NOTES Kinetics and Inhibition of Reverse Transcriptase from Human ...

ANTIMICROBIAL AGENTS

AND

CHEMOTHERAPY, Dec. 1988,

p.

1887-1890

Vol. 32, No. 12

0066-4804/88/121887-04$02.00/0 Copyright ©) 1988, American Society for Microbiology

NOTES Kinetics and Inhibition of Reverse Transcriptase from Human and Simian Immunodeficiency Viruses JOHN C.

WU,'*

MARLENE CHERNOW,1 RICHARD E.

BOEHME,l REBECCA T. SUTTMANN,l MARY JANE

McROBERTS,' ERNEST J. PRISBE,' THOMAS R. MATTHEWS,' PRESTON A. MARX,2 RONALD Y. CHUANG,2 AND MING S. CHEN' Department of Antimicrobial Research, Institute of Agriscience, Syntex Research, 2375 Charleston Road, Mountain View, California 94043,1 and California Primate Research Center and Department of Pharmacology, University of California, Davis, California 956162

Received 20 June 1988/Accepted 14 September 1988

Reverse transcriptase from the simian immunodeficiency virus (SIV) was found to have kinetic behavior similar to that of enzyme from the human immunodeficiency virus (HIV). Michaelis constants for the substrates TTP and dGTP and inhibition constants for the inhibitors 3'-azido-3'-deoxythymidine 5'-triphosphate, 2',3'-dideoxythymidine 5'-triphosphate, and 2'-3'-dideoxyguanosine 5'-triphosphate were obtained for SIV reverse transcriptase and were found to be similar to the corresponding values for HIV reverse transcriptase. Thus, the interaction of SIV reverse transcriptase with nucleotide analogs appears to be indistinguishable from that of the HIV enzyme, suggesting that SIV/simian acquired immunodeficiency syndrome (SAIDS) is a potentially good model of AIDS.

tases from SIV and HIV, we examined the kinetics and inhibition of enzymes from the two viruses. Radioactive [methyl-3H]TTP (25 Ci/mmol) was purchased from New England Nuclear Corp., and [1',2'-3H]dGTP (40 Ci/mmol) was purchased from Amersham Corp. Template primers poly(A)-oligo(dT)12_18 (1:1, A:dT ratio) and poly(C)oligo(dG)12_18 (1:1, C:dG ratio), 2',3'-dideoxythymdine 5'triphosphate (ddTTP), and 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP) were from Pharmacia-PL Biochemicals, Inc. The inhibitor AZT 5'-triphosphate (AZTTP) was synthesized by established methods (16, 17). Bovine serum albumin, Nonidet P-40, and dithiothreitol were purchased from Sigma Chemical Co. Single-stranded-DNA-agarose was from Bethesda Research Laboratories, Inc. HIV type 1 (human T-lymphotropic virus type III/lymphadenopathyassociated virus) and A3.01 cells were from Thomas Folks of the National Institutes of Health (10). SlVmac-infected HUT78 cells were obtained from Ron Desrosiers of the New England Regional Primate Research Center (9). For the preparation of HIV, A3.01 cells in log phase (-2 x 105/ml in RPMI 1640 with 10% fetal calf serum and 50 p.g of gentamicin per ml) were pelleted by low-speed centrifugation (3,000 x g; 15 min). The cells were suspended in fresh medium to a density of 2 x 106/ml, and supernatant from an 8-day HIV type 1-infected culture was added (1:4, infected: uninfected [by volume]). The cells were incubated at 37°C with agitation for 3 h, diluted with an equal volume of medium, and cultured at 37°C. The cell concentration was monitored daily and maintained at 106/ml by the addition of fresh medium. Viral replication was monitored by daily assays of reverse transcriptase activity in the supernatant. When peak reverse transcriptase activity was achieved (ca. day 8 postinfection), the medium was cleared of cells by centrifugation (3,000 x g; 15 min), and the virus in the supernatant was pelleted by centrifugation at 110,000 x g

The development of new compounds to inhibit the replication of human immunodeficiency virus (HIV) relies on valid tissue culture and animal models of acquired immunodeficiency syndrome (AIDS). HIV has been successfully cultured in cells of human origin and transmitted to chimpanzees (1, 10-12, 15, 18, 20). Although chimpanzees can be infected by HIV, the status of the animals as an endangered species and the fact that HIV does not appear to cause the fatal immunodeficiency syndrome in infected animals severely limit their usefulness (1, 11, 12). Alternative animal models for AIDS are necessary to complement available tissue culture systems. One animal model that resembles AIDS closely is the simian immunodeficiency disease (SAIDS) associated with a related retrovirus (SIV/simian T-lymphotropic virus type III) isolated from rhesus macaques (9). SIV has an approximately 40% nucleotide sequence homology with HIV type 1 (5), and it causes in infected monkeys a fatal immune deficiency disease that is clinically similar to AIDS (9). Thus, the SIV/macaque system represents an attractive animal model for the study of AIDS. The viability of SIV/SAIDS as an animal model of HIV/ AIDS depends on the similarity of the biochemical functions of SIV to those of HIV. The hallmark of the retroviral life cycle is expression of reverse transcriptase activity in infected cells. Inhibition of the retroviral enzyme RNA-directed DNA polymerase (reverse transcriptase) is one approach toward treatment of AIDS (2-4, 8, 14, 19, 22). An example of this approach is the drug 3'-azido-3'-deoxythymidine (AZT), which has clinical efficacy because in the nucleoside triphosphate form it preferentially inhibits viral reverse transcriptase (13, 19, 23). To compare the relative effects of nucleotides and their analogs on reverse transcrip*

Corresponding author. 1887

ANTiMICROB. AGENTS CHEMOTHER.

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-1.2

-0.8

0

-0.4

0.4

0.8

1.2

0.4

0.8

1.2

1/[dGTP] (gM 1)

-1.2

-0.8

0

-0.4

1/[dGTP] (gM

1)

FIG. 1. Lineweaver-Burke plots for inhibition of SIV and HIV reverse transcriptases by ddGTP. Poly(C)-oligo(dG)12_18 and [3H]dGTP were the substrates, and reactions were performed as described in the text. (Top panel) Kinetics and inhibition of SIV reverse transcriptase; (bottom panel) kinetics and inhibition of HIV reverse transcriptase. V (initial rate) is in disiritegrations per minute per minute of reaction titne. Symbols: *, no ddGTP; A, 0.05 ,uM ddGTP; O, 0.10 ,uM ddGTP. Inserts, Replots of slopes versus ddGTP concentrations to determine the Ki of ddGTP for each enzyme.

(30,000 rpm; 90 min; Beckman SW41 rotor). The virus pellet suspended in phosphate-buffered saline containing 2% Nonidet P-40 and 10% glycerol. For the preparation of SIV, SIVmac-infected HUT-78 cells were grown in RPMI 1640-10% fetal calf serum plus penicillin and streptomycin supplemented with glutamine at 100

was

mM. Cell cultures were seeded with infected/uninfected HUT-78 cells at a ratio of 3/2. Virus was harvested from spent media at 72 h postinfection. Cells were pelleted by centrifugation at 300 g for 10 min, and the supematant was further clarified by centrifugation at 10,000 x g in Beckman 45 Ti rotor. The SIV particles were pelleted at 95,000 x g for

TABLE 1. Kinetic constants for reverse Template primer

Substrate

Poly(A)-oligo(dT)12-18

[3H]dTTP

Poly(C)-oligo(dG)12-18

[3H]dGTP

x

trariscriptases from HIV and SIV

Km. (FLM) HIV

8.0 3.5 4.7

SIV 6.5 5.0 5.4

K1 (ILM)

Inhibitor HIV

I

SIV

AZTlP

0.020

0.020

ddTTP ddGTP

0.015

0.016

0.009

0.011

VOL. 32, 1988

1 h and suspended in phosphate-buffered saline containing 2% Nonidet P-40. Reverse transcriptases from the lysed preparations of HIV and SIV were purified by DNA-agarose chromatography. Lysates were diluted in column buffer (10 mM Tris-acetate [pH 7.0], 1 mM dithiothreitol, 10% glycerol) at 1:4 dilution for HIV (2-ml final volume) or 1:7 dilution for SIV (1.4-ml final volume) and applied to a single stranded-DNA-agarose column (1-ml packed volume equilibrated in column buffer). The enzyme activity was eluted with a gradient (100 ml) of 0 to 1.0 M NaCl in column buffer. Fractions (1 ml) were collected into tubes containing 200 ,ug of bovine serum albumin (10 ,ul of a 20-mg/ml solution), which improved enzymic stability. Activities, which eluted at 0.25 to 0.30 M NaCl, were collected and stored at -70°C. The assay for reverse transcriptase measured the incorporation of tritiated deoxynucleoside triphosphate into an acid-precipitable form. The assay mixture (80 pul) contained 50 mM Tris hydrochloride (pH 8.0), 5 mM MgCl2, 150 mM KCl, 5 mM dithiothreitol, and labeled deoxynucleoside triphosphate. The template-primer concentration for [PH] thymidine incorporation was 15 ,uM poly(A)-oligo(dT)12-18 (concentration in nucleotide base pairs); for [3H]deoxyguanosine incorporation, the concentration of poly(C)-oligo (dG)12_18 was 188 puM in nucleotide base pairs. The concentrations of the template primers were chosen to be saturating for the enzymes. The high concentration of poly(C)oligo(dG)12_18 needed to achieve Vmax was observed for both SIV and HIV enzymes. Inhibitors were added as required. After incubation at 37°C, 15-pdl samples were removed at various times and processed as described previously (7). Enzymes for this study were prepared from viral lysates by DNA-agarose chromatography. The final preparations of reverse transcriptases were free of nuclease activities and yielded linear reaction kinetics for up to 80 min of incubation time. The preparations were free of other viral proteins based on immunoblotting (Western hybridization) with HIV antisera. The nucleotides AZTTP, ddTTP, and ddGTP are potent inhibitors of HIV reverse transcriptase, with KmIKi ratios of 100 to 400 with respect to the corresponding deoxyribonucleoside triphosphates (6, 13, 21). Inhibition data for the SIV enzyme have not been reported. The kinetics of inhibition of reverse transcriptases from SIV and HIV by ddGTP are shown in Fig. 1. The data indicate the competitive nature of the inhibition by ddGTP with respect to dGTP and afford nearly equal Michaelis and inhibition constants for the two enzymes. The Kms of dGTP were 5.4 puM for the SIV enzyme and 4.7 puM for the HIV enzyme. The Kis of ddGTP were 0.011 and 0.009 puM for the SIV and HIV enzymes, respectively. The Km/Ki ratio for SIV reverse transcriptase was 490; for HIV, the ratio was 522. The complete data for two primer templates and three inhibitors are summarized in Table 1. For all substrates and inhibitors tested, the SIV reverse transcriptase had values of Km and Ki that were very similar to those of the HIV enzyme. The values determined for the enzyme from HIV were in good agreement with those reported previously (6, 13, 21). In addition to being potent competitive inhibitors of the enzymes, the compounds tested in this study are also chain terminators for the DNA polymerase reaction. Comparisons of chain termination for the two enzymes require further investigation. In summary, the results indicate that the kinetic properties of reverse transcriptase from SIV are virtually identical to

NOTES

1889

those of the enzyme from the human virus. In conjunction with data from animal studies, our data suggest that SIVinfected cells and animals are relevant models of AIDS and may be particularly suited to the development of drugs directed against HIV reverse transcriptase. We thank Diane Brodeur for assistance with cell culture and Agegnehu Gettie for technical assistance. P.A.M. and R.Y.C. were supported by Public Health Service grants Al 62559 and CA 33022 from the National Institutes of Health. LITERATURE CITED 1. Alter, H. J., J. W. Eichberg, H. Masur, W. C. Saxinger, R. Gallo, A. M. Macher, H. C. Lane, and A. S. Fauci. 1984. Transmission of HTLV-III infection from human plasma to chimpanzees: an animal model for AIDS. Science 226:549-552. 2. Baba, M., R. Pauwels, P. Herdewln, E. De Clercq, J. Desmyter, and M. Vandeputte. 1987. Both 2',3'-dideoxythymidine and its 2',3'-unsaturated derivative (2',3'-dideoxythymidinene) are potent and selective inhibitors of human immunodeficiency virus

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