site and suggested that the interaction spans -4 nucleotides on each strand (positions 6-9 of the DNA strand and 7-10 of the RNA strand).

Our fluorimetric approqch has also revealed important information regarding the consequences of inactivating mutations introduced into the RNase H domain of p66/p51 RT. Alteration of either or His539 in the 66-kDa RT polypeptide previously had the consequence of eliminating RNase H activity while minimally affecting polymerization (32). In this communication, we indicate that equivalent mutations in the isolated RNase H domain, although having little effect on the conformation of the polypeptide, have the consequence of severely influencing the interaction with its RNA/DNA substrate. While it would be desirable to apply fluorescence spectroscopy to study substrate binding to heterodimeric RT containing these RNase H mutations, the overabundance of tryptophan residues in both subunits makes this impractical.

The assay presented here may also be useful in studying the effect of inhibitors which have been suggested to specifically impair RNase H function of HIV-1 RT. Loya et al. (8) reported that illimaquinone, a secondary metabolite from the Red Sea sponge Smensopongia sp. , is an effective inhibitor at concentrations which do not severely impair polymerase function of either the retroviral enzyme or the mammalian DNA polymerase a. Similarly, Hafkemeyer et al. (9) presented evidence suggesting that HP 0.35, a degradation product prepared from the cephalosporin ceftazidim, displays prefer- ential activity against HIV-1 RNase H. Using the assay we have described here, it should be possible to determine whether these drugs impair binding of an RNA/DNA hybrid to the RNase H domain, or whether they allow binding but alter the conformation of the polypeptide. The equivalent approach might also be redesigned as a screening strategy for future drugs directed against this RT function. Finally, reten- tion of substrate binding but loss of enzymatic activity in our purified preparation of wild type HIV-1 RNase H suggests that additional domains of the 66-kDa polypeptide are required to achieve both functions. Contact between the RNase H domain and other portions of p66/p51 has been implied by both neutron diffraction (36) and x-ray crystallography (17). In addition, mutagenesis studies indicate a close relationship between the polymerase and RNase H domains (37-39). An interesting possibility might be inclusion of the portion of p66 RT between residues 398 and 414, which contains a periodic array of tryptophan residues. Currently, we are preparing recombinant RNase H polypeptides extended to include res-

Metzger, W., Hermann, T., Schatz, O., Le Grice, S. F. J., and -

Heumann, H. (1993) Proc. Natl. Acad. Sci. U. S. A., in press.

HIV-1 RNase HISubstrate Interactions 14749

idues 398-560. The data presented here with the 125-amino acid polypeptide will be useful in determining whether an interaction with the substrate is modulated with an N-ter- minally extended RNase H.

Acknowledgment-We are grateful to E. A. Arnold, CABM, Rutgers University, for providing crystallography data on HIV-1 RT prior to publication.

REFERENCES 1. Moelling, K., Bolognesi, D. P., Bauer, H., Busen, W., Plassmann, P., and

2. Keller, W., and Crouch, R. (1972) Proc. Natl. Acad. Sci. U. S. A. 69,3360- Hausen, P. (1971) Nature New Biol. 234,240-245

2 2 f i A

3. Leis, J., Berkower, I., and Hurwitz, J. (1973) Proc. Natl. Acad. Sci. U. S. A.

4. Schatz. 0.. Cromme. F.. Naas. T.. Lindemann. D.. Mous. J.. and Le Grice.

vyy .

70,466-470

S. F: J.'(1990) i n Okogenesis'and AIDS (Papas, T.,' ed) pp. 304-315; Portfolio Publishing, TX

5. Mizhrahi, V., Usdin, M. T., Harington, A,, and Dudding, L. R. (1990) Nucleic Acids Res. 18.5359-5363

6. Tisdale, M.. Schulze. T:, Larder, B. A,. and Moellina, K. (1991) J. Gen. Virol: 72,59-66

. . I

7. Moelling, K., Schulze, T., and Diringer, H. (1989) J. Virol. 63,5489-5491 8. Lova. S.. Tal. R.. Kashman. Y.. and Hizi. A. (19901 Antirnicrob. Agents

f%rnother.'34; 2009-2112 '

9. Hafiemeyer, P., Neftel, K., Hobi, R., Pfaltz, A,, Lutz, H., Luthi, K., Focher, F., Spadari, S., and Hubscher, U. (1991) Nucleic Acids Res. 1 9 , 4059- 4065

I , _ "

10. Kanaya, S., Kohara, A,, Miura, Y., Sekiguchl, A,, Iwai, S., Inoue, H., Ohtsuka, E., and Ikehara, M. (1990) J. Biol. Chem. 265,4615-4621

11. Kanaya, S., Kimura, S., Katsuda, C., and Ikehara, M. (1990) Biochern. J. 271,59-66

12. Kanaya, S., Katayanagi, K., Morikawa, K., Inoue, H., Ohtsuka, E., and Ikehara, M. (1991) Eur. J . Biochern. 198,437-440

13. Katayanagi, K., Mlyagawa, M., Matsushima, N., Ishikawa, M., Kanaya, S.,

309 Ikehara, M., Matsuzaki, T., and Morikawa, K. (1990) Nature 3 4 7 , 306-

14. Wang, W. Y., Hendrickson, W. A., Crouch, R. J., and Satow, Y. (1990)

15. Katayanagi, K., Miyagawa, M., Matsushima, M., Ishikawa, M., Kanaya, S., Science 2 4 9 , 1398-1405

Nakamura, H., Ikehara, M., Matsuzaki, T., and Morikawa, K. (1992) J. Mol. Biol. 223,1029-1052

16. Davies, J. F., Hostomska, Z., Hostomsky, Z., Jordan, S. R., and Matthews, D. A. (1991) Science 262,88-95

17. Kohlstaedt, L. A,, Wang, J., Friedman, M., Rice, P. A,, and Steitz, T. A. (1992) Science 256,1783-1790

18. Arnold, E. A., Jacobo-Molina, A., Nanni, R. G., Williams, R. L., Lu, X., Ding, J., Clark, A. D., Jr., Zhang, A., Ferris, A. L., Clark, P., Hizi, A.,

19. Stammers, D. K., Tisdale, M., Court, S., Parmar, V., Bradley, C., and Ross, and Hughes, S. H. (1992) Nature 357,85-88

20. Becerra, S. P., Clore, G. M., Gronenborn, A. M., Kasrlstrom, A. R., Stahl, C. K.. (1991) FEBS Lett. 283,298-302

21. Hostomsky, Z. Hostomska, Z., E d s o n , G. O., Moomaw, E. W., and Nodes, S. J., Wilson, S. H., and Win leld, P. T. (1990) FEBS Lett. 270,76-80

22. Evans, D. B., Brawn, K., Diebel, M. R., Jr., Tarpley, W. G., and Sharma,

23. Le Grice, S. F. J., Ette, R., Mills, J., and Mous, J. (1989) J. Biol. Chern.

24. Mizrahi, V., Lazarus, G. M., Miles, L. M., Meyers, C. A,, and Debouck, C.

25. Hostomska, Z., Matthews, %. A., Davies, J. F., 11, Nodes, B. R., and

26. Hochuli, E., Dobeli, H., and Schacher E. (1987) J. Chromatogr. 411,177-

27. Hochuli, E., Bannwarth, W., Dobeli, H., Gentz, R., and Stuber, D. (1988)

B. R. (1991jPrm. Natl. A C ~ . scl. u. s. A. 88,1148-1152

S. K. (1991) J. Bid. Chem. 266,20583-20585

2 6 4 , 14902-14908

(1989) Arch. Biochern. Bio hys 273,347-358

Hostomsky, 2. (1991) J . Biol. Chem. 266,14697-14702

184

28. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, H., Horn, G. BiolTechnology 6 , 1321-1325

T.. Mullis. K. B.. and Erlich. H. A. (1988) Science 239.487-491

30. Lakowitz, J. A. (1983) Principles of Fluorescence Spectroscopy, pp. 44-45,

31. McGhee, G., and von Hippel, P. H. (1974) J . Mol. Biol. 86,469-489 32. Schatz, 0.. Cromme, F., Griininaer-Leitch, F., and Le Grice, S. F. J. (1989)

Plenum Publishing Corp., New York

FERS I h t . 257. 311-314 ~

33. P o w ~ ~ s ~ R., Clore, G. M., Bax, A,, Garrett, D., Stahl, S. J., Wingfield, P.

34. Powers, R.. Clore, G. M.. Stahl. S. J.. Winafield. P. T.. and Gronenborn.

" ., ." ".

T., and Gronenborn, A. M. (1991) J. Mol. Biol. 221 , 1081-1090

A. (1992) Biochemistry 31,9150-9157 ~ '

J., an'd Heumann: H.'(1992) EMBOJ. 11, 1131-il39

3108

35. Schwarz, G. and Watanabe, F. (1983) J. Mol. Biol. 163,467-484 36. Lederer H.,'Schatz 0. May, R., Crespi, H., Darlix J. L., Le Grice, S. F.

37. Prasad, V., and Goff, S. P. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,3104-

38. Hizi, A., Barber, A,, and Hughes, S. H. (1989) Virology 170,326-329 39. Hizi, A,, Tal, R., and Hughes, S. H. (1991) Virology 1 8 0 , 339-346

fluorimetric analysis of recombinant p15 hiv- 1 ribonuclease h*

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