Subtype-Specific Differences in the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Connection Subdomain of CRF01_AE Are Associated with Higher Levels of Resistance to 3′-Azido-3′-Deoxythymidine

Abstract

We previously shown that mutations in the connection (CN) subdomain of human immunodeficiency virus type 1 (HIV-1) subtype B reverse transcriptase (RT) increase 3-azido-3-deoxythymidine (AZT) resistance in the context of thymidine analog mutations (TAMs) by affecting the balance between polymerization and RNase H activity. To determine whether this balance affects drug resistance in other HIV-1 subtypes, recombinant subtype CRF01_AE was analyzed. Interestingly, CRF01_AE containing TAMs exhibited 64-fold higher AZT resistance relative to wild-type B, whereas AZT resistance of subtype B containing the same TAMs was 13-fold higher, which in turn correlated with higher levels of AZT-monophosphate (AZTMP) excision on both RNA and DNA templates. The high level of AZT resistance exhibited by CRF01_AE was primarily associated with the T400 residue in wild-type subtype AE CN subdomain. An A400T substitution in subtype B enhanced AZT resistance, increased AZTMP excision on both RNA and DNA templates, and reduced RNase H cleavage. Replacing the T400 residue in CRF01_AE with alanine restored AZT sensitivity and reduced AZTMP excision on both RNA and DNA templates, suggesting that the T400 residue increases AZT resistance in CRF01_AE at least in part by directly increasing the efficiency of AZTMP excision. These results show for the first time that CRF01_AE exhibits higher levels of AZT resistance in the presence of TAMs and that this resistance is primarily associated with T400. Our results also show that mixing the RT polymerase, CN, and RNase H domains from different subtypes can underestimate AZT resistance levels, and they emphasize the need to develop subtype-specific genotypic and phenotypic assays to provide more accurate estimates of clinical drug resistance.

Identification of Novel Resistance-Related Polymorphisms in HIV-1 Subtype C RT Connection and RNase H Domains from Patients Under Virological Failure in Brazil

“…[23][24][25]30,34,[39][40][41] In this study, mutations in these portions were found in HIV-1B and C infecting patients under virological failure. Despite previous reports that have sporadically documented RT C-terminal mutations in non-B subtypes, 30,35,36 this is the first study specifically assessing differences of mutation occurrence in non-B subtype-specific contexts under the same therapeutic setting.…”

“…43 This could indicate that treatment-naive isolates of Brazilian HIV-1C would take more time to acquire drug resistance to AZT-containing regimens. Notwithstanding, this polymorphism potentiates drug resistance conferred by TAMs in HIV-1B and CRF02_AG viruses, 35,36,43,44 and further studies are warranted to understand the impact of these changes on viral fitness.…”

“…Mutations considered were N348I, A360V, T369I/V, A371V, A376S, A400T, D488E, Q509L, and Q547K for their recognized phenotypic role in drug resistance. [34][35][36][37][38] For the discovery of novel HIV-1C mutations associated with drug exposure, RT C-terminal sequences of this subtype were compared to global and Brazilian HIV-1C sequences from drug-naive subjects. Global consensus was obtained from the Los Alamos database, whereas the Brazilian consensus was inferred using sequences obtained from this study and additional 91 HIV-1C sequences of CN and 115 of RNH recently described by our group.…”

Patterns of Human Immunodeficiency Virus Type 1 Recombination Ex Vivo Provide Evidence for Coadaptation of Distant Sites, Resulting in Purifying Selection for Intersubtype Recombinants during Replication

“…Intersubtype recombinants in which epistatic pairs of mutations are separated from one another will thus have reduced fitness relative to either parent. It has also been shown that the polymorphism in the reverse transcriptase of the CRF-01_AE causes lower RNase H activity than that of NL4-3 subtype B viruses (13). Therefore, there may be inherent differences among proteins encoded by different subtypes.…”

A Novel Molecular Mechanism of Dual Resistance to Nucleoside and Nonnucleoside Reverse Transcriptase Inhibitors

“…We identified mutations in the CN from treatment-experienced patients that enhanced 3Ј-azido-3Ј-deoxythymidine (AZT) resistance (34,35), and closer examination of the CN and RH by us and others has identified additional mutations in this region that are associated with increased drug resistance (6,11,13,23,29,43,56,62). Several mutations in the CN of RT (N348I, T369I, and E399D) were identified by us and others to be associated with increases in both NRTI and NNRTI resistance (23,24,33,59).…”