First Report on a T69-ins Insertionin CRF06_cpx HIV Type 1

Young-Keol Cho,1 Ba-Reum Kim,1 Jung-Eun Kim,1 June-Hee Woo,2 and Brian T. Foley3

Abstract

Insertion mutations at codon 69 (T69-ins insertion) of the human immunodeficiency virus type 1 (HIV-1) reversetranscriptase confer full resistance to all approved nucleoside reverse transcriptase inhibitors. To date, nearly allreports on T69-ins insertions have described subtypes B and rarely subtypes A, C, and F of HIV-1. Here, weprovide the first report of a T69-ins insertion in circulating recombinant form (CRF) 06_cpx in a patient who hadbeen treated with a zidovudine/didanosine combination for 18 months and then shifted to lamivudine, sta-vudine, and nelfinavir for 76 months. Thereafter, the patient was additively administered Korean red ginseng.This is the first report on the appearance of the T69-ins insertion mutation in CRF HIV-1.

The introduction of highly active antiretroviral thera-py (HAART) for HIV-1-infected patients has markedly

reduced the rates of mortality and morbidity related to thisdisease. Although HAART suppresses the replication of HIV-1, its effectiveness is often limited by the emergence of anti-retroviral drug-resistant mutants. The replication of drug-re-sistant viruses during HAART is a major cause of treatmentfailure in HIV-1-infected patients.1

Insertion mutations at codon 69 (T69-ins insertion) of theHIV-1 reverse transcriptase (RT) confer full resistance to allapproved nucleoside reverse-transcriptase inhibitors (NRTIs).2,3

The T69-ins insertion mutation seems to arise after the admin-istration of didanosine (ddI), zalcitabine (ddC), or stavudine(d4T) treatments, although prior or concurrent zidovudine(AZT) treatment is a prerequisite for the development of T69-ins insertion mutations.4–7 However, this insertion mutationhas never been observed in patients receiving AZT mono-therapy8,9 or in its circulating recombinant forms (CRFs).CRF06_cpx most probably emerged in Burkina Faso in 1979,10

and CRF06-cpx (BFP90) was isolated for the first time inAustralia in 1996.11 To date, nearly all reports on T69-ins in-sertions have described subtypes B and rarely subtypes A, C,or F.12,13 Here, we provide the first report of a T69-ins inser-tion in CRF06_cpx in a patient who had been treated with anAZT/ddI combination and later shifted to HAART. There-after, this patient additionally took Korean red ginseng(KRG),14 and the T69-ins insertion mutation disappeared for asignificant period of time (Fig. 1).

Patient A was born a female (body weight 3.2 kg) by ce-sarean section without perinatal problems or any informationregarding the maternal HIV infection status in December 31,

1997. Patient A was breastfed for 10 months and then ad-mitted for acute otitis media (AOM) and pneumonia to theAsan Medical Center (AMC) via a local clinic on April 27,1999. Upon admission, she was diagnosed with aspirationpneumonia, acute gastroenteritis, AOM, oral thrush, andfailure to thrive. She was diagnosed with HIV-1 infection onMay 13, 1999. She was later admitted for bacterial pneumoniafrom December 16, 2000 through February 14, 2001 at a ter-tiary teaching hospital in Seoul. Patients B and C (father andmother of patient A, respectively) were diagnosed with HIV-1infection in May 1999 following the diagnosis in patient A.

Patient A was administered AZT/ddI combination therapyfor 18 months from June 1999 through to December 2000. Shewas then shifted to 3TC, stavudine (d4T), and nelfinavirtherapy in December 2000. Her HIV RNA plasma level was5.7 · 105 copies/ml on January 3, 2001. In July 2011, the pa-tient was shifted to abacavir, lamivudine, and atazanavirtherapy. Self-reported compliance with HAART was 100%.Patients B and C’s treatment regimens are described in Fig. 1.Patient B began to take HAART in November 1999 and shiftedto other drugs. His self-reported compliance was 98–100%.Patient C took AZT during the third trimester ( June–August1999) of her pregnancy with her second baby who was bornon August 14, 1999. Her second baby was not infected withHIV-1. Patient C took Combivir and abacavir in February2009 and shifted to Combivir and Kaletra (LPV/r) in early2012. Self-reported compliance with HAART was about 90%,and her plasma RNA was always positive during HAART(Fig. 1). The three patients first visited our laboratory in orderto receive KRG on May 2, 2007. Patient A began to take acombination therapy with KRG (1.2 g/day for the first 15

Departments of 1Microbiology and 2Internal Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea.3Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, New Mexico.

AIDS RESEARCH AND HUMAN RETROVIRUSESVolume 29, Number 7, 2013ª Mary Ann Liebert, Inc.DOI: 10.1089/aid.2013.0013

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FIG. 1. Immunological andvirological changes followingtreatment with antiretroviraltherapy and Korean red gin-seng (KRG). Patient A dem-onstrated T69-ins-VT in May2007, although earlier sam-ples were unavailable. De-spite 100% compliance withHAART, patient A was posi-tive for plasma RNA prior tothe initiation of KRG intake inMay 2007. Under the sametreatment regimen (which in-cluded KRG), the plasmaRNA copy level significantlydecreased from 5,500 copies/ml in May 2007 to < 20 cop-ies/ml in February 2008, andthis was maintained belowthe detection limit for about44 months. In associationwith the viral suppression,T69-ins insertions dis-appeared in the two samplesobtained in 2009 and 2010.Patient B also demonstratedgood compliance for HAARTand KRG. In patient C, theRNA copy number was con-tinuously positive because ofpoor compliance for HAART.The open, semiclosed, andclosed circles indicate wildtype only, mixtures of wild-type and T69-ins insertions,and T69-ins insertions only,respectively. HAART, highlyactive antiretroviral therapy;3TC, lamivudine; d4T, stavu-dine; NFV, nelfinavir; ABC,abacavir; ATV, atazanavir;LPV/r, Kaletra; EFV, efavirenz.

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FIG. 2. Sequences were alignedwith the HIV-1 subtype referenceset from the HIV Sequence Data-base (hiv-web.lanl.gov/content/hivdb/Subtype_REF/align.html)and phylogenetic trees were builtusing the PHYLIP DNAdist (F84model, Ts:Tv 1.7) plus Neighbor,DAMBE maximum likelihood, andBEAST Bayesian maximum likeli-hood programs. Based on the re-sults of the phylogenetic treeanalyses, the viruses of patients A( JSE), B ( JIG), and C (YYH) wereclassified as CRF06_cpx.

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months and then 1.8 g/day) on May 2007. Patient B began totake KRG (3.8–5.0 g/day) on May 2, 2007. His self-reportedcompliance with KRG therapy was 98–100%. Patient C beganto take KRG on May 2, 2007. Self-reported compliance withKRG (3.0 g/day) was 90%.

Peripheral blood mononuclear cells (PBMCs) were col-lected for 6 months beginning on May 2, 2007. Amplificationof the pol gene was performed as described previously.15,16

Briefly, the pol gene was amplified using nested polymerasechain reaction (PCR) with TaKaRa R-Taq (TaKaRa Bio, Inc.,Otsu, Shiga, Japan) in two reaction tubes at the same time, andfull sequences were obtained by the overlap of three PCRproducts. The subsequent amplicons were directly sequencedusing Applied Biosystems 3730XL. The Roche Cobas Ampli-Prep/Cobas TaqMan HIV-1 kit (version 2.0; Roche Diag-nostics Systems, Branchburg, NJ) was used to determine theplasma RNA copy level. Full-length pol sequences werealigned with the HIV-1 subtype references in the HIV Se-quence Database (http://hiv-web.lanl.gov/content/hidb/

Subtype_REF/align.html), and phylogenetic trees were builtusing the DNAML maximum likelihood method, PAUPmaximum parsimony, PHYLIP Dnadist (F84 model, Ts:Tv1.7), and other neighbor programs (Fig. 2).16

The three subject patients’ full-length sequences of the polgene were classified as CRF06-cpx (Fig. 2). We detected aT69-ins insertion in patient A, within a family consisting offour individuals. In total, we retrospectively obtained 19sequences over 5 years from patient A. Of these 19 se-quences, 14 sequences demonstrated major resistance mu-tations. Of these 14 sequences, 11 demonstrated a T69-VTinsertion. The first sequences obtained in May 2007 dem-onstrated the T69-ins insertion (all with the same GTTACTsequences, except one sequence that was GTAACT) togetherwith six major resistance mutations in the protease and RT.This was determined 95 months after the initiation of AZTand ddI (Table 1). However, it is speculated that the T69-insinsertion might have developed prior to the first visit to thelaboratory in May 2007. Except for the T69-ins insertion, the

Table 1. Alignment of Major Resistance Mutations in pol Gene and Viral Load

Protease codon Reverse transcriptase codon

46 88 90 41 62 67 68 69 70 184 215Date

Numberof PCRa

Monthsfrom ART M N L M A D S T Ins Ins K M T

RNAcopy (/ml)

Patient AJun 06 84 Unavailable 1,100May 07 1 95 . . M . . . . . — — . ns ns 5,500

1 . S M . V . . S V T . ns nsAug 08 1 110 . . . . . . . . — — . . . < 20

1 . S M . V . . S V T . V Y1 I S M L . . . . — — . V Y

Feb 09 1 116 . . . . . . . . — — . . . < 201 I S M L . . . . — — . V Y

Aug 10 1 134 . . . . . . . . — — . . . 441 I S M L . . . . — — . V Y

Jan 11 1 139 . . . . . . . . — — . . . < 201 ns ns ns . . . . S V T . . Y1 . S M . . . . S V T . V Y

Sep 11 1 147 I S M . V . . S V T . V Y 100Feb 12 1 152 I S M . V E . S V T . V . 14,500

1 I S M . V E . S V T . V Y1 I S M . V E . S V T . ns ns1b ns ns ns ns V . . S V T . V Y

Jul 12 2 157 I S M . V E . S V T . V Y 23,200

Patient BMay 07 1 90 . . . . . . . . — — . . . < 20Feb 02 1 111 . . . . . . . . — — . . . < 20Feb 12 1 147 . . . . . . . . — — . . . < 20

1b 147 ns ns ns ns ns ns ns ns ns ns ns I . < 20Jul 12 1 152 . . . . . . . . — — . . . < 20

Patient CMay 07 1 - 21 . . . . . . . . — — . . . 101,000Jul 09 1 5 . . . . . . . . — — . . . 714Jan 11 1 23 . . . . . . . . — — . . . PositiveFeb 12 1 36 . . . . . . . . — — . . . PositiveJul 12 1 41 . . . . . . . . — — . . . 1,392

aNumber of PCR amplicons.bStop codons caused by G-to-A hypermutation (KC680836-37, KC680840, and KC680845). ART, antiretroviral therapy; —, absence of T69-

ins insertion; ns, no sequences. Dots indicate sequence identity with wild type.

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number of resistance mutations gradually increased fromfour to eight (Table 1).

In contrast, patients B and C have never taken ddI orddC. Differing from her parents’ regimens, patient A wastreated with AZT and ddI prior to HAART. This might beconsistent with exposure to ddI as the driving factor in thedevelopment of insertions, as reported for subtypes otherthan subtype B.4–7

In February 2012, patients A and B each revealed prema-ture stop codons in the pol gene. We have routinely usedPBMCs for genotyping and HIV-1 sequencing because ofeasiness rather than RT-PCR using serum or plasma, althoughplasma virus is the typical source for genotyping.

Although our observation of T69-VT insertion is inter-esting, we have to evaluate and characterize any further rolethis mutation may play in CRF06_cpx HIV-1. This prelimi-nary observation should be used to provide phenotypicdata on the role of T69-ins in nucleoside reverse transcrip-tase inhibitor (NTRI) resistance in the presence or absence ofother related mutations. Site-directed mutagenesis could beused for phenotypic characterization of CRF06 and otherrecombinant HIV-1 back bones and elucidate their roles indrug resistance.

In Korea, we have not previously detected a T69-ins in-sertion in any long-term AZT patients,14,15,17 as reported byLarder et al.2 Among the different T69-ins insertion types, T69-VT is a very rare amino acid pattern that has been reportedpreviously in only three patients who had highly resistantviruses.18,19 However, despite its rarity, it is interesting thatpatient A revealed the T69-VT insertion rather than T69-SG,T69-SS, T69-ST, or any other.

Here, the presence of the T69-ins insertion in CRF06-cpxsuggests that the same insertion will also occur in other CRFs,although many studies on T69-ins have been performed onHIV-1 subtypes A, B, C, and F only.11,12

Sequence Data

The GenBank accession numbers are as follows: KC680822–KC680862.

Acknowledgments

This work was supported by a grant from the KoreanSociety of Ginseng, which was funded by the Korea GinsengCorporation (2011–2012).

Author Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Young-Keol Cho

Department of MicrobiologyUniversity of Ulsan College of Medicine

88 Olympic-ro 43-gilSongpa-gu

Seoul 138-736Korea

E-mail: ykcho2@amc.seoul.kr

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