Purification and kinetic characterization of equine infectious anemia virus reverse transcriptase

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November 14, 1991

Purification and Kinetic Characterization of Equine Infectious Anemia Virus Reverse Transcriptase Deborah A. Thomast* and Phillip A. Furman* tDepartment of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514 *Division of Virology, Burroughs Wellcome Co., 3030 Cornwallis Rd., Research Triangle Park, NC 27709 Received September 23, 1991

Summarv: The reverse transcriptase of Equine Infectious Anemia Virus (EIAV) was partially purified from virus particles and appeared to be a heterodimer with subunit molecular masses of 70 kdal and 59 kdal. The polymerase activity of this enzyme had an absolute requirement for a divalent cation, preferring Mg++ over Mn++. Addition of a monovalent cation to the reaction mixture enhanced, but was not required for enzyme activity. Kinetically, the reverse transcriptase of EIAV is similar to the reverse transcriptase of Human Imunodeficiency Virus Type 1 (HIV-1). Both enzymes have similar Km values for 2'-deoxynucleoside-5'-triphophates on the synthetic template/primers tested, both exhibit substrate inhibition, and both are inhibited to similar extents by most nucleoside-triphosphate analogs. The results of this study suggest that the reverse transcriptase of EIAV may be a good model for studying structure/function relationships of retroviral reverse transcriptases. ~ 1991Academic Press,

Inc.

Equine infectious anemia virus (EIAV) is a lentivirus of the family retroviridae(1-5). EIAV infection is characterized by recurring episodes of fever, complement and noncomplement hemolytic anemia, weight loss, and an immune-complex glomerulonephritis. Unlike most lentivirus infections, symptoms of equine infectious anemia appear rapidly. However, in the majority of cases, chronic infection and viral persistence occur despite host immune response, a hallmark of lentivirus infections.(6) EIAV reverse transcriptase (RT) exhibits homology with the RTs of other lentiviruses, particularly with the RT of human immunodeficiency virus type 1(HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). In a comparison of the RT coding regions of these two viruses, 59% sequence similarity and 43% sequence identity are found at the amino acid level. Such high levels of similarity with HIV-1 RT suggest that EIAV RT may be a good model for examining drug resistance and structure/function relationships in this class of enzymes. We report here the partial purification (85%) of EIAV RT from virus particles by anion exchange chromatography. The optimal pH and monovalent and divalent cation

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concentrations for assaying the polymerase activity of this enzyme have been determined. Further, kinetic constants for incorporation of nucleoside substrates into homopolymeric template-primers have been determined. Finally, the effects of several inhibitors on the authentic, wild-type EIAV RT are discussed.

MATERIALS AND METHODS

Chemicals and Reagents: Poly(rA).p(dT)12-1e, poly(rC).p(dG)12.1e, poly(dA).p(dT)lo, and the ultrapure tetrasodium salts of dTTP, dGTP, ddTTP,and ddGTP were from Pharmacia LKB Biotechnology (Piscataway, NJ); [3H]dTTP and [3H]dGTP from either ICN Chemical and Radioisotope Division (Irvine, CA), or from Dupont-New England Nuclear (Boston, MA); Trizma base, dithiothreitol(DTT), aprotinin, leupeptin, tryptic soy inhibitor and PMSF from Sigma Chemical Co. (St. Louis, MO); Glycerol, MgCI2, MnCI2 NaCI and KCI from Mallinckrodt Specialty Chemical Co. (Paris, KY); Centriprep-30 concentrators from Amicon Division, W.R. Grace and Co.-Conn. (Beverly, MA). Enzyme Purification: Culture fluid (750 ml) from EIAV infected equine dermal fibroblasts was clarified by centrifugation at 1000 RPM for 10 min. at 4C. The virus was precipitated with 10% polyethylene glycol(PEG) and 100 mM NaCI for 2 hours on ice. Precipitated virus was pelleted by centrifugation for 20 min. at 500 xg. The virus pellet was suspended in 10 mM Tris-HCI, pH 7.6, 50 mM NaCI, pelleted by centrifugation for 1 hour at 35,000 xg, and resuspended in the same buffer and repelleted. The enzyme was extracted by incubating the pellet on ice for 30 min. in buffer A [50 mM Tris-HCI, pH 7.9; 0.25% NP-40; 5% (vol./vol.) glycerol; 0.5% deoxycholate; 500 mM KCI; 20 mM DTT; 0.1 mM PMSF; 10 I~g/ml each of soybean trypsin inhibitor, leupeptin, and aprotinin]. After centrifugation for 10 min. at 10,000 xg, the supernatant was dialyzed against buffer B [50 mM Tris-HCI, pH 8.8; 10% glycerol, 0.5 mM DTT] and applied to an FPLC preparative mono Q anion exchange column equilibrated in the same buffer. Enzyme was eluted from the column with a 0-600 mM NaCI gradient in buffer B. Fractions were collected and assayed for RT activity, and those fractions having peak activity were pooled. The protein was concentrated, and the elution buffer exchanged for storage buffer [50 mM Tris-HCI, pH 7.9, 5 mM MgCI2, 10 mM KCI, 0.5 mM DTT, 20% glycerol]. The protein was judged to be approximately 85% pure by silver stain SDS-PAGE. Determination of optimal reverse transcriptase assay conditions: (i). Monovalent cation assays: The reaction mixture (100 ~1) contained 50 mM TrisHCI, pH 7.6, 5 mM MgCI2, 0.5 mM DTT, 10 I~M [3H]dTTP, 80 p.g/ml poly(rA).p(dT)12.18, and indicated concentrations of KCI. Reactions were performed at 37C and started by addition of enzyme. Serial aliquots (20 ~1) of the reaction mixture were taken, spotted immediately on Whatman DE-81 paper, and processed as previously described.(7) (ii). Divalent cation assays: The reaction mixture (100 I~1)was the same as above except that the mixture contained 100 mM KCI. MgCI2 and MnCI2 were present at the indicated concentrations. (iii). pH assays: The reaction mixture was as above and included 10 mM MgCI2. The pH of the buffer was varied from 6.0 to 9.5. Enzymes assays for kinetic analyses: The final assay buffer conditions, based on the above experiments, were 50 mM Tris-HCI, pH 7.6; 10 mM MgCI2, 100 mM KCI, 0.5 mM DTT. The template-primer concentrations were 80 ~g/ml poly(rA).p(dT)12.18, 200 Ilg/ml poly(dA)-p(dT)lo, and 1 mg/ml poly(rC).p(dG)12.1e, dNTP substrates and inhibitors were used at the indicated concentrations. Serial aliquots (15 I~1)were taken from 150 I~1 reaction mixtures, spotted immediately on Whatman DE-81 paper, and processed as previously described.(7) 1366

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RESULTS Characterization of EIAV reverse transcriptase activity EIAV RT was partially purified from virus particles by anion exchange chromatography. Two major protein bands with molecular masses of 70 kdal and 59 kdal and accounting for 85% of the total protein were observed by SDS gel electrophoresis. This is consistent with a heterodimeric quaternary structure for the enzyme. The enzyme catalyzed the incorporation of [3H]dTMP into the template-primer poly(rA)-p(dT)12.18. Rates were linear for 30 min. and a divalent cation, either Mg ++ or Mn ++, was required for this activity. The rate observed at the optimal Mn ++ concentration (0.25 mM) was approximately four-fold lower than that observed with optimal Mg ++ concentrations (10-15 mM) (Fig. 1). The enzyme also remained highly active over a wide range of Mg ++ concentrations. These results are consistent with those from endogenous assays using permeabilized EIAV (3,5). Similar results are seen with HIV-1 RT, however, HIV-1 RT is more sensitive to increased ionic strength and efficiently polymerizes dTTP over a narrower range of Mg++ concentrations(8). Addition of a monovalent cation increased the activity of the EIAV RT by approximately 50 percent, but was not a strict requirement, with the optimal concentrations of KCI being from 50-100 mM. Finally, EIAV RT was active over a broad pH range, with a maximum at 7.5- 8.5. Assay conditions for further kinetic characterization of the enzyme were selected based on the optimal conditions stated above and were presented in the Materials and Methods section. Determination of Substrate Kinetic Constants Apparent Km values of 6.1 + 0.3 ~M for dTTP and 8.0 + 0.9 I~M for dGTP were obtained using the template-primers poly(rA).p(dT)12.18 and poly(rC).p(dG)12.18, respectively.

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40

60

80

100

120

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1

2

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Fiq. 1. Divalent cation requirements [Mg++ (A) or Mn÷÷ (B)] for EIAV reverse transcriptase purified from virus particles. Reaction mixtures (1001~1)contained 50 mM Tris, pH 7.6; 0 5 mM DTT, 100 mM KCI, 80 ~g/ml poly(rA)-p(dT)12.18, and 10 IIM [3H]dTTP (1367 dpm/pmol). Mg÷+ concentrations were varied from 0 to 100 raM. Mn++ concentrations were varied from 0 to 5 mM. 1367

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Table 1.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Apparent K m and Ki Values for Nucleoside-5'-Triphosphate Substrates Km

Enzyme EIAV HIV FIV AMV EIAV HIV EIAV HIV

Substrate dTTP dTTP dl-rP d-I-rP dl-rP dTTP dGTP dGTP

Temolate-Primer

uM

Ki uM

poly(rA).p(dT)12-18 poly(rA).p(dT)lo poly(rA).p(dT)lo poly(rA).p(dT)lo poly(dA).p(dT)12-18 poly(dA).p(dT)12-18 poly(rC).p(dG)12.18 poly(rC)-p(dG)12.18

6.1 + 0.3 6.6 + 0.5 a 1.8 + 0.2 a 48.5 + 10.0 a 26 + 1 5.8 + 0.8 a 8.0 + 0.9 2.2 a

1000 + 200 195 + 37 a 620 + 70 a N.D. *a N.D.* N.D. *a 900 + 100 189 + 32 a

* Substrate Inhibition Not Detected at 600 I~M dTTP. aFurman et al(9)

An apparent Km for dTTP of 26 + 1 I~M was obtained using poly(dA).p(dT)lo as the template-primer (Table 1). These values are similar to the apparent Km values for substrate incorporation using HIV-1 RT with these template-primers (Table 1). Additionally, substrate inhibition was observed with EIAV RT, as evidenced by an upward deflection in the 1/V vs 1/S plot at high substrate concentrations (Fig. 2). Substrate inhibition has already been documented for HIV-1 RT and for feline immunodeficiency virus (FIV) RT (9). EIAV RT is much less susceptible to substrate inhibition than is HIV-1 RT, as demonstrated by an approximately five-fold difference in Ki values for both dTTP and dGTP with their respective template-primers (Table 1 ). Instead, substrate inhibition of EIAV RT more closely resembles that observed with FIV RT (Table 1).

0.4

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0.0 0.0

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Fig. 2. Lineweaver-Burk Plot of d-l-rP incorporation into poly(rA).p(dT)12_18, catalyzed by EIAV reverse transcriptase purified from virus particles. Reaction mixtures were as described in Materials and Methods• Concentrations of dTTP (1367 dpm/pmol) were 600,400,200, 100, 50, 25, 12.5, and 6.25 I~M. 1368

Vol. 180, No. 3, 1991 Table 2.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Apparent Ki Values for Dideoxynucleosidetriphosphate Inhibitors of Reverse Transcriptase Activity

Enzyme EIAV HIV EIAV HIV EIAV HIV HIV

Inhibitor ddTTP dd-I-rP dd]-I-P dd-l-I-P ddGTP ddGTP ddGTP

TemDl~,tt~-Prim~r Poly(rA).p(dT)~2.18 Poly(rA)'p(dT)lo P°Iy(dA)'p(dT)I o Poly(dA)'p(dT)lo Poly(rC).p(dG)12_18 Poly(rC)'p(dG)12-18 Poly(rC)'p(dG)12-18

Ki, (uM) 0.051 + 0.003 0.03 a 61 + 2 46 b 0.0202 + 0.0009 0.009 c 0.03a

aChen and Oshana (13) bMartin et al. (14) cWu et al. (15)

Inhibition of EIAV reverse transcriDtase bv nucleoside-5'-triDhosDhate analoos Inhibition of EIAV RT by 2',3'-dideoxythymidine-5'-triphosphate (ddTTP) and 2',3'dideoxyguanosine-5'-triphosphate (ddGTP) was examined. Apparent Ki values for ddTTP of 51 + 3 nM and 61 + 2 I.tM were determined using the template-primers poly(rA).p(dT)12.18 and poly (dA).p(dT)lo, respectively. With poly(rC)-p(dG)12-18 as the template-primer and ddGTP as the inhibitor, an apparent Ki of 20.2 + 0.9 nM was observed (Table 2). These values are similar to those observed for ddTTP and ddGTP with HIV-1 RT (Table 2). AZT-TP was a competitive inhibitor, with respect to dTTP, of EIAV RT. Compared to HIV-1 RT, the levels of inhibition of EIAV RT by AZT-TP differed significantly. When poly(rA)-p(dT) was the template-primer, HIV-1 RT was found to be three to four-fold more sensitive to AZT-TP than was EIAV RT (Table 3). Even more significant was the twelve fold decrease in sensitivity of EIAV RT to AZT-TP as compared to HIV-1 RT on the template primer poly(dA).p(dT) (Table3). These differences in apparent Ki values between EAIV RT and HIV-1 RT could account for the difference in sensitivity of the two viruses to inhibition by AZT in cell culture as demonstrated by an ID5o of approximately 1 I.tM for EIAV versus an ID5o of 0.05 I.tM for HIV-l.(Unreported data,10).

Table 3.

Enzvme EIAV HIV EIAV HIV

Apparent Ki Values for AZTTP

Inhibitor AZ'I-iP AZ'I-I'P AZI-FP AZ-I-FP

Template-Primer Poly(rA).p(dT)12.18 Poly(rA)'p(dT)lo Poly(dA).p(dT)l o Poly(dA)p(dT)lo

a Furman et al. (16) bMartin et al. (14) 1369

K~.(~M~ 0.143 + 0.005 0.04 ± 0.002. a 230 + 60 11.8 b

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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

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Fig. 3. Lineweaver-Burk Plot of the inhibition of EIAV reverse transcriptase by carbovir triphosphate. Reaction mixtures (150 I.tl) were as described in Materials and Methods. For each set of reactions the concentrations of dGTP (1596 dpm/pmol) were 50, 25, 12.5, 6.25, and 3.125 I.tM. Concentraions of carbovir-triphosphate were i , 250; o, 125; o, 62.5; c!, 31.25; and., 0 nM.

Carbovir-5'-triphosphate (carb-TP) was examined for its ability to inhibit EIAV RT on poly(rC)'p(dG)12-18.

Carb-TP appeared to cause a linear, mixed-type inhibition, with

respect to dGTP, exhibiting an apparent Kis of 27 + 4 nM and an apparent KII of 180 + 20 nM (Fig.3). These data can be explained several ways. The simplest explanation would be that the enzyme/inhibitor complex has a lower affinity for substrate than does the enzyme alone and that the enzyme/inhibitor/substrate complex does not result in product formation. This type of inhibition is a mixture of partial competitive and pure noncompetitive inhibition. A second explanation suggests the presence of two separate binding sites for carb-TP on EIAV RT. Depending on the mechanisms of the different binding reactions, the inhibition pattern could be described as a mixture of pure competitive and pure noncompetitive or a mixture of pure competitive and pure uncompetitive inhibitions(11). This mixed-type inhibition of EIAV RT is very different from the inhibition observed with HIV-1 RT which exhibits competitive inhibition with respect to carb-TP (12). There is no simple explanation for this apparent difference in the inhibition of these two similar enzymes by carb-TP. However, the nonhomogeneous nature of the EIAV RT preparation may have contributed to this apparent difference in activity. In summary, EIAV RT has been partially purified. The enzyme exhibits the same divalent and monovalent cation requirements, and pH optimum as the RT activity observed in permeabilized viral preparations of EIAV. Additionally, the Km values reported here for incorporation of nucleotide substrates by EIAV RT, along with the K i values for ddTTP and ddGTP are very similar to those reported for HIV-1 RT. EtAV RT also exhibits substrate inhibition. Substrate inhibition may therefore be a phenomenon unique to lentivirus reverse transcriptases since all tested lentivirus reverse transcriptases exhibit substrate inhibition, whereas the RT of avian 1370

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myeloblastosis virus (AMV), a non-lentivirus does not. These results suggest that the similarity between the EIAV RT and HIV-1 RT extends from the structural level to the functional level. However, differences in the patterns and levels of inhibition of EIAV RT compared to those seen with HIV-1 RT in the presence of AZT-TP, carb-TP and in the presence of elevated levels of the natural 2'-deoxynucleoside-5'-triphosphates, suggest that the functional similarity of these two proteins, while significant, is not absolute. If these differences in RT activity can be correlated with differences in the structures of these two proteins through studies of drug resistance, it may be possible to establish a structure-function relationship for substrate binding that may help in the design of antiviral chemotherapeutics against this major target of retroviral infection. Acknowledgment8 We would like to thank Drs. Susan Deluge and Sam Hopkins for providing us with the carbovir-5'-triphosphate, Dr. Fred Fuller for the EIAV infected equine dermal cells, Drs. George Painter and John Reardon for critical review of the manuscript and J. Louise Martin et al. for providing us with data prior to publication.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Yaniv, A., Dahlberg, J., Gazit, A., Sherman, L., Chiu, I.M., Tronick, S.R., and Aaronson, S.A. (1986)Virology. 154, 1-8. Stephens, R.M., Casey, J.W., and Rice, N.R. (1986) Science. 589-594. Archer, B.G., Crawford, T.B., McGuire, T.C., and Frazier, M.E. (1977) J. Virol. 22, 16-22. Cheevers, W.P., Archer, B.G., and Crawford, T.B. (1977) J. Virol. 24, 489-497. Charman, H.P., Bladen, S., Gilden, R.V., and Coggins, L. (1976) J. Virol. 19, 1073-1079. McGuire, T.C., and Henson, J.B. (1973) Perspect. Virol. 8, 229-247. Altman, S. and Lerman, LS. (1970) J. Mol. Biol. 50,235-261. Wondrak, E.M., LOwer, J., and Kurth, R. (1986) J. Gen Virol. 67, 2791-2797. Furman, P.A., Painter, G., Wilson, J.E., Cheng, N., and Hopkins, S. (1991) Proc. Natl. Acad. Sci. USA 88, 6013-6017. Mitsuya, H., Weinhold, K.J., Furman, P.A., St. Clair, M.H., Lehrman, S.N., Gallo, R.C., Bolognesi, D., Barry, D.W., and Broder S. (1985) Proc. Natl. Acad. Sci. USA 82, 7096-7100. Segel, I.H. (1975) Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. pp. 170-178. John Wiley and Sons, Inc., New York, NY. Parker, W.B., White, E.L., Shaddix, S.C., Ross, L.J., Buckheit, Jr.,R.W., Germany, J.M., Secrist III, J.A., Vince, R., and Shannon, W.M. (1991) J. Biol. Chem. 266, 1754-1762. Chen, M.S., and Oshana, S.C. (1987) Biochem. Pharmac. 36, 4361-4362. Martin, J.L, Wilson, J., Hopkins, S., and Furman, P.A. (1991) Manuscript in Preparation. Wu, J.C., Chernow, M., Boehme, R.E., Suttman, R.T., McRoberts, M.J., Prisbe, E.J., Matthews, T.R., Marx, P.A., Chaung, R.Y., and Chen, M.S. (1988) Antimicrob. Agents Chemother. 32, 1887-1890. Furman, P.A., Fyfe, J.A., St. Clair, MH., Weinhold, K., Rideout, J.L., Freeman, G.A., Lehrman, S.N., Bolognesi, D.P., Broder, S., Mitsuya, H., and Barry, D.W. (1986) Proc. Natl. Acad. Sci. USA 83, 8333-8337. 1371