Crosslinking of substrates occurs exclusively to the p66 subunit of heterodimeric HIV-1 reverse transcriptase

Vol. 174, No. 2, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

January 31, 1991 Pages 785-789

Crosslinkin R

of Substrates Occurs Exclusively to the p66 Subunit of eterodimeric HIV-1 Reverse Transcriptase

Nancy Cheng*, George R. Painterr, and Phillip A. Furman*

Divisions of Virology* and Organic Chemistryt Burroughs Wellcome Co., 3030 Cornwallis Rd.,

Research Triangle Park, NC 27709

Received December 13. 1990

Summary: Photoaffinity labeling of the hetero- and homodimeric forms of HIV-1 reverse transcriptase has been carried out using [32P]rAr2-r8.dTrr) as a representative template-primer and [a-32PIdTTP as a representative 2’-deoxynucleoside- S-triphosphate. UV irradiation produces stable, covalent crosslinks between each of the reactants and both the hetero- (~661~51) and homodimeric (p66/p66, p51/p5l) forms of the enz

J me. In the case of the p66/p51 heterodimer, the form of the

enzyme believe to be involved in viral replication, crosslinking occurs exclusively to the p66 subunit. These results suggest that the polymerase activity of the heterodimer resides on ~66. g ~1 Academic preSS, Inc.

The reverse transcriptase (RT, E.C.2.7.7.49) encoded by Human

lmmunodeficiency Virus Type 1 (HIV-l) is essential for the replication of the virus.

Like the RT of other retroviruses, HIV-1 RT catalyzes the synthesis of a duplex DNA

copy of the viral RNA genome. The functional protein complex purified from virions

consists of two polypeptides of molecular weight 66,000 and 51,000 (1,2). Both

polymerase and RNase H activities reside on the 66-kD subunit. The 51-kD subunit is

generated by C-terminal cleavage of the 66-kD polypeptide at a protease-sensitive

site between the polymerase and RNase H domains and posseses only polymerase

activity (3,4).

A bireactant, biproduct mechanism similar to that described for several other

well characterized DNA polymerases has been proposed for DNA synthesis by HIV-1

RT (5). In this mechanism, binding of the reactants is ordered, with the template-

primer binding first followed by the binding of the 2’-deoxynucleoside-

5’-triphosphate (dNTP) having Watson-Crick complementarity to the next

appropriate template residue. There are two potential binding sites for each of the

reactants on both the heterodimeric (p66/p51) and homodimeric (p66/p66, p51/p5l)

forms of the enzyme (3). However, Hill coefficients for the binding of template-

primers and dNTPs to both the hetero- and homodimers indicate that the number of

binding sites occupied by each of the substrates is one (6,7).


As part of an effort to establish a structure-function relationship for HIV-l RT,

we carried out photolabeling experiments on both the hetero- and homodimeric

forms of the enzyme using [a- 32P]dlTP as a representative dNTP and [32Pl

rA12-1a.dTlo as a representative template-primer. Numerous amino acid residues

including tyrosine, methionine, cysteine, phenylalanine, serine, and histidine have

been shown to form stable, covalent adducts with nucleic acid (8) and nucleotides

(9) when irradiated with UV light. Consequently, site specific photochemical

crosslinking to proteins has been used to identify those segments of the primary

sequence involved in nucleic acid (10) and nucleotide (11) binding. In the case of the

HIV-l RT heterodimer, the form of the enzyme believed to be operative in viral

replication, our results show that substrate adducts are formed only to ~66. This

observation suggests the polymerase activity of the heterodimer resides on ~66.

Experimental Procedures

Materials. dlTP, dCTP, rAr2-18, dTlo, and polynucleotide kinase were purchm Pharmacia and used without further purification. [y-32P]ATP (3000 Ci/mmol), [aJzP]dTTP (600 Ci/mmol), and [methyl-3HldTTP (20.5 Ci/mmol) were obtained from DuPont-New England Nuclear. DE-81 filter paper was purchased from Whatman Co.

Enzyme Purification. HIV-1 RT was expressed in E. co/i using the recombinant olasmid moRT4 (12). Homodimeric ~51 was exoressed in E. co/i usina the olasmid CTRTl (13). The‘purification procedure for thep66/p51 heterodime;and the p51 homodimer is as follows. E. co/i paste (10 g) was resuspended in 30 ml of buffer containing 50 mM Tris, pH 7.5; 0.1 mM PMSF; 1 mM pepstatin; and 0.5 mM aprotinin. This solution was treated with lysozyme for 5 minutes at 220 C, and then wassonicated for three 10s intervals using a model W350 sonicator from Branson Sonic Power Co. After centrifugation at 26,000 x g for 20 minutes the supernatant was precipitated with 35-50% ammonium sulfate. The precipitate was dissolved in a solution of 50 mM Tris, pH 7.5; 150 mM NaCI; and 1 mM DTT and was applied to a Superose-12 column (Pharmicia) equilibrated with the same buffer solution. The fractions containing RT activity were pooled and dialyzed against a solution containing 10 mM TAPS, pH 8.0; 5% glycerol; 1 mM DTT (buffer A). The dialyzed RT solution was then applied to a Mono Q-10 column (Pharmicia) equilibrated with buffer A. RT activity was eluted from the column with a gradient of O-l M NaCl in 150 ml of buffer A at a flow rate of 4 ml/min. The p66/p51 heterodimer eluted with 180 mM NaCl whereas the p66 homodimer eluted with 150 mM NaCI. The fractions containing predominantly p66 homodimer were pooled and the ~66 was further purified by ammonium sulfate trituration. The protein was greater than 99% pure as judged from silver stained SDS gels. The concentration of RT was determined by amino acid analysis.

Reverse Transcriptase Assay. Enzyme assays were performed at 370 in a buffer containing 50 mM HEPES, pH 7.0; 5 mM MgCl2; 1 mM DTT; 100 mg/ml poly(rA)5oo.p(dT)to; and 10 mM [3H]dlTP (1 mCi/mmol). The assays were initiated by the addition of enzyme. At designated times, 10 pL aliquots of the reaction mixture were removed, spotted onto DE-81 paper (2 cmz), and processed as previously described (14).

Affinit Labelin -

. [32P] end-labelled rAl2-la.dTlo was prepared by labeling the rA12-18 stran using y-32P]ATP and polynucleotide kinase. The labeled strand (0.1 A260 units) was purified using a NAPS column (Pharmacia) and was annealed to 0.16A260 unitsof dTlo. A standard 100 ml crosslinking reaction for both [aJzP]dlTP and [32P]rA12-18.dTlo contained 5 pg of heterodimeric or homodimeric RT, 5 mM MgCl2,ZmM MnCl2, 1 mM DTT, and 50 mM HEPES (pH 7.0). The reaction mixture was placed in a 96-well microtiterplate and irradiated for 10 minutes with a 254-nm hand held UV light (Model UVGL-25, UVP, Inc.) at a distance of 8 cm. The reaction was

786


0 0

B

14

0.030.060.23 0.71 1.42 42 126

-66K

c51K

0 0

0

5 10 15

dTTP OJM) rA 12.18 l dT,, WV

Figure 1. The effect of increasing concentrations of (A) [a-3zPldTTP and (B) [32P]rAlz-la.dTlo on the extent of UV-induced crosslinking to the p66/p51 heterodimer. Crosslinking reactions were performed as described in Materials and Methods: Followin

9, SDS-PAGE of an aliquot of the reaction

mixture, bands corresponding tot e enzyme were excised and counted by Cerenkov counting. The number of picomoles of 3zP bound to the enzyme was then plotted versus the total concentration of the reactant. The inserts in panels A and B show autoradiographs of the SDS-PAGE as a function of reactant concentration.

kept on ice during the procedure. Samples ot the reaction mixture were analyzed by SDS-PAGE and autoradiography to determine if crosslinking had occurred. The extent of 32P incorporation wasdetermined by excising the radiolabeled bands from the gel and Cerenkov counting. Crosslinking of both reactants increased with increasing irradiation time up to 10 minutes, after which no further crosslinking was observed. The extent of crosslinking is dependent on the concentration of the reactants, asshown in Figure 1. Exposure of a solution of the enzyme to UV light in the absence of reactants results in a 20% reduction in enzyme activity.

Results and Discussion

The results of crosslinking [32P]dTTP and [32P] end-labelled rA12-1a.d(T)jo to the

reverse transcriptase are shown in Figure 2. Insert A of Figure 2 shows the silver-

stained SDS-PAGE of the homodimeric and heterodimericforms of the enzyme. The

p66 homodimer is shown in lane 1, the p51 homodimer in lane 2, and the p66/p51

heterodimer in lane 3. Inserts B and C of Figure 2 are autoradiographs made from

the SDS-PAGE of the heterodimer and homodimers crosslinked to [32P]dTTP and to

[32P]rA12-18.d(T)lo, respectively. Mg + 2 was required for crosslinking of [32P]dlTP

but was not required for crosslinking of [32P]rA12-lB.dTlo. In the case of the

heterodimer, crosslinking of both substrates occurs exclusively to the p66 subunit.

787


- 66K + 66K

-5lK +51K

---- Comperltor AZTTP cabTP dCTP

0 3 Competitor % Cont. Photolabel (PM) - 100 76 5 20 50 100 50 66 10 63 50

Figure 2

Figure 3.

Crosslinking of [a-3zP]dTTP and [3zP]rAlz.1a.dTlo to the heterodimeric p66/p51 and the homodimeric p51 and p66 forms of HIV-1 RT. (A) Silver- stained SDS-PAGE of the p66 homodimer (lane l), the p51 homodimer (lane 2), and the p66/p51 heterodimer (lane 3). Molecular wei ht markers are shown to the left of panel A. (B) Autoradiograph of [a-32P P dlTP crosslinked to the p66 and the p51 homodimers (lanes 1 and 2, respectively) and the p66/p51 heterodimer (lane 3). (C) Autoradiograph of [3zP]r(A)1z-1a.dTlo crosslinked to the p66 and p51 homodimers (lanes 1 2, respectively) and the ~661~51 heterodimer (lane 3).

Autoradiographs of a polyacrylamide gel showin carb-TP, and dCTP on the extent of crosslinking o 3

the effects of AZTTP, [a-azP]dT-TP to the

p66/p51 heterodimer. All samples contained 20 pM [a-azP]dTTP and were irradiated for 10 minutes in the presence of the indicated concentration of AZTTP, carb-TP or dCTP. The extent of photolabeling is expressed as a percentage of labeling observed in the absence of competitor.

This can be clearly seen in lane 3 of Figures 2B and 2C. The results of crosslinking to

the p66 and p51 homodimers are shown in lanes 1 and 2 of panels B and C. Because

the labeling efficiency was less than 100% (8% for dTTP, 27% for rA12-1a.dTlo), it

was impossible to establish whether or not crosslinking occured to one or both of

the subunits of the homodimers.

A slight decrease in the electrophoretic mobility of the enzyme was detected in

the autoradiographs upon crosslinking to rA12-18.dTlo, which can be attributed to

the increased size of the protein-nucleic acid complexes (Figure 1C). In addition, all

of the rAl2-t8.dTlo-polypeptide complexes formed in thisstudy migrate as doublets.

Reverse phase HPLC analysis indicates that annealing of dTlo to rA12-18 produces two

major nucleic acid species and each crosslinks with the protein to form an adduct

with a different mobility.

To determine whether the crosslinking reactions occurred at a unique site or

were nonspecific, the heterodimer was heated to 60” in the presence of 0.1% SDS,

conditions which destroy all enzyme activity, before being irradiated in the presence

of a substrate. No crosslinking of [a-32PldlTP or [32P]rA12-18. dTlo to enzyme

treated in this fashion was observed. The specificity of dTTP crosslinking was further

tested by including alternative nucleotide or nucleotide analog substrates in the

reaction mixture. Figure 3 shows the effect of dCTP, carbovir-5’-triphosphate

(carb-TP), and zidovudine-5’-triphosphate (AZTTP) on [a-32PldTTP crosslinking. The

results are expressed in terms of percent photolabeling compared to a reaction


carried out without alternative substrate. AZllP was the most effective at blocking

dTTP crosslinking, with 20 PM compound causing a 50% reduction in labeling. dCTP

was less effective, reducing dTTP crosslinking by 40% at a concentration of 50 ‘mM.

Both AZTTP and dCTP have been shown to competitively inhibit the binding of dTTP

to heterodimeric HIV-l RT (67). Although carb-TP appears to be utilized as a

substrate by HIV-1 RT (5. Hopkins, Burroughs Wellcome Co., personnel

communication), it does not block dTTP crosslinking at concentrations up to 250 PM.

In summary, dTTP and rA1z-la.dTlo will crosslink to both the hetero- and

homodimeric forms of HIV-I RT. Crosslinking of the reactants is only observed on the

p66 subunit of the p66/51 heterodimer, suggesting that template-primer and dNTP

binding, and therefore polymerase activity, resides on this portion of the enzyme.

Although the stoichiometry of dTTP and rA12-18.dTle crosslinking to the

homodimeric forms of the enzyme could not be established in this study,

fluorescence binding assays indicated that only one of the two binding sites

available to either substrate is occupied (6,7). The crosslinking of dlTP to the

heterodimer is specific, as indicated both by the requirement that the enzyme be In a

native state, and the observation that substrates known to competitively inhibit

dTTP binding will block crosslinking of [a-QP] dlTP.

References

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Crosslinking of substrates occurs exclusively to the p66 subunit of heterodimeric HIV-1 reverse transcriptase