In VitroSelection of HIV-1 CRF08_BC Variants Resistant to Reverse Transcriptase Inhibitors

Abstract

Human immunodeficiency virus type 1 (HIV-1) circulating recombinant form 08_BC (CRF08_BC), carrying the recombinant reverse transcriptase (RT) gene from subtypes B and C, has recently become highly prevalent in Southern China. As the number of patients increases, it is important to characterize the drug resistance mutations of CRF08_BC, especially against widely used antiretrovirals. In this study, clinically isolated virus (2007CNGX-HK), confirmed to be CRF08_BC with its sequence deposited in GenBank (KF312642), was propagated in human peripheral blood mononuclear cells (PBMCs) with increasing concentrations of nevirapine (NVP), efavirenz (EFV), or lamivudine (3TC). Three different resistance patterns led by initial mutations of Y181C, E138G, and Y188C were detected after the selection with NVP. Initial mutations, in combination with other previously reported substitutions (K20R, D67N, V90I, K101R/E, V106I/A, V108I, F116L, E138R, A139V, V189I, G190A, D218E, E203K, H221Y, F227L, N348I, and T369I) or novel mutations (V8I, S134N, C162Y, L228I, Y232H, E396G, and D404N), developed during NVP selection. EFV-associated variations contained two initial mutations (L100I and Y188C) and three other mutations (V106L, F116Y, and A139V). Phenotypic analyses showed that E138R, Y181C, and G190A contributed high-level resistance to NVP, while L100I and V106L significantly reduced virus susceptibility to EFV. Y188C was 20-fold less sensitive to both NVP and EFV. As expected, M184I alone, or with V90I or D67N, decreased 3TC susceptibility by over 1,000-fold. Although the mutation profile obtained in culture may be different from the patients, these results may still provide useful information to monitor and optimize the antiretroviral regimens.

Novel Mutations L228I and Y232H Cause Nonnucleoside Reverse Transcriptase Inhibitor Resistance in Combinational Pattern

“…In the previous study, 14 increasing NVP pressure. The results suggested that these two mutations conferred moderate-to-high-level resistance to NVP in combination with other mutations.…”

“…Additionally, we also assessed the drug susceptibility to NNRTIs of Y188C and found that Y188C conferred a high-level resistance to NVP and a moderate-level resistance to EFV, which was consistent with previous reports from our and others' studies. [37][38][39] Modeling study showed that a hydrogen bond was formed between L228 and Y188 in WT RT. When only a single mutation L228I emerged, it would not affect this hydrogen RCs of these variants were tested by a single-cycle cell culture assay (i).…”

“…Viral susceptibility to drugs was expressed as the fold changes in IC 50 of mutant virus compared to that of the WT reference virus. To compare the results in this study with those of previous work in the same criterion, the levels of resistance are divided into four groups based on fold changes as described previously, 14,27 that is, high-level resistance (>20-folds), moderatelevel resistance (4-to 20-folds), low-level resistance (2-to 4-folds), and susceptive (0-to 2-folds).…”

“…14 Several novel mutations (L228I, Y232H, and D404N) in RT were observed in that study to confer moderate-to-high-level NVP resistance either alone or in combination. 14 In a later study, we demonstrated that D404N in the RT connection subdomain was an NNRTI resistance-related mutation that displayed cross-resistance to NVP, EFV, rilpivirine (RPV), and AZT. 16 In this study, we extended our research to the other two novel mutations, L228I and Y232H.…”

“…Circulating recombination form 08_BC (CRF08_BC) is one of the current predominant subtypes in China containing a recombinant reverse transcriptase (RT) gene derived from subtypes Thailand B and Indian C. 13,14 More than 40% of HIV infections in Chinese blood donors were found to be the CRF08_BC subtype from 2007 to 2010. 15 However, few data are available for the drug resistance mutations in this subtype in treatment-exposed patients.…”