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1278
Predominance of Codon 215 Mutation in Reverse
Transcriptase-Coding Region of 3'-azido-3'-
deoxythymidine (AZT)-Resistant HIV-1
Isolates after Long-Term
AZT Therapy
Makiko KONDO
Department of Virology, Kanagawa Prefectural Public Health Laboratory,Department of Bacteriology, Yokohama City University, School of Medicine
(Received: June 16, 1995)(Accepted: August 7, 1995)
Key words: AZT, drug resistance, HIV-1, PCR, reverse transcriptase
Abstract
Among human immunodeficiency type 1 viruses (HIV-1) isolated during long-term 3'-azido-
3'-deoxythymidine (AZT) therapy, 4 condons (Asp67, Lys70, Thr215, and Lys219) in the HIV-1 reversetranscriptase (RT)-coding region have been considered to be related to the AZT resistance of HIV-
1. Therefore we determined these mutation patterns in HIV-1 isolates from patients undergoing
long-term AZT treatment. In 41 clones of HIV-1 from 7 patients, the Thr215 mutation was the most
predominant (97.6%), and more frequent than Asp67 (48.8%), Lys70 (31.7%) and Lys219 (9.8%)mutations.
All 22 clones from isolates cultured in the presence of 1 ,u M AZT showed Thr215 mutation; such
a high frequency was not found for the other 3 codon mutations.In a clinical follow-up study, Thr215 mutation appeared in the late stage of AZT treatment in
parallel with the emergence of AZT insusceptibility. It is worthnoting that this Thr215 mutation toTyr or Phe is a 2-nucleotide mutation in contrast to the 1-nucleotide mutations seen in the other 3codons. Isolates with the single amino acid change of Thr215 of the RT-coding region obtained after
long-term AZT treatment grew in the presence of 1 ,u M AZT. This single amino acid mutation in the
Thr215 codon is the most important factor in AZT resistance.
Introduction
For prevention and therapy of acquired immunodeficiency syndrome (AIDS), the hypervariability
of human immunodeficiency type 1 virus (HIV-1) has raised many obstacles. Hypervariability in the
envelope coding region has hindered the development of an HIV vaccine and immunoprophylacticmeasures. Hypervariability in the reverse transcriptase (RT)-coding region has raised concerns
regarding long-term anti-retroviral therapy with 3'-azido-3'-deoxythmidine (AZT). Because the
RT-coding region of HIV-1 is the target of nucleoside analogues such as AZT, multiple mutations in
HIV-1 RT are thought to be correlated with a high level of resistance to AZT. Comparativenucleotide sequence analysis of the RT-coding region from AZT-sensitive and AZT-resistant HIV-1
Correspondence to: Makiko KONDO
Department of Virology, Kanagawa Prefectnral Public Health Laboratory, 52- 2 Nakao-cho Asahi-ku Yokohama,
241, Japan
感染症学雑誌 第69巻 第11号
Mutation in AZT-resistant HIV-1 1279
isolates identified 4 predicted amino acid substitutions among resistant strains (Asp67•¨Asn, Lys70
→Arg , Thr215→Tyr or Phe, Lys 219→Gln)1)~3).
To determine the mechanism of AZT resistance after long-term AZT treatment in hopes of
improving anti-retroviral therapy, we isolated HIV strains from patients treated with AZT at various
times during therapy and compared the mutation patterns in amino acid residues in the RT region of
those strains with the patterns of the AZT-susceptible wild type HIV-1 strains.
In this paper we report 1) the prevalence of mutation at each of the 4 codons in isolates obtainedafter long-term AZT therapy, 2) amino acid mutation patterns of these HIV-1 isolates grown in the
presence or absence of AZT, 3) comparison of HIV-1 mutations over time, and 4) changes innucleotide mutation patterns in individuals over the course of AZT treatment and the susceptibility
of each isolate to AZT.
Materials and Methods
1) Subjects
The viruses used in this study were obtained from 9 patients, 6 with hemophilia, 2 with heterosex-
ual contact and 1 with unknown risk factor. They were in clinical stages II to IV as defined by the
U. S. Centers for Disease Control and Prevention.
2) AZT treatment
All patients were treated with 400 mg of AZT per day for longer than 6 months, except patient
107 who was treated with 800 mg/day.
3) Isolation of HIV-1
Viruses were isolated from patients' peripheral blood mononuclear cells (PBMC) by co-
cultivation with phytohemagglutination (PHA)-stimulated PBMC from healthy individuals4'5)
4) HIV-1 infection marker
During cultivation, HIV-1 infection of the co-cultured cells was confirmed by 3 steps: (1) syncytial
formation by cultured cells; (2) HIV-1-related antigen appearance, detected by the indirect immuno-
fluorescent antibody test using anti-HIV-1 antibody-positive serum; (3) p24 antigen detection in
culture fluid by ELISA (Abbott).
5) AZT resistance test in vitro
Isolates obtained from patients before AZT treatment, and 2 and more than 6 months after
commencing treatment with AZT were cultured in the presence of AZT (0.1 and 1 ƒÊ M) or in its
absence. After 7 days of cultivation, the culture supernatants were transferred to PHA-stimulated
PBMC from healthy donors. This procedure was carried out 4 times and the cultures were examined
for infection markers.
6) Nested PCR for RT region of HIV-1 isolates
To determine the mutation patterns in the RT-coding region gene, DNA was extracted from the
HIV-1 isolate obtained from each patient, and was amplified by the nested PCR6'7. A region en-
compassing codons 67, 70, 215, and 219 was amplified by using oligonucleotide primers that anneal to
conserved sequences flanking this regin. The first PCR was carried out in 50 ƒÊl of reaction mixture
(200 ƒÊmol each of dATP, dGTP, dCTP, dTTP; 50 mM of KC1; 10 mM of tris-HC1 pH 8.8; 1.5 mM
of MgCl2, and Triton X-100, 0.1%), to which were added 2.5 U of Taq DNA polymerase (Wako) and
0.5 ƒÊmol each of KH55 (5'-GACTCAGATTGGTTGCAGTTT-3') and KH52 (5'-
CTTATCTATTCCATCTAGAAATAGT-3')8' as the first PCR primer. The second PCR was conduct-
ed with 5 ƒÊl of the sample amplified by the first PCR and the second PCR primer, 0.5 ƒÊmol each of
SAl (5'-CCATACAATACTGCAGTATTTGC-3') and SA3 (5'-AGTGCTTTGGATCCTCTAAGGAG-
3')7) in the same reaction mixture. Primers SA1 and SA3 were designed to contain Pst 1 and BamHI
平成7年11月20日
1280 Makiko KONDO
restriction enzyme-susceptible regions respectively. Samples were subjected to 30 cycles of denatura-
tion for 1 minute at 92•Ž, primer annealing for 1.5 minutes at 54•Ž and DNA synthesis for 3 minutes
at 71•Ž with a Taitec Thermo processor TR-100. The PCR products of the RT-coding region were
analyzed on 2% agarose gels and a 705-base pair product was identified by ethidium bromide staining.
7) Sequencing of PCR products
Amplified products of the RT-coding region were digested with restriction enzymes Pst 1 and
BamHI (Takara). The fragments were ligated into bacteriophage M13mp18 DNA predigested with
Pst 1 and Barn HI. The ligation solution was used to transform Escherichia coli (strain TG1).
Recombinant M13 clones containing the RT-coding region were screened for expression of an
approximately 700-base pair RT, and single-stranded DNA was prepared from these constructs for
nucleotide sequencing. The dideoxy nucleotide chain termination method') was used for sequencing
(United States Biochemical Co.).
Results
1) Prevalence of amino acid mutants in the RT-coding region of HIV-1 isolates from patients treatedwith AZT for longer than 6 months.
HIV-1 isolates were obtained from 9 patients treated with AZT for longer than 6 months. From
these, 48 HIV-1 RT-coding clones were obtained and sequenced. Fourty-one of the 48 clones showedmutation in the RT-coding region at 1 or more of codons 67, 70, 215, or 219. These mutants were
isolated from 7 of 9 patients (77.8%).
Among the 41 mutant clones, mutations at codons 67, 70, 215, and 219 were observed in 48.8, 31.7,
97.6 and 9.8 % respectively (Table 1) . It is worthynoting that almost all mutants showed a mutationat codon 215.
2) Amino acid mutation patterns of 22 HIV-1 RT-coding clones from isolates grown in the presence
Table 1 Prevalence of amino acid mutations in the RT-coding region of
HIV-1 isolates from patients with long-term AZT treatment
Table 2 Amino acid mutation patterns of 22 clones from 4 HIV-1 strains grown in the presence of 1,uM AZT for
4 weeks and 17 clones from 4 HIV-1 strains grown without AZT
Underline indicates amino acid mutation
感染症学雑誌 第69巻 第11号
Mutation in AZT-resistant HIV-1 1281
of 1 ƒÊM of AZT for 4 weeks, and 17 HIV-1 RT-coding clones from isolates grown without AZT.
From 4 patients (043-2, 107, 091-1 and 202, treated with AZT for 6, 13, 14, and 31 months
respectively), 22 clones from isolates grown in vitro in the presence of 1ƒÊM AZT and 17 clones from
isolates grown without AZT were obtained.
Sequence analysis of these clones is shown in Table 2. The 22 clones from isolates grown in the
presence of 1ƒÊM AZT all showed mutation in at least one of codons 67, 70, 215, and 219, with
mutation frequencies for each codon of 18.2, 36.4, 100 and 31.8% respectively. It should be noted that
mutation was uniformly observed in codon 215. Fourteen of these 22 clones showed only a single
codon, 215, mutation from Thr to Tyr. For codon 215 in these 22 clones, 18 mutated from Thr (ACC)
to Tyr (TAC) and 4 from Thr (ACC) to Phe (TTC). These are all mutations of 2 nucleotides. Mutation
in Asp67 (GAC) to Ser (AGC); Lys70 (AAA) to Arg (AGA); and Lys 219 (AAA) to Gln (CAA) was
observed in 18.2, 36.4, and 31.8% of these clones respectively. Mutation at these 3 codons was much
less frequent than at codon 215, and the changes all involved 1-nucleotide substitution.
Of the 17 clones obtained from isolates grown in the absence of AZT, mutationat codon 215 was
also observed in 100% of the clones. The frequencies for codons 67, 70, and 219 were 58.8, 52.9 and
52.9%, respectively. All the particular mutation patterns in RT-coding clonesfrom isolates grown in
the presence of 1ƒÊM AZT were observed also in RT-coding clones from isolatesgrown without AZT.
One additional pattern, Ser67, Lys70, Phe215, Glu219 was observed only among the isolates grown
without AZT. Two clones of Asp67, Arg70, Thr215, Lys219 (215 codon not mutated) were obtained
from isolates grown in the presence of 0.1ƒÊM AZT (data not shown) but not 1ƒÊM AZT, and they
are considered to be weakly resistant to AZT.
3) Comparison of HIV-1 mutations over time.
HIV-1 RT-coding clones from isolates obtained at various times during AZT treatment were
sequenced and compared: 12 clones established before treatment, 19, 2 months after starting treat-
ment, 10, 6-7 months after starting, and 12, more than 8 months after starting. Results are shown in
Table 3.
All 12 clones from HIV-1 isolates before AZT treatment were wild type ,and the isolates did not
grow in the presence of 1ƒÊM AZT. After 2 months of AZT treatment, 9 out of 19 clones (47.4%)
showed mutation. Among these, all 9 showed mutation at codon 70 (47.4%), 1 atcodon 215 (0.1%),
and 1 at codon 219 (0.1%). After 6-7 months of AZT treatment, all 10 clones (100%) showed mutation:
4 at codon 67 (40%), 5 at codon 70 (50%) and 9 at codon 215 (90%). After AZT therapy longer than
8 months, all 12 clones were mutants: 1 at codon 67 (8.3%), 1 at codon 70 (8.3%) and all 12 at codon
215. As shown in Fig. 1, the frequency of individual mutant patterns among total clones changed over
the period of AZT treatment. After treatment for 8 months or longer, clones of all isolates were
mutants clones. In the early stage, codon 70 mutants were more frequent but these decreased in later
stages. On the other hand, mutation at codon 215 appeared rarely in the earlystage but later became
Table 3 HIV mutations at 4 codons over time in patients treated with AZT
平成7年11月20日
1282 Makiko KONDO
Fig. 1 Frequency of HIV-1 mutations at 4 codons in patients treated with AZT
over time.*M
/ T=mutant clones (M) per total clones (T) isolated , 67, 70, 215, 219 =codons in RT,
N = number of clones
before therapy (N=12) after 2 monthsof AZT therapy
(N=19)
after 6-7 monthsof AZT therapy
(N=10)
after 8 monthsof AZT therapy
(N=12)
Table 4 Nucleic acid changes in HIV-1 RT-coding region codons and AZT sensitivity of
HIV-1 isolates in the course of AZT treatment of 2 patients
Patient 038
Underline indicates nucleotide mutation.
感染症学雑誌 第69巻 第11号
Mutation in AZT-resistant HIV-1 1283
predominant.
4) Changes in the HIV-1 RT-coding genome obtained from individual patients inthe course of AZT
treatment and AZT sensitivity of HIV-1 isolates.
From 2 patients (038, 005) HIV-1 RT-coding clones were obtained from HIV-1 isolated before
therapy, and 2 months and 8 months or 12 months after AZT treatment. The mutation patterns in
codons 67, 70, 215 and 219 are shown in Table 4. The susceptibility of these isolates to AZT at each
time was tested in vitro. The results are shown in Table 4. Twelve clones from isolates from these
2 patients before AZT treatment were all wild type and did not grow in the presence of 0 .1 ,uM and
1 At A4 AZT.
Among clones from isolates cultured after 2 months of therapy, 3 of 5 from patient 038 and 5 of
10 from patient 005 showed mutation from AAA to AGA at codon 70. Isolates from 005 did not grow
in the presence of 0.1 ,u M or 1 ,uM AZT.
All 7 clones from the isolate from patient 038 after 8 months of AZT therapy showed codon 215
mutation from ACC to TAC. The isolate grew in the presence of 0.1 ,uM and 1 ,uM AZT.
Among the 5 clones of the isolate from patient 005 after 12 months of AZT therapy, 4 clones
showed mutation at codon 215 from ACC to TAC only, and 1 clone also showed mutation at codon
67 (GAC AAC) and codon 70 (AAA•¨AGA). This isolate grew in the presence of 0.1,u M and 1,u M
AZT.
Discussion
We found 41 HIV-1 RT-coding clone mutants among 48 clones obtained from 10 isolates from 9
patients who underwent long-term AZT therapy. As it has been reported that Asn67, Lys70, Thr215,and Lys219 RT mutations are associated with AZT resistance1-3), we determinedthe occurrence ofthese 4 codon mutations among our HIV-1 mutants. Of these 4 mutant amino acid, Thr215 was
observed quite frequently (40 of 41 or 97.6%) compared with other codon mutations. Asp67 occurred
in only 48.8% of the clones, Lys70 in 31.7%, and Lys219 in 9.8%. These results showed the possibility
of predominance of codon 215 mutation in AZT resistance after long-term AZT therapy.
We tried to culture HIV-1 from patients treated with AZT for 6 to 31 months in the presence of1 ,uM AZT. Assuming that the concentration of AZT in the blood 4 hours after a 250 mg dose (given
every 4 hours) is 0.94-1 ,umol/L10), 1 ,uM in vitro represents an appropriateconcentration to
determine AZT resistance in vivo. Twenty-two HIV-1 clones were obtained from these long-termAZT-treated patients by culture with AZT; their amino acid mutation patterns in the RT-coding
region were compared with those of clones obtained from the same patients by culture without AZT.
Among 22 clones with AZT and 17 clones without AZT, the codon 215 (Thr) mutation was observedin all isolates. By contrast, of the clones cultured with AZT, Asn67, Lys70, and Lys219 mutation was
observed in only 18.2, 36.4 and 31.8% respectively. These 3 amino acid mutation patterns were more
frequently found in isolates grown in the absence of AZT (52.9-58.8%) than inthose grown in its
presence (18.2-36.4%). These results also demonstrate that the codon Thr215 mutation in theRT-coding region plays a more important role in AZT resistance than the other3 amino acid
mutations. Asp67 mutation to Asn, which was observed in isolates cultured without AZT, was not
observed in isolates cultured with AZT. These findings suggest a rather minorrole for Asp67
mutation in AZT resistance.
In 14 of 22 (63.6%) mutant clones grown in the presence of AZT, and in 7 of 17 (41.2%) grownwithout AZT, the single amino acid substitution (Thr215 to Tyr) was the only mutation found. This
finding underlines the predominance of this particular mutation of HIV-1 in relation to AZT resist-
ance in long-term AZT treatment. In contrast to this single amino acid mutation among AZT-
平成7年11月20日
1284 Makiko KONDO
resistant isolates, the other Thr215 mutants (Thr215 to Phe) are all associated with 2 or 3 additionalamino acid mutations in Asp67, Lys70 or Lys219.
The appearance of HIV-1 mutations in these 4 codons was investigated over thecourse of AZT
treatment. Growth of HIV-1 isolates cultured in the presence of AZT was not observed before of AZT
therapy, but the virus appeared after 6-7 months. The low rate of Thr215 mutation in the early stage
of AZT treatment and extremely high rate of Thr215 mutation in later stages, such as after 6 monthsof AZT therapy, demonstrates the predominant role of Thr215 mutation in AZT resistance. This is
in contrast to Lys70 mutation which was frequently observed in the early stage but decreased in later
stages.The nucleotide sequences of 2 patients individually followed up clinically are shown in Table 4.
After 2 months of AZT treatment, the observed mutations had only 1 nucleotideshift, codon 70 from
AAA to AGA or codon 215 from ACC to CCC, and these isolates were not resistant to AZT. However,
the codon 215 mutation from ACC to TAC (Tyr) or TTC (Phe) observed in AZT-resistant isolates
after 8 or 12 months of therapy are 2-nucleotide mutations. This may explain the late appearance ofthe predominant mutation pattern of Thr215 to Phe or especially to Tyr in relation to AZT resist-
ance. Single nucleotide mutations such as codon 70 from AAA to AGA which appear in the early
stages of AZT therapy may not play such a significant role in acquisition of AZT resistance by HIV-1.
Acknowledgments
I am deeply indebted to Professor Kenji Okuda (Department of Bacteriology, Yokohama City
University, School of Medicine), for his guidance in the study. Thanks are also due to Dr. Mitsunobu
Imai and Mr. Takayuki Saito (Department of virology, Kanagawa Prefectural Public Health
Laboratory), Dr. Akira Ito (Division of Clinical Laboratory, Yokohama City University, School ofMedicine), Dr. Shinnichi Oka (Department of Infectious Disease, Institute of Medical Science,
University of Tokyo), and Dr. Kusuya Nishioka (Viral Hepatitis Research Foundation of Japan), for
their advice and encouragement; and Ms. Carson Gleberman for careful reading of the manuscript.This study was supported by a research grant from Kanagawa Prefecture and theMinistry of
Health and Welfare, HIV Epidemiology Project.
References
1) Larder, B. A., Darby, G. & Richman, D. D.: HIV with reduced sensitivity tozidovudine (AZT) isolated duringprolonged therapy. Science, 243: 1731-1734, 1989.
2) Larder, B. A. & Kemp, S. D.: Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance tozidovudin (AZT). Science, 246: 1151-1158, 1989.
3) Boucher, C. A. B., Tersmette, M., Lange, J. M. A., Kellam, P., Goede, R. E. Y., Mulder, J. W., Darby, G., Goudsmit, J.& Larder, B. A.: Zidovudine sensitivity of human immunodeficiency viruses from high-risk symptom-free individ-uals during therapy. Lancet, 336: 585-590, 1990.
4) Barre-Sinoussi, F., Ghermann, J. C., Ray, F., Nugeyre, M. T., chamaret, S., Gruest, J., Dauguet, C. & Axler-Blin, C.:Isolation of T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS).Science, 220: 868-871, 1983.
5) Saito, T., Kondo, M., Ito, A., Nagao, T. & Imai, M.: Investigation of AZT sensitivity of HIV-1 isolated from
patients treated with AZT. JJA Inf D, 67: 311-314, 1993.6) Kondo, M, Saito, T., Ito, A., Oka, S., Hamamoto, K. & Imai, M.: Reverse transcriptase gene analysis of HIV-1
mutants cultured in the Presence of AZT. JJA Inf D, 67: 992-997, 1993.7) Kondo, M., Saito, T., Ito, A., Oka, S,,Takata, N. & Imai, M.: Mutation of HIV-1 RT gene isolated from patients
treated with AZT. JJA Inf D, 67: 185-189, 1993.8) St. Clair, M. H., Martin, J. L., Tudor-Williams, G., Bach, M. C., Vavro, C. L., King, D. M., Kellam, P., kemp, S. D. &
Larder, B. A.: Resistance to ddI and sensitivity to AZT induced by a mutationin HIV-1 reverse transcriptase.Science, 253: 1557-1559, 1991.
感染症学雑誌 第69巻 第11号
Mutation in AZT-resistant HIV-1 1285
9) Sanger, F., Nicklen, S. & Coulson, A.R.: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad SciUSA, 74: 5463-5467, 1977.
10) Yarchoan, R., Berg, G., Brouwers, P., Fischel, M. A., Spitzer, A. R., Wichman, A., Grafman, J., Thomas, R. V., Safai,B., Brunetti, A., Perno, C. F., Schmidt, P.J., Larson, S. M., Myer, C. E. & Broders, S.: Response of humanimmunodeficiency-virus-associated neurological disease to 3'-azido-3'-deoxythymidine. Lancet. 1: 132-5, 1987.
AZT長 期投与後 出現 したAZT耐 性HIV-1分 離株 に見 られる
逆転写酵素遺伝子の変異に関する研究
神奈川県衛生研究所・ウイルス部,横浜市立大学医学部細菌学講座
近 藤 真規子
要 旨
6カ 月 以 上3'-azido-3'-deoxythymidine(AZT)
を 投 与 し た 患 者9名 か らhuman immuno-
de丘ciencytype-1virus(HIV-1)を 分 離iし,AZT
耐 性 に 関与 す る逆 転 写 酵 素(RT)遺 伝 子 の4カ 所
の ア ミノ 酸(Asp67,Lys70,Thr215,Lys219)に い
て 解 析 した.そ の結 果,9名 中7名 に これ ら4カ
所 の ア ミノ 酸 い ず れ か が 変 異 して い る ク ロ ー ンが
検 出 さ れ た.こ れ ら7名 か ら得 ら れ た 合 計41ク
ロー ン の 内40ク ロー ン(97.6%)に215番 ア ミノ酸
のTyrあ るい はPheへ の 変 異 が 認 め られ,こ の
変 異 は他 の3カ 所 の ア ミ ノ酸 の変 異 に比 べ 最 も高
率 で あ っ た.
ま た,AZT1μMの 存 在 下 で 培 養 で き たAZT
耐 性 株 か ら得 られ た 合 計22個 の ク ロ ー ン も4カ 所
の ア ミノ酸 い ず れ か が 必 ず 変 異 して お り,特 に215
番 ア ミノ 酸 の 変 異 は全 て の ク ロー ン に 認 め ら れ
た.
次に一人の患者のAZT投 与前から投与後 にか
けて経時的にHIV-1を 分離し,RT遺 伝子 を解析
した.AZT投 与前に得 られた分離株には4カ 所の
アミノ酸いずれにも変異は認 められなかったが,
投与2カ 月後 には70番 ア ミノ酸がArgに 変異 し
たクローンが優勢 となった.AZT投 与6カ 月以上
を経過するとほ とん どのクローンで215番 ア ミノ
酸がTyrに 変異 してお り,こ れら分離株はAZT
1μM存 在下で培養が可能であった.
215番 アミノ酸の変異 は,他の3カ 所のア ミノ酸
の変異が1コ ドン内1塩 基置換であるのに対 し2
塩基置換であり,比 較的確率の低 い変異にもかか
わ らず,全 てのAZT耐 性株 に認 められることか
ら,こ のア ミノ酸の変異はAZT耐 性 に最 も重要
であることが確認 された.
平成7年11月20日