The S68G polymorphism is a compensatory mutation associated with the drug resistance mutation K65R in CRF01_AE strains

The rate of S68G mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase has increased and is closely related to the K65R mutation among CRF01_AE-infected patients who failed treatment. We aimed to explore the temporal association of S68G and K65R mutations and disclose the role of the former on susceptibility to nucleotide/nucleoside reverse transcriptase inhibitor (NRTI) and viral replication with the K65R double mutations among CRF01_AE-infected patients who failed treatment.

The occurrence of S68G and K65R mutations was evaluated among HIV-1 of various subtypes in the global HIV Drug Resistance Database. The temporal association of S68G and K65R mutations was analyzed through next-generation sequencing in four CRF01_AE-infected patients who failed treatment with tenofovir/lamivudine/efavirenz. The impact of the S68G mutation on susceptibility to NRTI and replication fitness was analyzed using pseudovirus phenotypic resistance assays and growth competition assays, respectively.

The frequency of the S68G mutation increased by 1.4–9.7% in almost all HIV subtypes and circulating recombinant forms in treatment-experienced patients, except subtype F. The S68G mutation often occurred in conjunction with the K65R mutation among RTI-treated patients, with frequencies ranging 21.1–61.7% in various subtypes. Next-generation sequencing revealed that the S68G mutation occurred following the K65R mutation in three of the four CRF01_AE-infected patients. In these three patients, there was no significant change detected in the half maximal inhibitory concentration for zidovudine, tenofovir, or lamivudine between the K65R and K65R/S68G mutations, as demonstrated by the phenotypic resistance assays. Virus stocks of the K65R and K65R/S68G mutations were mixed with 4:6, 1:1, and 9:1 and cultured for 13 days, the K65R/S68G mutants outgrew those of the K65R mutants irrespective of the input ratio.

S68G may be a natural polymorphism and compensatory mutation of K65R selected by NRTIs in the CRF01_AE strain of HIV-1. This mutation does not affect susceptibility to NRTI; however, it improves the replication fitness of K65R mutants. This study deciphers the role of the S68G mutation in the HIV reverse transcriptase of the CRF01_AE strain and provides new evidence for the interpretation of drug-resistant mutations in non-B subtypes of HIV-1.