ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1990, p. 1505-1507
Vol. 34, No. 8
0066-4804/90/081505-03$02.00/0 Copyright ©D 1990, American Society for Microbiology
Direct Comparisons of Inhibitor Sensitivities of Reverse Transcriptases from Feline and Human Immunodeficiency Viruses THOMAS W. NORTH,* RICHARD C. CRONN, KATHRYN MARTIN REMINGTON, AND ROLF T. TANDBERG Division of Biological Sciences, University of Montana, Missoula, Montana 59812 Received 15 March 1990/Accepted 5 June 1990
The sensitivities of reverse transcriptases (RTs) from feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (IRV) were directly compared. The two enzymes had similar sensitivities to three analogs of dTTP, namely, 3'-azido-3'-deoxythymidine 5'-triphosphate, 2',3'-dideoxythymidine 5'triphosphate, and 2',3'-dideoxy-2',3'-didehydrothymidine 5'-triphosphate. Each of these analogs demonstrated competitive inhibition of both enzymes. Ki values for inhibition of FIV RT by these three inhibitors were 3.3, 6.7, and 1.8 nM, respectively; Ki values for inhibition of the HIV enzyme were 6.5, 5.9, and 8.3 nM, respectively. Ratios of the Ki for the inhibitor to the Km for the substrate were also determined for each inhibitor, and no differences between the two enzymes greater than threefold were observed. Inhibition constants for 3'-amino-3'-deoxythymidine 5'-triphosphate and 3'-fluoro-3'-deoxythymidine 5'-triphosphate were determined for FIV RT, and these were similar to published values for HIV RT. The activities of three dideoxynucleoside 5'-triphosphates against FIV RT were determined; ddGTP was slightly more potent than ddTTP, whereas both were much more effective than ddCTP. The activity of a noncompetitive inhibitor, phosphonoformate, was also examined with the FIV enzyme; it was much more active with poly(rA)-oligo(dT) as the template-primer than with poly(rC)-oligo(dG) or poly(rI)-oligo(dC).
Feline immunodeficiency virus (FIV) is a lentivirus that immune deficiency in domestic cats that is very similar to acquired immune deficiency syndrome (AIDS) in humans (13). FIV is similar to human immunodeficiency virus type 1 (HIV) in morphology, protein composition, gene arrangement, and tropism to T lymphocytes and macrophages (13, 17). These features make FIV a valuable animal model for studies of AIDS (5). In our laboratory, it has recently been shown that reverse transcriptase (RT) obtained from detergent-lysed virions of FIV is inhibited by the 5'-triphosphates of 3'-azido-3'-deoxythymidine and 2',3'-dideoxythymidine and by phosphonoformate (PFA) (12). The inhibition constants determined for these inhibitors were similar to values that had been published for HIV RT (3, 16). In contrast, FIV RT was much different from avian myeloblastosis virus RT in sensitivity to these inhibitors (12). Moreover, Take et al. (15) have shown that the HIV and avian myeloblastosis virus RTs are different in their sensitivities to many inhibitors. We subsequently purified FIV RT and showed that it is also similar to HIV RT in physical properties, template specificities, and requirements for Mg2+ (11). Of central importance to the use of FIV as a model for chemotherapy of AIDS is the degree of similarity between its RT and HIV RT, particularly in sensitivity to active forms of antiviral drugs. Our previous work described preliminary comparisons in which FIV RT from detergent-treated virions was compared with data reported by other laboratories for HIV RT. This report describes more detailed studies with purified FIV RT and direct comparisons of purified FIV and HIV -RTs. causes an
MATERIALS AND METHODS
Chemicals. 2', 3' Dideoxynucleoside 5' triphosphates (ddTTP, ddCTP, and ddGTP) were purchased from Pharmacia LKB, Piscataway, N.J. PFA and 2'-deoxyribonucleoside -
*
Corresponding author.
-
5'-triphosphates (dTTP, dCTP, and dGTP) were purchased from Sigma Chemical Co., St. Louis, Mo. The 5'-triphosphate of 3'-azido-3'-deoxythymidine was provided by Wayne Miller of the Burroughs Weilcome Co., Research Triangle Park, N.C.; 2',3'-dideoxy-2',3'-didehydrothymidine 5'-triphosphate was provided by H.-T. Ho of BristolMyers Squibb Co., Wallingford, Conn; 3'-amino-3'-deoxythymidine 5'-triphosphate and 3'-fluoro-3'-deoxythymidine 5'-triphosphate were provided by Y.-C. Cheng, Yale University, New Haven, Conn. Poly(rA)-oligo(dT)1o and the RNA homopolymers and DNA primers used to make poly(rI)-oligo(dC)10 and poly (rC)-oligo(dG)1o were purchased from Pharmacia LKB. The latter two template-primers were prepared as previously described (3, 11). Isotopes. Radioactive deoxyribonucleotides, [methyl-3H] dTTP, [5-3H]dCTP, and [8-3H]dGTP, were purchased from Dupont, NEN Research Products, Boston, Mass. Enzymes. RT was purified from virions of FIV. The Petaluma strain of FIV was grown in Crandell feline kidney cells as previously described (12, 17). Purification of FIV RT was recently reported (11). Briefly, this involves disruption of virions with Triton X-100, followed by chromatography on DEAE-cellulose and phosphocellulose. Analysis of this RT by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 67,000 (11). The HIV RT used in these studies was provided by Stephen H. Hughes, National Cancer Institute, Frederick, Md. This enzyme is produced from a construct of the gene for HIV RT cloned into Escherichia coli (7). It was shown to be indistinguishable from virion-associated HIV RT in sensitivity to several antiretroviral agents (14). Enzyme assays. Conditions for assay of RT with the templates used in these experiments were described previously (11). Reaction mixtures contained 0.05 to 0.25 U of RT. One unit of RT is the amount that catalyzes incorporation of 1 nmol of dTMP per h into poly(rA)-oligo(dT)10 at 1505
1506
NORTH ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Inhibition of FIV and HIV RTs by analogs of dTTP Mean K. + SD
Inhibitor'
(nM)b HIV
FIV
N3dTTP ddTTP D4TTP 3'-F-dTTP 3'-NH2-dTTP
3.3 6.7 1.8 3.1 1.6
± ± ± ± ±
1.6 3.4 0.7 0.1 0.5
(6) (5) (6) (3) (3)
6.5 5.9 8.3 5 6
± ± ± ± ±
1.8 (4) 4.2 (4) 0.8 (6) lc 2c
Mean
KIKm ratio
FIV
0.0010 0.0020 0.0005 0.0009 0.0005
HIV
0.0011 0.0010 0.0014
TABLE 3. Inhibition of FIV RT by PFA Template-primer
Substrate
Mean Ki -+- SD (>LM)a
poly(rA)-oligo(dT)jO
dTTP dGTP dCTP
0.18 + 0.05 (8) 2.2 ± 0.6 (4) 2.6 ± 1.6 (3)
poly(rC)-oligo(dG)1O poly(rI)-oligo(dC)1O
a Numbers in parentheses indicate numbers of determinations.
0.0010C 0.0012C
a N3dTTP, 3'-Azido-3'-deoxythymidine 5'-triphosphate; D4TrP, 2',3'-dideoxy-2',3'-didehydrothymidine 5'-triphosphate; 3'-F-dTTP, 3'-fluoro3'deoxythymidine 5'-triphosphate; 3'-NH2-dTTP, 3'-amino-3'-deoxythymidine S'-triphosphate. b Numbers in parentheses indicate numbers of determinations. c From Cheng et al. (3).
37°C. Kinetic parameters (Km and K,) were determined as previously reported (12), by using intercept values calculated from double-reciprocal plots. Ratios of Ki to Km were calculated by using the mean values of Ki and Km. RESULTS AND DISCUSSION Inhibition of FIV and HIV RTs by analogs of dTTP. We examined inhibition of FIV RT by five analogs of dTTP, and for three of these, inhibition of HIV RT was also examined (Table 1). These experiments were performed with poly(rA)oligo(dT)1o as the template-primer; to enable direct comparisons, the assay conditions were identical for the two enzymes. Double-reciprocal plots of our data demonstrated that each of these analogs inhibits in a manner that is competitive with respect to dTTP. The FIV and HIV RTs were similar in sensitivity to 3'-azido-3'-deoxythymidine 5'-triphosphate, ddTTP, and 2', 3'- dideoxy - 2', 3'- didehydrothymidine 5'- triphosphate. The K, values determined for inhibition of the two enzymes ranged from nearly identical values obtained for inhibition of the two enzymes by ddTTP to a 4.6-fold difference for inhibition by 2',3'-dideoxy-2',3'-didehydrothymidine 5'-triphosphate (lower for FIV RT). The Km values for dTTP in the uninhibited reactions were 3.4 + 0.2 ,M for FIV RT and 6.0 + 1.9 ,uM for HIV RT. This is in agreement with our previous report that FIV RT has a slightly lower Km for dTTP in this reaction than does HIV RT (11). Comparison of the K/lKm ratios revealed that the difference between the two enzymes in inhibition by any of these three analogs was less than threefold (Table 1). We examined two other analogs of dTTP, 3'-fluoro-3'deoxythymidine 5'-triphosphate and 3'-amino-3'-deoxythymidine 5'-triphosphate, which were previously shown to inhibit HIV RT (3). These also inhibited FIV RT, and the K,/Km ratios obtained were very similar to those reported by Cheng et al. for inhibition of HIV RT (3). Inhibition of FIV RT by dideoxynucleoside 5'-triphosphates. FIV RT utilizes poly(rC)-oligo(dG)j0 and poly(rI)oligo(dC)10 as template-primers under the assay conditions
used, but it does not use poly(rG)-oligo(dC)10 or poly (rU)-oligo(dA)10 (11). We used the former two templates to
we
analyze inhibition of FIV RT by ddGTP and ddCTP, respectively, and to compare these with inhibition by ddTTP. All three of these dideoxynucleotides were competitive inhibitors of FIV RT; the values determined for Ki and K/,Km are shown in Table 2. Of the three, ddGTP was the most potent inhibitor and ddTTP was only slightly less effective, whereas ddCTP was considerably less potent than the other two when assayed under these conditions. We do not believe that the lower activity of ddCTP was due to use of poly(rI)oligo(dC)10 in these assays, because the Km for dCTP in this reaction was only slightly higher than the Km for dTTP in the reaction with poly(rA)-oligo(dT)10. This relative order of activity is similar to that reported by Chen and Oshana (2) for inhibition of HIV RT. However, absolute values of the inhibition constants we determined are different from those of Chen and Oshana, possibly because of different assay conditions. Inhibition of FIV RT by PFA. We also examined inhibition of FIV RT by PFA with each of the three templates described above. PFA was previously shown to be a noncompetitive inhibitor of partially purified FIV RT when assayed with poly(rA)-oligo(dT)10 (12) and also of HIV RT (16). Inhibition of purified FIV RT by PFA was also noncompetitive with the other template-primers, poly(rC)-oligo (dG)1o and poly(rI)-oligo(dC)10 (data not shown). Ki values for inhibition of the three reactions by PFA are shown in Table 3. These data show that PFA was considerably more potent as an inhibitor of the reaction with poly(rA)oligo(dT)10 than with either of the other two templateprimers. We also made a direct comparison of the PFA sensitivities of FIV and HIV RTs in the reaction with poly(rA)-oligo(dT)10. The Ki values for inhibition of the FIV and HIV RTs were 0.18 and 0.72 ,uM, respectively. This value for FIV RT is slightly lower than that which we previously determined with the partially purified enzyme (12). The value we obtained for inhibition of HIV RT is slightly higher than the value reported by Vrang and Oberg, who used different assay conditions (16). The availability of FIV and HIV RTs in pure form has enabled direct comparisons of their sensitivities to inhibitors. These direct comparisons confirm the earlier conclusions that the FIV and HIV RTs are very similar in sensitivity to the active forms of several important antiviral agents and that FIV may be useful as a model for studies of AIDS chemotherapy. These data also provide basic infor-
TABLE 2. Inhibition of FIV RT by dideoxynucleoside 5'-triphosphates
Mean Km ± SD Mean K__± SD Substrate Template-primer ~~~~(nM)a (~~~LM)' Inhibitor 3.4 ± 0.1 (10) dTTP ddTTP 6.7 + 3.3 (6) poly(rA)-oligo(dT)jO dGTP 1.1 ± 0.4 (5) ddGTP 1.2 ± 0.6 (8) poly(rC)-oligo(dG)jO dCTP 5.8 ± 0.3 (3) ddCTP 101 ± 39 (5) poly(rI)-oligo(dC)10 a Numbers in parentheses indicate numbers of determinations.
K/Km ratio 0.0020 0.0011 0.0176
VOL. 34, 1990
mation needed before initiation of chemotherapeutic studies of FIV infection in cats. We have identified several antiviral compounds whose active forms are potent inhibitors of FIV RT. Work is in progress to evaluate the activities of their corresponding nucleosides against replication of FIV RT in cell culture. We believe that the FIV systems will enable coordination of in vivo and in vitro experiments and that these studies can provide information that will be important in the design of chemotherapeutic strategies for AIDS in humans. On the basis of the results of this investigation and the proven anti-HIV activities of 3'-azido-3'-deoxythymidine (4, 10) and 2',3'-dideoxy-2',3'-didehydrothymidine (1, 6, 8, 9), these two compounds look particularly attractive for further studies with the FIV model. ACKNOWLEDGMENTS This work was supported by Public Health Service grant Al 28189 from the National Institute of Allergy and Infectious Diseases. We thank Niels C. Pedersen and Y.-C. Cheng for much helpful discussion. LITERATURE CITED 1. Baba, M., R. Pauwels, P. Herdewijn, 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 replication in vitro. Biochem. Biophys. Res. Commun. 142: 128-134. 2. Chen, M. S., and S. C. Oshana. 1987. Inhibition of HIV reverse transcriptase by 2',3'-dideoxynucleoside triphosphates. Biochem. Pharmacol. 36:4361-4362. 3. Cheng, Y.-C., G. E. Dutschman, K. F. Bastow, M. G. Sarngadharan, and R. Y. C. Ting. 1987. Human immunodeficiency virus reverse transcriptase. General properties and its interaction with nucleoside triphosphate analogs. J. Biol. Chem. 262: 2187-2189. 4. Fischel, M. A., D. D. Richman, M. H. Grieco, M. S. Gottlieb, P. A. Volberding, 0. L. Laskin, J. M. Leedom, J. E. Groopman, D. Mildvan, R. T. Schooley, G. G. Jackson, D. T. Durack, D. King, and the AZT Collaborative Working Group. 1987. The efficacy of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS-related complex. N. Engl. J. Med. 317: 185-191. 5. Gardner, M. B., and P. A. Luciw. 1989. Animal models of AIDS. FASEB J. 3:2593-2606. 6. Hamamoto, Y., H. Nakashima, T. Matsui, A. Matsuda, T. Ueda, and N. Yamamoto. 1987. Inhibitory effect of 2',3'-didehydro2',3'-dideoxynucleosides on infectivity, cytopathic effects, and replication of human immunodeficiency virus. Antimicrob.
FIV REVERSE TRANSCRIPTASE
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Agents Chemother. 31:907-910. 7. Hizi, A., C. McGill, and S. H. Hughes. 1988. Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants. Proc. Natl. Acad. Sci. USA 85:1218-1222. 8. Lin, T.-S., R. F. Shinazi, and W. H. Prusoff. 1987. Potent and selective in vitro activity of 3'-deoxythymidine-2'-ene (3'deoxy-2',3'-didehydrothymidine) against human immunodeficiency virus. Biochem. Pharmacol. 36:2713-2718. 9. Mansuri, M. M., J. E. Starrett, I. Ghazzouli, M. J. M. Hitchcock, R. Z. Sterzycki, V. Brankovan, T.-S. Lin, E. M. August, W. H. Prusoff, J.-P. Sommadossi, and J. C. Martin. 1989. 1-(2,3Dideoxy-1-D-glyceropent-2-enefuranosyl)thymidine (d4T). A highly potent and selective anti-HIV agent. J. Med. Chem. 32:461-466. 10. Mitsuya, H., K. J. Weinhold, P. A. Furman, M. H. St. Clair, S. N. Lehrman, R. C. GaUo, D. Bolognesi, D. W. Barry, and S. Broder. 1985. 3'-Azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphoadenopathyassociated virus in vitro. Proc. Natl. Acad. Sci. USA 82: 7096-7100. 11. North, T. W., R. C. Cronn, K. M. Remington, R. T. Tandberg, and R. C. Judd. 1990. Characterization of reverse transcriptase from feline immunodeficiency virus. J. Biol. Chem. 265:51215128. 12. North, T. W., G. T. North, and N. C. Pedersen. 1989. Feline immunodeficiency virus, a model for reverse transcriptasetargeted chemotherapy for acquired immune deficiency syndrome. Antimicrob. Agents Chemother. 33:915-919. 13. Pedersen, N. C., E. M. Ho, M. L. Brown, and J. K. Yamamoto. 1987. Isolation of a T-lymphotropic virus from domestic cats with an immunodeficiency-like syndrome. Science 235:790-793. 14. Schinazi, R. F., B. F. H. Eriksson, and S. H. Hughes. 1989. Comparison of inhibitory activities of various antiretroviral agents against particle-derived and recombinant human immunodeficiency virus reverse transcriptases. Antimicrob. Agents Chemother. 33:115-117. 15. Take, Y., Y. Inouye, S. Nakamura, H. S. Aflaudeen, and A. Kubo. 1989. Comparative studies of the inhibitory properties of antibiotics on human immunodeficiency virus and avian myeloblastosis virus reverse transcriptases and cellular DNA polymerases. J. Antibiot. 42:107-115. 16. Vrang, L., and B. Oberg. 1986. PP1 analogs as inhibitors of human T-lymphotropic virus type III reverse transcriptase. Antimicrob. Agents Chemother. 29:867-872. 17. Yamamoto, J. K., E. Sparger, E. W. Ho, P. R. Andersen, T. P. O'Connor, C. P. Mandell, L. Lowenstine, R. Munn, and N. C. Pedersen. 1988. Pathogenesis of experimentally induced feline immunodeficiency virus infection in cats. Am. J. Vet. Res. 49:1246-1258.
Vol. 34, No. 8
0066-4804/90/081505-03$02.00/0 Copyright ©D 1990, American Society for Microbiology
Direct Comparisons of Inhibitor Sensitivities of Reverse Transcriptases from Feline and Human Immunodeficiency Viruses THOMAS W. NORTH,* RICHARD C. CRONN, KATHRYN MARTIN REMINGTON, AND ROLF T. TANDBERG Division of Biological Sciences, University of Montana, Missoula, Montana 59812 Received 15 March 1990/Accepted 5 June 1990
The sensitivities of reverse transcriptases (RTs) from feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (IRV) were directly compared. The two enzymes had similar sensitivities to three analogs of dTTP, namely, 3'-azido-3'-deoxythymidine 5'-triphosphate, 2',3'-dideoxythymidine 5'triphosphate, and 2',3'-dideoxy-2',3'-didehydrothymidine 5'-triphosphate. Each of these analogs demonstrated competitive inhibition of both enzymes. Ki values for inhibition of FIV RT by these three inhibitors were 3.3, 6.7, and 1.8 nM, respectively; Ki values for inhibition of the HIV enzyme were 6.5, 5.9, and 8.3 nM, respectively. Ratios of the Ki for the inhibitor to the Km for the substrate were also determined for each inhibitor, and no differences between the two enzymes greater than threefold were observed. Inhibition constants for 3'-amino-3'-deoxythymidine 5'-triphosphate and 3'-fluoro-3'-deoxythymidine 5'-triphosphate were determined for FIV RT, and these were similar to published values for HIV RT. The activities of three dideoxynucleoside 5'-triphosphates against FIV RT were determined; ddGTP was slightly more potent than ddTTP, whereas both were much more effective than ddCTP. The activity of a noncompetitive inhibitor, phosphonoformate, was also examined with the FIV enzyme; it was much more active with poly(rA)-oligo(dT) as the template-primer than with poly(rC)-oligo(dG) or poly(rI)-oligo(dC).
Feline immunodeficiency virus (FIV) is a lentivirus that immune deficiency in domestic cats that is very similar to acquired immune deficiency syndrome (AIDS) in humans (13). FIV is similar to human immunodeficiency virus type 1 (HIV) in morphology, protein composition, gene arrangement, and tropism to T lymphocytes and macrophages (13, 17). These features make FIV a valuable animal model for studies of AIDS (5). In our laboratory, it has recently been shown that reverse transcriptase (RT) obtained from detergent-lysed virions of FIV is inhibited by the 5'-triphosphates of 3'-azido-3'-deoxythymidine and 2',3'-dideoxythymidine and by phosphonoformate (PFA) (12). The inhibition constants determined for these inhibitors were similar to values that had been published for HIV RT (3, 16). In contrast, FIV RT was much different from avian myeloblastosis virus RT in sensitivity to these inhibitors (12). Moreover, Take et al. (15) have shown that the HIV and avian myeloblastosis virus RTs are different in their sensitivities to many inhibitors. We subsequently purified FIV RT and showed that it is also similar to HIV RT in physical properties, template specificities, and requirements for Mg2+ (11). Of central importance to the use of FIV as a model for chemotherapy of AIDS is the degree of similarity between its RT and HIV RT, particularly in sensitivity to active forms of antiviral drugs. Our previous work described preliminary comparisons in which FIV RT from detergent-treated virions was compared with data reported by other laboratories for HIV RT. This report describes more detailed studies with purified FIV RT and direct comparisons of purified FIV and HIV -RTs. causes an
MATERIALS AND METHODS
Chemicals. 2', 3' Dideoxynucleoside 5' triphosphates (ddTTP, ddCTP, and ddGTP) were purchased from Pharmacia LKB, Piscataway, N.J. PFA and 2'-deoxyribonucleoside -
*
Corresponding author.
-
5'-triphosphates (dTTP, dCTP, and dGTP) were purchased from Sigma Chemical Co., St. Louis, Mo. The 5'-triphosphate of 3'-azido-3'-deoxythymidine was provided by Wayne Miller of the Burroughs Weilcome Co., Research Triangle Park, N.C.; 2',3'-dideoxy-2',3'-didehydrothymidine 5'-triphosphate was provided by H.-T. Ho of BristolMyers Squibb Co., Wallingford, Conn; 3'-amino-3'-deoxythymidine 5'-triphosphate and 3'-fluoro-3'-deoxythymidine 5'-triphosphate were provided by Y.-C. Cheng, Yale University, New Haven, Conn. Poly(rA)-oligo(dT)1o and the RNA homopolymers and DNA primers used to make poly(rI)-oligo(dC)10 and poly (rC)-oligo(dG)1o were purchased from Pharmacia LKB. The latter two template-primers were prepared as previously described (3, 11). Isotopes. Radioactive deoxyribonucleotides, [methyl-3H] dTTP, [5-3H]dCTP, and [8-3H]dGTP, were purchased from Dupont, NEN Research Products, Boston, Mass. Enzymes. RT was purified from virions of FIV. The Petaluma strain of FIV was grown in Crandell feline kidney cells as previously described (12, 17). Purification of FIV RT was recently reported (11). Briefly, this involves disruption of virions with Triton X-100, followed by chromatography on DEAE-cellulose and phosphocellulose. Analysis of this RT by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 67,000 (11). The HIV RT used in these studies was provided by Stephen H. Hughes, National Cancer Institute, Frederick, Md. This enzyme is produced from a construct of the gene for HIV RT cloned into Escherichia coli (7). It was shown to be indistinguishable from virion-associated HIV RT in sensitivity to several antiretroviral agents (14). Enzyme assays. Conditions for assay of RT with the templates used in these experiments were described previously (11). Reaction mixtures contained 0.05 to 0.25 U of RT. One unit of RT is the amount that catalyzes incorporation of 1 nmol of dTMP per h into poly(rA)-oligo(dT)10 at 1505
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ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Inhibition of FIV and HIV RTs by analogs of dTTP Mean K. + SD
Inhibitor'
(nM)b HIV
FIV
N3dTTP ddTTP D4TTP 3'-F-dTTP 3'-NH2-dTTP
3.3 6.7 1.8 3.1 1.6
± ± ± ± ±
1.6 3.4 0.7 0.1 0.5
(6) (5) (6) (3) (3)
6.5 5.9 8.3 5 6
± ± ± ± ±
1.8 (4) 4.2 (4) 0.8 (6) lc 2c
Mean
KIKm ratio
FIV
0.0010 0.0020 0.0005 0.0009 0.0005
HIV
0.0011 0.0010 0.0014
TABLE 3. Inhibition of FIV RT by PFA Template-primer
Substrate
Mean Ki -+- SD (>LM)a
poly(rA)-oligo(dT)jO
dTTP dGTP dCTP
0.18 + 0.05 (8) 2.2 ± 0.6 (4) 2.6 ± 1.6 (3)
poly(rC)-oligo(dG)1O poly(rI)-oligo(dC)1O
a Numbers in parentheses indicate numbers of determinations.
0.0010C 0.0012C
a N3dTTP, 3'-Azido-3'-deoxythymidine 5'-triphosphate; D4TrP, 2',3'-dideoxy-2',3'-didehydrothymidine 5'-triphosphate; 3'-F-dTTP, 3'-fluoro3'deoxythymidine 5'-triphosphate; 3'-NH2-dTTP, 3'-amino-3'-deoxythymidine S'-triphosphate. b Numbers in parentheses indicate numbers of determinations. c From Cheng et al. (3).
37°C. Kinetic parameters (Km and K,) were determined as previously reported (12), by using intercept values calculated from double-reciprocal plots. Ratios of Ki to Km were calculated by using the mean values of Ki and Km. RESULTS AND DISCUSSION Inhibition of FIV and HIV RTs by analogs of dTTP. We examined inhibition of FIV RT by five analogs of dTTP, and for three of these, inhibition of HIV RT was also examined (Table 1). These experiments were performed with poly(rA)oligo(dT)1o as the template-primer; to enable direct comparisons, the assay conditions were identical for the two enzymes. Double-reciprocal plots of our data demonstrated that each of these analogs inhibits in a manner that is competitive with respect to dTTP. The FIV and HIV RTs were similar in sensitivity to 3'-azido-3'-deoxythymidine 5'-triphosphate, ddTTP, and 2', 3'- dideoxy - 2', 3'- didehydrothymidine 5'- triphosphate. The K, values determined for inhibition of the two enzymes ranged from nearly identical values obtained for inhibition of the two enzymes by ddTTP to a 4.6-fold difference for inhibition by 2',3'-dideoxy-2',3'-didehydrothymidine 5'-triphosphate (lower for FIV RT). The Km values for dTTP in the uninhibited reactions were 3.4 + 0.2 ,M for FIV RT and 6.0 + 1.9 ,uM for HIV RT. This is in agreement with our previous report that FIV RT has a slightly lower Km for dTTP in this reaction than does HIV RT (11). Comparison of the K/lKm ratios revealed that the difference between the two enzymes in inhibition by any of these three analogs was less than threefold (Table 1). We examined two other analogs of dTTP, 3'-fluoro-3'deoxythymidine 5'-triphosphate and 3'-amino-3'-deoxythymidine 5'-triphosphate, which were previously shown to inhibit HIV RT (3). These also inhibited FIV RT, and the K,/Km ratios obtained were very similar to those reported by Cheng et al. for inhibition of HIV RT (3). Inhibition of FIV RT by dideoxynucleoside 5'-triphosphates. FIV RT utilizes poly(rC)-oligo(dG)j0 and poly(rI)oligo(dC)10 as template-primers under the assay conditions
used, but it does not use poly(rG)-oligo(dC)10 or poly (rU)-oligo(dA)10 (11). We used the former two templates to
we
analyze inhibition of FIV RT by ddGTP and ddCTP, respectively, and to compare these with inhibition by ddTTP. All three of these dideoxynucleotides were competitive inhibitors of FIV RT; the values determined for Ki and K/,Km are shown in Table 2. Of the three, ddGTP was the most potent inhibitor and ddTTP was only slightly less effective, whereas ddCTP was considerably less potent than the other two when assayed under these conditions. We do not believe that the lower activity of ddCTP was due to use of poly(rI)oligo(dC)10 in these assays, because the Km for dCTP in this reaction was only slightly higher than the Km for dTTP in the reaction with poly(rA)-oligo(dT)10. This relative order of activity is similar to that reported by Chen and Oshana (2) for inhibition of HIV RT. However, absolute values of the inhibition constants we determined are different from those of Chen and Oshana, possibly because of different assay conditions. Inhibition of FIV RT by PFA. We also examined inhibition of FIV RT by PFA with each of the three templates described above. PFA was previously shown to be a noncompetitive inhibitor of partially purified FIV RT when assayed with poly(rA)-oligo(dT)10 (12) and also of HIV RT (16). Inhibition of purified FIV RT by PFA was also noncompetitive with the other template-primers, poly(rC)-oligo (dG)1o and poly(rI)-oligo(dC)10 (data not shown). Ki values for inhibition of the three reactions by PFA are shown in Table 3. These data show that PFA was considerably more potent as an inhibitor of the reaction with poly(rA)oligo(dT)10 than with either of the other two templateprimers. We also made a direct comparison of the PFA sensitivities of FIV and HIV RTs in the reaction with poly(rA)-oligo(dT)10. The Ki values for inhibition of the FIV and HIV RTs were 0.18 and 0.72 ,uM, respectively. This value for FIV RT is slightly lower than that which we previously determined with the partially purified enzyme (12). The value we obtained for inhibition of HIV RT is slightly higher than the value reported by Vrang and Oberg, who used different assay conditions (16). The availability of FIV and HIV RTs in pure form has enabled direct comparisons of their sensitivities to inhibitors. These direct comparisons confirm the earlier conclusions that the FIV and HIV RTs are very similar in sensitivity to the active forms of several important antiviral agents and that FIV may be useful as a model for studies of AIDS chemotherapy. These data also provide basic infor-
TABLE 2. Inhibition of FIV RT by dideoxynucleoside 5'-triphosphates
Mean Km ± SD Mean K__± SD Substrate Template-primer ~~~~(nM)a (~~~LM)' Inhibitor 3.4 ± 0.1 (10) dTTP ddTTP 6.7 + 3.3 (6) poly(rA)-oligo(dT)jO dGTP 1.1 ± 0.4 (5) ddGTP 1.2 ± 0.6 (8) poly(rC)-oligo(dG)jO dCTP 5.8 ± 0.3 (3) ddCTP 101 ± 39 (5) poly(rI)-oligo(dC)10 a Numbers in parentheses indicate numbers of determinations.
K/Km ratio 0.0020 0.0011 0.0176
VOL. 34, 1990
mation needed before initiation of chemotherapeutic studies of FIV infection in cats. We have identified several antiviral compounds whose active forms are potent inhibitors of FIV RT. Work is in progress to evaluate the activities of their corresponding nucleosides against replication of FIV RT in cell culture. We believe that the FIV systems will enable coordination of in vivo and in vitro experiments and that these studies can provide information that will be important in the design of chemotherapeutic strategies for AIDS in humans. On the basis of the results of this investigation and the proven anti-HIV activities of 3'-azido-3'-deoxythymidine (4, 10) and 2',3'-dideoxy-2',3'-didehydrothymidine (1, 6, 8, 9), these two compounds look particularly attractive for further studies with the FIV model. ACKNOWLEDGMENTS This work was supported by Public Health Service grant Al 28189 from the National Institute of Allergy and Infectious Diseases. We thank Niels C. Pedersen and Y.-C. Cheng for much helpful discussion. LITERATURE CITED 1. Baba, M., R. Pauwels, P. Herdewijn, 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 replication in vitro. Biochem. Biophys. Res. Commun. 142: 128-134. 2. Chen, M. S., and S. C. Oshana. 1987. Inhibition of HIV reverse transcriptase by 2',3'-dideoxynucleoside triphosphates. Biochem. Pharmacol. 36:4361-4362. 3. Cheng, Y.-C., G. E. Dutschman, K. F. Bastow, M. G. Sarngadharan, and R. Y. C. Ting. 1987. Human immunodeficiency virus reverse transcriptase. General properties and its interaction with nucleoside triphosphate analogs. J. Biol. Chem. 262: 2187-2189. 4. Fischel, M. A., D. D. Richman, M. H. Grieco, M. S. Gottlieb, P. A. Volberding, 0. L. Laskin, J. M. Leedom, J. E. Groopman, D. Mildvan, R. T. Schooley, G. G. Jackson, D. T. Durack, D. King, and the AZT Collaborative Working Group. 1987. The efficacy of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS-related complex. N. Engl. J. Med. 317: 185-191. 5. Gardner, M. B., and P. A. Luciw. 1989. Animal models of AIDS. FASEB J. 3:2593-2606. 6. Hamamoto, Y., H. Nakashima, T. Matsui, A. Matsuda, T. Ueda, and N. Yamamoto. 1987. Inhibitory effect of 2',3'-didehydro2',3'-dideoxynucleosides on infectivity, cytopathic effects, and replication of human immunodeficiency virus. Antimicrob.
FIV REVERSE TRANSCRIPTASE
1507
Agents Chemother. 31:907-910. 7. Hizi, A., C. McGill, and S. H. Hughes. 1988. Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants. Proc. Natl. Acad. Sci. USA 85:1218-1222. 8. Lin, T.-S., R. F. Shinazi, and W. H. Prusoff. 1987. Potent and selective in vitro activity of 3'-deoxythymidine-2'-ene (3'deoxy-2',3'-didehydrothymidine) against human immunodeficiency virus. Biochem. Pharmacol. 36:2713-2718. 9. Mansuri, M. M., J. E. Starrett, I. Ghazzouli, M. J. M. Hitchcock, R. Z. Sterzycki, V. Brankovan, T.-S. Lin, E. M. August, W. H. Prusoff, J.-P. Sommadossi, and J. C. Martin. 1989. 1-(2,3Dideoxy-1-D-glyceropent-2-enefuranosyl)thymidine (d4T). A highly potent and selective anti-HIV agent. J. Med. Chem. 32:461-466. 10. Mitsuya, H., K. J. Weinhold, P. A. Furman, M. H. St. Clair, S. N. Lehrman, R. C. GaUo, D. Bolognesi, D. W. Barry, and S. Broder. 1985. 3'-Azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphoadenopathyassociated virus in vitro. Proc. Natl. Acad. Sci. USA 82: 7096-7100. 11. North, T. W., R. C. Cronn, K. M. Remington, R. T. Tandberg, and R. C. Judd. 1990. Characterization of reverse transcriptase from feline immunodeficiency virus. J. Biol. Chem. 265:51215128. 12. North, T. W., G. T. North, and N. C. Pedersen. 1989. Feline immunodeficiency virus, a model for reverse transcriptasetargeted chemotherapy for acquired immune deficiency syndrome. Antimicrob. Agents Chemother. 33:915-919. 13. Pedersen, N. C., E. M. Ho, M. L. Brown, and J. K. Yamamoto. 1987. Isolation of a T-lymphotropic virus from domestic cats with an immunodeficiency-like syndrome. Science 235:790-793. 14. Schinazi, R. F., B. F. H. Eriksson, and S. H. Hughes. 1989. Comparison of inhibitory activities of various antiretroviral agents against particle-derived and recombinant human immunodeficiency virus reverse transcriptases. Antimicrob. Agents Chemother. 33:115-117. 15. Take, Y., Y. Inouye, S. Nakamura, H. S. Aflaudeen, and A. Kubo. 1989. Comparative studies of the inhibitory properties of antibiotics on human immunodeficiency virus and avian myeloblastosis virus reverse transcriptases and cellular DNA polymerases. J. Antibiot. 42:107-115. 16. Vrang, L., and B. Oberg. 1986. PP1 analogs as inhibitors of human T-lymphotropic virus type III reverse transcriptase. Antimicrob. Agents Chemother. 29:867-872. 17. Yamamoto, J. K., E. Sparger, E. W. Ho, P. R. Andersen, T. P. O'Connor, C. P. Mandell, L. Lowenstine, R. Munn, and N. C. Pedersen. 1988. Pathogenesis of experimentally induced feline immunodeficiency virus infection in cats. Am. J. Vet. Res. 49:1246-1258.
