Increased Multinucleoside Drug Resistance and Decreased Replicative Capacity of a Human Immunodeficiency Virus Type 1 Variant with an 8-Amino-Acid Insert in the Reverse Transcriptase

Abstract

Resistance to antiretroviral drugs is generally conferred by specific amino acid substitutions, rather than insertions or deletions, in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The exception to these findings is the amino acid insertions found in the ␤3-␤4 loop of the RT enzyme in response to treatment with nucleoside reverse transcriptase inhibitors. This insert consists most commonly of two amino acids, but we describe in detail the evolution of a variant with an 8-amino-acid (aa) insert in a patient treated with zidovudine (ZDV) and 2-3-dideoxycytidine (ddC). The 24-nucleotide insert is a partial duplication of local sequences but also contains a sequence segment of unknown origin. Extensive sequence analysis of longitudinal patient samples indicated that the HIV-1 population prior to the start of therapy contained not the wild-type amino acid 215T in RT but a mixture with 215D and 215C. Treatment with ZDV and subsequent ZDV-ddC combination therapy resulted in the evolution of an HIV-1 variant with a typical ZDV resistance genotype (41L, 44D, 67N, 69D, 210W, 215Y), which was slowly replaced by the insert-containing variant (41L, 44D, insert at position 69, 70R, 210W, 215Y). The latter variant demonstrated increased resistance to a wide range of drugs, indicating that the 8-aa insert augments nucleoside analogue resistance. The gain in drug resistance of the insert variant came at the expense of a reduction in replication capacity when assayed in the absence of drugs. We compared these data with the resistance and replication properties of 133 insertcontaining sequences of different individuals present in the ViroLogic database and found that the size and actual sequence of the insert at position 69 influence the level of resistance to nucleoside analogues.

The Remarkable Frequency of Human Immunodeficiency Virus Type 1 Genetic Recombination

“…Such an "ectopic duplication" of viral sequences caused one reported drug resistance-associated mutation (194), and sequence analyses suggest other instances of ectopic viral sequence insertions (314,329). The development of improved computational approaches for identifying transposed genome segments would likely reveal ectopic duplication to be a common means of lentiviral insert generation.…”

Insertions in the Human Immunodeficiency Virus Type 1 Protease and Reverse Transcriptase Genes: Clinical Impact and Molecular Mechanisms

“…Clinical T69SϩXX insert-containing isolates show reduced susceptibilities to all NRTIs (12,31,34,39,62,63,67). The median fold changes in susceptibility for the 144 clinical isolates phenotyped in six different published studies are as follows: zidovudine (AZT), Ͼ500ϫ; stavudine (d4T), 20ϫ; didanosine (ddI), 5ϫ; zalcitabine (ddC), 14ϫ; lamivudine (3TC), Ͼ250ϫ; abacavir (ABC), Ͼ10ϫ; tenofovir (TFV), Ͼ10ϫ.…”

“…The NRTI treatment regimens that select for T69SϩXX insert-containing strains can be identified from well-documented treatment history data. The treatment histories of 36 patients from 12 different publications show that initial AZT monotherapy, followed by combination therapy with AZT plus other nucleosides (usually ddI or ddC), is the most common course of treatment in patients with T69SϩXX insert-containing strains (8,9,11,12,31,35,53,57,59,62,63,67). Patients who have received at least 1 year of AZT monotherapy showed development of T69SϩXX insert-containing strains as early as 6 months after they switched to combination therapy with AZT-ddC or AZT-ddI (67).…”

“…The most commonly inserted amino acids (XX) found in patient-derived strains are SG, SS, and SA, although at least 22 other combinations of codon 69 mutations and insert amino acids have been reported (40,67). A small number of single amino acid inserts at this position have been reported, as have inserts of 5, 8, 11, and 15 amino acids (34,39,40,62). At the nucleotide level, inserts appear to be duplications of neighboring genetic sequences (67), likely generated during reverse transcription by RT stalling and slippage (3,20), although one report suggested that a 15-amino-acid insert was generated through intramolecular recombination (34).…”

Functional Correlation between a Novel Amino Acid Insertion at Codon 19 in the Protease of Human Immunodeficiency Virus Type 1 and Polymorphism in the p1/p6 Gag Cleavage Site in Drug Resistance and Replication Fitness

“…Recently, the emergence of variants containing amino acid insertions in RT or PR has also been reported. The insertions in RT emerge between codons 69 and 70 or near codon 103 (7,21,22,27,37,38,39). The insertion of 2, 5, 8, 11, or 15 amino acids at these positions has been reported.…”