COPY VIROLOGICAL ASPECTS OF HIV-1 INFECTION

Identification of mutations that encode drug resistance in the polymerase gene of the human immunodeficiency virus ZHENGXIAN Gu MD, HENGSHENG FANG MD, HORACIO SALOMON PhD, Q IN G GAO MD, MARK A W AINBERG PhD

Z Gu, HFANG, H SALOMON, Q GAO, MA WAINBERG. Identification of mutations that encode drug resistance in the polymerase gene of the human immunodeficiency virus. Can J Infect Dis 1994;5(Suppl E):29E-33E. In vitro selection in MT-4 cells was used lo generate human immunodeficiency virus-type 1 (111v 1) vai·iant.s lhal are resislanl lo 2',3'-clicleoxycylidine (ddC). 2'.3'-diclcoxyinosinc (ddl) and lhc (-) cnanliomer of 2' .3' -dicleoxy-3' -lhiacylidine (3TC). The comp le le reverse l1·anscriplase open reading frames of lhesc viruses. a nd portions of flanking prolease a nd inlegrase wilhin lhe pol gene. were cloned and sequenced by polymerase chain reac tion (PC'R) lechniques. Mulalions were observed al each of am ino acid s ilcs 65 (Lys -, Arg: AAA -, AGA) and 184 (M el -, Va l: ATG -, GTG) when dclC was used in lhis prolocol. and al s ite 184 only when either 3TC' or cldl was employed. These mulalions were inlroduced into lhe pol gene of infectious rcco mbinanl HXB2-D DNA by sile-direcled mutagenesis lo confirm. by viral replicalion assay, lheir importance in conferring res islance against lhese drugs. A recombinant virus conlaining the silc 65 mutation only possessed greater lhan IO-fo ld resistance againsl dclC co mpared wilh parental HXB2- D. Moreover. cross-resistance of about 20-folcl a nd lhreefolcl . respectively. was cleleclable against 3TC and dell but not against 3'-azido-3'-deoJs.')'1.hym idine (AZr). When U1c 65 and 184 mulalions were combined into I LXB2-D . lhe resulta nt conslruct did not possess highe r levels of resislance lo any of these drugs lhan observed wilh lhe silc 65 or 184 mulalion alone. These mu la lions were furth er demonslraled by PCH analysis of periph eral blood mononuclear cells from l O patients on long term ddC therapy. a IU1 ough variab le pallcrns were observed in le nns of which of lhe lwo mulalions or bolh were present. Sometimes . lhe wi ld-type s ile 65 codon was a lso d elecled. indicating lhc presence of mixtures of viral quasi -sp ecies. Direct cloning and sequ encing revealed lhe s ile 65 mutation in viruses isola ted from palienls on prolonged ddC lherapy. (Pour res ume. uoir page 30E)

Key Words: 3TC. AZI'. ddC. ddl. Human immunodeficiency uirus-type 1. Res istance

Lacly Dauis Institute-Jewish General Hospital and McGill Uniuersily AJDS Centre. Montreal. Quebec Correspondence and reprints: Dr Zhengxian Gu. McG ill Uniue,·sity AIDS Centre. J ewis h General Hospital. 3755 Chemin Cote Ste-Catherine. Montreal. Quebec 113T 1E2

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Identification des mutations codant la resistanc e aux medicaments dons le gene de la polymerase du virus de l'immunodeficience humaine RESUME : La selecUon in vitro de ccllules MT-4 devail gene1-cr des varianles du virus de l'immunodC'ficience

humaine de type I (v111 - l) qui soicnl resislanles a la 2'.3'-dideoxycyUdine (ddC). a la 2' .3'-dideoxyinosinc (ddl) el a l'cnanliomerc de la 2'.3' -didem,-y-3'-lhiacylidine (3TC). Les cadres de lecture ouvcrls des transcriplascs inverses completes de ces vii-us cl !es portions des proteases avoisinanles cl de rinlegrase a l'inlcricur du gene pol onl etc clones el mis en sequence a l'aide de techniques de rcacUons de polymerisations en chaine (PCR). Des mutations onl ele obscnrecs pour chaquc silc des acides amines 65 (Lys ~ Arg: AM ~ AGAJ el 184 (Mel ~ Val: ATG ~ GTGJ lorsque le ddC a cle utilise dans ce prolocolc el au site 184. sculcmcnl lorsque soil le 3TC ou le dell onl ele cmployes. Ccs mutations onl ele inlroduilcs dans le gene pol de l'ADN recombinant I !XB2-D par une mutagcnese dirigcc scion la localisation af1n de con nrmcr par replication viralc leur crtkacile a conlere1- une resistance a l'endroil de ccs medicaments. Un vii-us recombinant rcnfennanl unc mutation au site 65 ne possedail qu\mc resistance decuplee a l'endroit du ddC en comparaison avcc le HXB2- D parental. De plus. une resistance croisce multipliec par des faclcurs de 20 cl de 3 respectivemcnl a ele decelee a l'cndroit du 3TC ct du dell el non pas a l'endroil de la 3'-azido-3'-dcoxyU1 imidine (AZr). Lorsquc les mutations 65 e l 184 onl etc combinees au t lXB2- D. la slrnclure resultanle nc s·esl pas revelee posscder des laux de resistance a l'un ou l'aulre de ces medicaments plus elevcs que ccux obsenres aux sites 65 cl 184 des mulalions seulcs. Ccs mutations onl en oulre etc connrmees par analyse Pc11 des ccll ulcs sanguines mononuclcaircs pc,;phcriques de 10 patients sous lrailemenl prolonge par ddC. bicn qu·une cerlainc variabilile ail ele observee quanl a la presence de l'unc ou de l'aulre ou encore des dcux mutations. Parfois. un codon du site 65 de type sauvage a cgalement etc deccle. ind iquanl la presence de melanges de quasi -cspeccs viralcs. Le clonage direct el le scqucrn;agc ont revele des mutations au site 65 clans des virus isoles de patients sous lrailemenl prolongc par ddC.

D

RUG -RE:SIST/\NT Vi\RIANTS OF IIIV ARE COMMONLY PREsent in the circulation of patients receiving prolonged chemotherapy with nucleoside compounds lhal block viral reverse transcriplase (rn·) activity (1-3). Resistant 111v variants can also be selected in tissue culture by gradually increasing the concentrations of antiviral drugs in the medium (4.5). Resistance has also been demonslraled against non-nucleoside antagonists of viral Rl' (6. 7). Although U1e appearance of drug-resistant viruses may be predictive of clinical progression. it is still unclear whether treatment failure is causally related to the emergence of viral drug resistance (8-10). Nucleosicle antagon ists of the 111v RT act by blocking lhe synthesis of provira l DN/\ (11). Since the IIIV RT is error-prone, mutations occur frequently in the viral genome during replication (12). Thus. mutations that confer drug resistance are amplified under drug pressure. Mutations in the pol genes of 111v -1 strains that arc resistant to 3'-azido-3'-deoxythymicline (AZT) (13). 2' ,3'dideo:>..-yinosine (ddl) (3, 14). 2',3'-dideoxy-3'-thiacylidine (3TC) (15, 16) and 2'.3'-clicleoxycyt.icline (ddC) (17) have been identified. ddC has been used extensively to treat 111v-infecled individuals who are intolerant to either i\ZT or ddl; it is also used in combination \.vith AZT Lo try to prevent or delay the development of drug resistance (18). dell is being used increasingly Lo treal patients who have previously received AZT for one year or more, and 3TC is a promising drug now in clinical trials. It is important to ask whether these compounds will be likely to encounter problems of drug resistance and Lo identify the mutation sites responsible. This paper reports on novel mutations at siles 65 and 184 oflhe 111v-1 RT U1at confer resistance against both ddC and 3TC. and lo a lesser 30E

cxlenl against ddl. depending on the type of host cell employed in the drug sensitivity analysis.

MATERIALS AND METHODS Ce lls an d viruses: MT-4 cells were used lo propagate both wild -type and resistant variants of 111v-1 in suspen sion culture in RPMI-1640 medium (Gibco Laboratories). supplemented with 2 mM L-glutamine, 100 U/mL penicillin. 100 ~tg/mL streptomycin, and 10% fetal calf serum (Flow Laboratories). Variants resistant lo each or cldC. dell and 3TC lhal were selected under conditions of in vitro passage, as well as the 11iv-111s laboratory strain of 111v-1 (supplied by Dr RC Gallo, National Institutes of Health. Maryland) were studied extensively (15). The I lXB2-D clone of infectious DNA was used as a control for assaying the viral replication (19). Five micromolar cldC (Sigma Laboratories Inc, Missouri) was routinely used for propagation of viruses possessing a clc!C-resistance phenotype (approximately 10-fold tile usual 50% effective concentration [E:C50] for ddC-sensilivc strains). Other procedures have been previously described (14.15). Viruses that had inilially been grown in MT-4 cells were passaged onlo phytohemagglutinin (Pl Ii\) pres timulated cord blood lymphocytes (CBL) (donated by the Department of Obstetrics of the authors' hospital) or peripheral blood mononuclear cells (PBMC) donated by healthy individuals (14). Samples of Cl3L or PBMC (5x 105 cells/mL) were pretreated with concentrations of ddC between O and 50 µM for 4 h and inoculated with CBL-grown 11iv-1 at a multiplicity of infection or 1.0 (as determined by plaque assay on MT-4 cells). using the same concentration of drug as used for pretreatment. Fresh medium, containing ddC at appropriate concen -

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Mutations that encode HIV drug resistance

TABLE l Drug sensitivity of HIV- 1 variants ECso (pM)

Host cell

Variant

ddC

3TC

MT-4

HXB2-D HXB2-D65 HXB2-Dl84 HXB2-D65+ 184 HXB2-D HXB2-D65 HXB2-Dl84 HXB2-D65+ 184 HXB2-D HXB2-D65 HXB2-Dl84 HXB2-D65+ 184

0.45 5.6 2.5 5.8 0.35 3.9 1.8 4.2 0.4 0.7 0.6 7.8

0.76 18.4 1032 1033 0.9 21 .6

Cord blood lymphocytes

PBMC

552 631 0.7 23.5 478 512

---

ddl

7.0 22.l 37.6 34.2 8.5 24.3 26.5 21.0 6.4 26.5 5.7 21.3

AZT

0.012 0.011 O.Dl5 0.013 0.01 0.03 0.02 0.01 0.02 0.01 0.02 0.04

ECsos were obtained from plots of amounts of p -24 detected in c ulture fluids as a function of drug c oncentration. Eac h value is the average of three separate determinations (± the standard mean deviation)

trations. was added three times per week along with fresh PHA prestimulated CBL. Similar studies were perfom1ed with ddl and 3TC at concentrations between 0 and 500 µM , and AZT at concentrations between O and 10 µM. Cloning and sequencing: Cellular DNA was obtained from about 2xl06 MT-4 cells that had been infected with ddC-resistant variants of 1-11v-1 derived from 1-1rv-n1s by tissue culture selection as described (14). Polymerase chain reaction (PCR) methodology was used to amplify a 1742-bp segment containing the complete RT-coding sequence plus portions of the 3' end of protease and the 5' end of integrase (14). Site-directed mutagenesis: The construction of mu tated HXB2-D containing either U1e codon 65 (Lys ~ Arg) or 184 (Met ~ Val) mutations of the RT was as described to produce HXB2-D65 and HXB2-D184 (14). We also generated a recombinant that contained both these changes termed HXB2-D65 + 184. The presence of these substitutions was confirmed by DNA sequencing. Transfections and viral resistance assays: MT-4 cells were transfected by electroporation, and fresh MT-4 cells were added to cultures as soon as cytopathic effects were seen to generate viral stock. Culture fluids were clarified and frozen at -70°C until study. Assays of HIV susceptibility to drugs, RT assays and p-24 antigen capture assays were performed as described (20). Viruses were isolated from patients on prolonged nucleoside therapy. using a protocol similar to that recently published (21), except that CBL were used in place of adult PBMC as Hrv targets. Calculation ofEC50 values was on the basis ofp-24 antigen levels in culture Ouids (14).

RESULTS Cloning and sequencing of ddC-resistant HTV-IIIB, selected in tissue culture, revealed that mutations at codons 65 (Lys ~ Arg) and 184 (Met ~ Val) were CAN

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frequently present. Similar findings were obtained using 1-1rv-1 selected for resistance against ddl and 3TC under in vitro conditions. Site-directed mutagenesis was employed to determine tl1e potential biological significance of these mutations. Arg-encoding AGA was introduced in place of AAA at position 65 of the RT gene of IlXB2-D to yield the recombinant clone HXB2-065. In addition. a construct, HXB2-065+184, was generated that contained both this change as well as the 184 alteration associated with high level resistance against 3TC and low level resistance against both ddl and ddC (14-16). Drug susceptibilities of each of these constructs were initially assessed by viral replication in MT-4 cells. EC50 values were determined from levels of p-24 antigen in culture fluids. Table 1 demonstrates that the AGA substitution at position 65 caused a significant diminution in susceptibility to both ddC and 3TC. However. l-1XB2D65+ 184 did not possess higher levels of drug resistance than those obtained with either mutation alone . Similar observations were obtained on the basis of RT levels in culture fluids as well as indirect immunoOuorescence assays for p-24 antigen in infected cells (not shown). Table 1 presents a summary of findings that show that the Arg-65 mutation caused a more than 10-fold loss in sensitivity to ddC and 20-fold resistance against 3TC, without affecting sensitivity to AZT. A small degree of cross-resistance to ddl (approximately threefold) was also noted . In contrast, the site 184 mutation conferred resistance levels against 3TC of 500 to 1000 times, but only low level resistance (approximately fivefold) against each of ddl and dc!C . Furthermore, certain of these resistance patterns were dependent on the host cell type used for each assay. While the site 65 mutation conferred resistance to both ddC and 3TC in each of MT-4 cells, CBL and PBMC, the site 184 mutation d id not result in diminished sensitivity to ddl and ddC in PBMC. 31E

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TABLE 2 Information on patients who received ddC Patient number

Months treated with ddC

Genotype codon 65

codon 184

WT

mutated

WT

+

+

+

+

+

+

+

+

6

+

+

6

+

6

0

l

15

2

7

3

11

4 5

codon 69

mutated

WT

mutated

1.9

+ +

+ +

+

+ +

+

ECso for ddC

+

1.6

+

2.1

+

5.3

+

2.2

+

0.4

ND

7

18

+

+

8

2

+

+

9

10

+

+

10

0

+

+

+

0.4

11

16

+

+

+

0.6

12

5

+

+

13

8

+

+

+ +

+

0.2

+

ND

+

+

0.2

ND

WT Wild-type; ND No t determined

Table 2 includes dala on lhe therapeutic regimens received by 11 ddC-lrcaled and lwo unlrealed patients. and the detection of resistance-conferring mulalions as analyzed by PC H. All palienls had received AZT for vari ous limes before changing lo ddC for reasons of AZT intolerance or lrealmenl failure. Prelrealmenl isolates from these individuals were nol available. Viruses from palienls whose cells contained lhe sile 65 mulalion were resislanl lo ddC. as delermined in tissue cullure CBL assays. The range of EC50 values for these isolates. ie, approximately five lo 15 limes above average. arc consislenl with previous studies on resistance to dclC ( 17). Sequencing of isolates from palienls on prolonged ddC confirmed the presence of the sile 65 mulalion. All samples were also lesled by PCR lo clelccl lhe Val- 184 subslilulion. Four samples possessed Val - 184 and one possessed bolh Arg-65 and Val - 184. None of lhe sam ples contained a previously described Asp-69 substitu tion.

DISCUSSION resistance to antivira l compounds has become an important issue, and is clue lo lhe inficlelily of the 111v 1 RT and drug selection pressure. Mutations in the RT gene U1al encode resistance lo each of AZT. dell. clclC, 3T C and olhcr drugs have been described ( 13- 1 7). Sometimes. combinations of mutations may yield higher resistance levels lhan obtained wilh single mutations alone. while in olher cases such combinations may help lo restore sensitivity lo AZT (3.22). We have shown lhal mutation al codon 65 (Lys -. Arg) and 184 (Mel -. Val) are associated wilh JJIV resistance lo clclC and 3TC. A mutation responsible for resistance lo cldC has been previously identified al codon 69 (14.17). Interestingly, bolh lhe 65 and 69 mutations are localed in lhe 65 lo 70 am ino acid sequence of lhe viral rn. known lo be associated wiU1 lhe active site of this 111v

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enzyme. Monoclonal anlibodie against 111v RT enzy matic activity map lo this region (23). Bolh dclC and 3T possess 2',3'-dideoxy moieties. which may account for lhe cross-resistance between these drugs reported here. Consislenl with lhis is lhal neither the Arg-65 nor Val- 184 ub lilutions are present in 111v variants selected in AZT. A mulalion al Val-74 thal confers resistance against dell and crossresislance lo ddC has a lso been described (3). l 1XB2-D. grown in CBL. onlinued lo po sess the codon 65 or 184 mulalions and lo maintain resistance lo bolh ddC and 3TC. Ilence, lhis mutation can persist in cells other than MT-4. However. resistance against ddC and dell cou ld no longer be deleclcd when 1IXB2D 184 was grown in Pl3MC. indicating lhe hosl celldependenl nature of some of these results. Palicnls whose isolates contained lhe ddC 65 mulalion had a ll received therapy with ddC for al least six months. Similar results were obtained wilh each of 11 individu als. although an absence of the ddC 65 mulalion was noted in s ven other cases who a lso received prolonged cldC. Since neither U1e Val-184 nor Arg-65 ubslilulions conferred resistance lo AZT, these mulalions may affect different regions of lhe RT. Lys-65 is on lhe 'fingers· ubclomain of lhe RT crystal structure, llioughl to play a role in enzyme-template interaction, while Mel- 184 is on lhe 'palm' region related lo primer-template binding aclivily (24) . This may be why U1ese lwo mutation . in concert. did nol yield synergistic or antagonistic effects with regard lo resistance lo ddC and 3T . We used specific PCR to amplify the Arg-65 substilu lion from clinical isolates obtained from 10 111 v I in fected palienls on prolong cl dd therapy. The Sile 65 mulalion was present in each of four such individuals and absenl in U1e other six. Of the four people who were positive for the Arg-65 codon, lhree contained viruses

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12. 13.

14.

ACKNOWLEDGEM ENTS: This research was supported by grants from I lcallh and Welfare Canada and the Medical Re search Council of Canada. We thank Ms F' Busschacrt for preparation or the manuscript.

15.

REFERENCES l. Larder B. Darby G. Richman DD. I !IV with reduced sensitivity lo zidovudine (AZT) isolated during prolonged therapy. Science 1989:243: 1731 -4. 2 . Rooke R. Tremblay M. Soudcyns I-I. cl al. Isolation of drug-resistant va1ianls of I IIV- 1 from pa lien ls on long-lem1 zidovudinc (AZT) therapy. /\IDS 1989:3:41 1-5. 3. St Clair Ml I. Marlin JL. Tudor-Williams G. cl al. Resistance lo dell and sensitivity lo AZT induced by a mutation in HIV- I reverse lransc1iplase. Science 1991 :253: 1557-9. 4 . Larder BA. Coates 1,E. Kemp SD. Zidovudine- rcsistanl human immunodcl1eiency virus selected by passage in ecll culture. J Virol l 991:65:5232 -6. 5. Gao Q. Gu Z. Pamiak MA. Li X. Wainberg MA. In vitro sclcelion or va1·ianls of human immunodefieieney vi1·us type I resistant lo 3'-azido-3'-dcoxylh_vmidinc and

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2'.3'-didcoxyinosinc. J Virol 1992:66: 12-9. NunbcrgJII. SchlcifWA. Boots EJ. cl al. Viral resistance to human immunodeficiency virus type 1-spccif1c pyridinonc reverse lranscriplasc inhibitors. J Virnl 1991 :65:4887-92. Richman D. Shih C - K. Lowy I. et al. I l uman immunodcfkicncy virus type l mutants resi s tant lo nonnuclcoside inhibitors or reverse lransn·iptasc arise in tissue culture. Proe Nall Acad Sci USA 1991 :88: 11241 - 5. Tudor-Williams G. St Clair Ml I. McKinney RE. cl al. HIV- I sensitivity to zidovudinc and clinical outcome in children. Lancet 1992:339: 15-9. Monlancr JSG. Singer J , Schechter MT. cl al. Clinical correlates or in vitro I-IJV- 1 1·csislancc to zidovudinc. Results of the Mulliccntrc Canadian AZr trial. AIDS 1993:7: 189-96. Kozal MJ. Shafer RW. Winters MA . Kalzenslcin DA. Merigan TC. A mutation in human immunodeficiency virus reverse transcriptasc and decline in CD4 lymphocyte numbers in long-lem1 zidovudine recipients. J In feel Dis 1993: 167:526-32. F'Lll·man PA. F'yfc JA. Sl C lair Ml-I. cl al. Phosphorylation of 3' -azido-3'-deoi-.ylhymidine and selective interaction of lhe 5' -lriphosphalc wilh human immunodefkicncy virus

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~ i o n s that encode HIV drug resistance

reverse transcriplasc. Proc N.:111 Acad Sci US/\ l 986:83:8333 7. Preston BD . Poicsz l3J. Loeb LA. F'idclily of l l!V 1 rewrsc lrans(-i-iplase. Science 1988:242: 1 l 68 71. Larder BA. Kemp SD. Mulliplc mulalions in I IIV- 1 reverse lranscriplasc confer high -level resistance to zidovudinc (AZT). Science 1989:246: I I 55-8. Gu Z. Gao Q. Li X. Parniak Ml\. Wainbcrg Ml\. Novel mutation in the human immunodeficiency virus type reverse transcriptasc gene that encodes cross resistance lo 2'.3' -clidcoxyinosinc and 2'.3' didcoxyeyticlinc. J Virol 1992:66: 7128 -35. Gao Q. Gu Z. Parniak MJ\. ct al. The same rnutalion !!tat encodes low-levcl human immunoclcfkicncy virus type I resistance to 2' 3' -dicleox-yinosinc ancl 2' 3' -dicleoxy cyticlinc conlc1·s high -level resistance to the (- ) cnantiomcr or 2'.3' dideoxy -3' -t hiacyt icline. 1\111 i111icrob Agents Chcrnothcr 1993:37: 1390-2 . Shinazi RF'. Lloyd RM Jr. Nguyen M -11. cl al. Characterization or human immunoclclkiency viruses resistant lo oxalhiolanc -cytosinc nuclcosicks. /\ntimicrob Agents C hcmot her 1993:37:875 81. Fitzgibbon JG. I lowcll RM. I labc1·zctlle Cl\. Sperber SJ. Gocke DJ. Dubin DT. I luman immunodeficiency virus type I pol gene mutations which cause clccn' asccl susceptibility lo 2'.3' didcoxycylidinc. l\ntimicrob Agents Chcmothc r 1992:36: 153-7. Meng T -C. Fish! MA. 13oota MM. cl al. Combination therapy with ziclov11dinc and clidcxoycytidine in patients with advanced human imm1111odcl1cicncy virus infection. A phase 1/11 study. Ann Intern Med 1992:116:13-20. F'ishcr AG. Collalti E. Ratner L. Gallo RC. Wong Staal F' . J\ molecular clone of I ITLV- 111 with biological activity. Nature 1985:316:262-5. Rooke R. Tremb lay M. Wa i nberg MA. J\7;r (ziclovuclinc) may act poslintegrationally to inhibit generation of I !IV- I progeny virus in chronically inlcctccl cells. Virology 1990: 176:205- 15. Japour AJ. Mayers DL. Johnson VI\. cl al. Stanclarclizcd peripheral blood mononuclear cell culture assay fo1· determination of drug susccplibililics or c linical human immunoclcficicncy virus type l isolates. Antimicrob Agrnl s Chcmolher 1993:37: I 095 - 10 I. Tisdale M. Kemp SD. Pan-y NR. Larclc1· 13A. Rapid in vitro sclcclion or human immunodcl1cicney virus type I resistant to 3 '- thiacytidine inhibitors clue lo a mutation in the YMDD region of 1-cvc1·sc lranscriptasc. Proc Nall l\cacl Sci USA 1993:90:5653-6. Wu J, Amandoron E. Li X. Wainbcrg MA. Parniak Ml\. Monoclonal ant ibody- mcdialecl inhibition of I IIV- 1 reverse lransc1·iptase polymerase act ivily. J 13iol Chem 1993:268:9980-5. Kohlslaedl LA. Wang J. F1·ieclman JM, Rice Pl\. Steitz Tl\. C1-ystal sln.1clurc at 3.5 A resolution or I IIV- 1 reverse transc1·iptasc complcxccl with an inhibitor. Science 1992:256: l 783 -90.

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