18th International HIV Drug Resistance Workshop Basic Principles & Clinical Implications June 9–13 2009, Fort Myers, Florida, USA

SUBTYPE-SPECIFIC AMINO ACID POLYMORPHISMS IN THE HIV-1 REVERSE TRANSCRIPTASE CONNECTION SUBDOMAIN OF CRF01_AE ARE ASSOCIATED WITH HIGHER 3´-AZIDO-3´-DEOXYTHYMIDINE RESISTANCE

Antivir Ther 2009; 14 Suppl 1:A31 (abstract no. 29)

KA Delviks-Frankenberry¹, GN Nikolenko¹, F Maldarelli², S Hase³, Y Takebe³ and VK Pathak^1
1Viral Mutation Section, Host-Virus Interaction Branch, HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, MD, USA; ²Host-Virus Interaction Branch, HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, MD, USA; ³Laboratory of Molecular Virology and Epidemiology, AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan

BACKGROUND: We previously showed that mutations in the connection subdomain (cn) of HIV-1 subtype B reverse transcriptase (RT) increase 3´-azido3´- deoxythymidine (AZT) resistance in the context of thymidine analog mutations (TAMs) by altering the balance between nucleotide excision and template RNA degradation, thereby providing more time for RT to carry out NRTI excision. To determine whether the balance between polymerization and RNase H activity affects drug resistance in other HIV-1 subtypes, AZT resistance in CRF01_AE was analysed.

METHODS: Extensive RT subdomain swapping and mutagenesis was used to determine AZT susceptibility of wild-type and treatment-experienced CRF01_AE. The ability of wild-type and mutant RTs to excise AZT-monophosphate (AZTMP) from a blocked primer was determined in an excision-extension assay using a 19/42mer RNA/DNA and DNA/DNA hybrid. Primary RNase H cleavages were quantified in an RNase H assay using an 18/18-mer RNA/DNA hybrid substrate.

RESULTS: Interestingly, CRF01_AE-containing TAMs exhibited 64-fold higher AZT resistance (relative to wild-type B) in comparison to subtype B containing the same TAMs (13-fold), which in turn correlated with higher levels of AZTMP excision on both RNA and DNA templates. The high AZT resistance exhibited by CRF01_AE was primarily associated with the T400 residue, which is present in wild-type CRF01_AE CN. A400T substitution in subtype B increased AZTMP excision on both DNA and RNA templates and reduced RNase H cleavage. Substituting the T400 in CFR01_AE with alanine restored AZT sensitivity and reduced AZTMP excision on both DNA and RNA templates, suggesting that the T400 increases AZT resistance in CRF01_AE at least in part by directly increasing the efficiency of AZTMP excision.

CONCLUSIONS: These results demonstrate that the amino acid composition of CRF01_AE in the background of TAMs exhibits higher AZT resistance than subtype B containing TAMs, and this resistance is associated with the T400 amino acid in the CRF01_AE CN. Our results show that mixing the RT pol, CN and RH domains from different subtypes, specifically the CRF01_AE pol and the subtype B CN and RH, can underestimate AZT resistance levels. These observations indicate the need to develop subtype-specific genotypic/phenotypic assays to provide more accurate estimates of drug resistance.

2009-06-09
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