Connection Domain Mutations N348I and A360V in HIV-1 Reverse Transcriptase Enhance Resistance to 3′-Azido-3′-deoxythymidine through Both RNase H-dependent and -independent Mechanisms

Abstract

Thymidine analogue-associated mutations (TAMs) in reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) cause resistance to 3-azido-3-deoxythymidine (AZT) through excision of the incorporated monophosphate. Mutations in the connection domain of HIV-1 RT can augment AZT resistance. It has been suggested that these mutations compromise RNase H cleavage, providing more time for AZT excision to occur. However, the underlying mechanism remains elusive. Here, we focused on connection mutations N348I and A360V that are frequently observed in clinical samples of treatment-experienced patients. We show that both N348I and A360V, in combination with TAMs, decrease the efficiency of RNase H cleavage and increase excision of AZT in the presence of the pyrophosphate donor ATP. The TAMs/N348I/A360V mutant accumulates transiently formed, shorter hybrids that can rebind to RT before the template is irreversibly degraded. These hybrids dissociate selectively from the RNase H-competent complex, whereas binding in the polymerase-competent mode is either not affected with N348I or modestly improved with A360V. Both connection domain mutations can compensate for TAM-mediated deficits in processive DNA synthesis, and experiments with RNase H negative mutant enzymes confirm an RNase H-independent contribution to increased levels of resistance to AZT. Moreover, the combination of diminished RNase H cleavage and increased processivity renders the use of both PP i and ATP advantageous, whereas classic TAMs solely enhance the ATP-dependent reaction. Taken together, our findings demonstrate that distinct, complementary mechanisms can contribute to higher levels of excision of AZT, which in turn can amplify resistance to this drug.Human immunodeficiency virus type 1 (HIV-1) 4 replicates using a virally encoded reverse transcriptase (RT), which contains a catalytically active large subunit (p66) and a smaller p66-derived subunit (p51). The p66 subunit comprises the DNA polymerase (residues 1-321), connection (residues 322-440), and RNase H (residues 441-560) domains (1, 2). The RNase H activity, which degrades the RNA of DNA⅐RNA hybrids, is essentially required to convert the single-stranded viral RNA genome into double-stranded proviral DNA (3).Due to its key role in viral replication, HIV-1 RT represents a major therapeutic target (4, 5). Approved RT inhibitors belong to two distinct classes: nucleoside analogue and nonnucleoside analogue RT inhibitors (NRTIs and NNRTIs, respectively). NRTIs are synthetic derivatives of the natural deoxynucleosides. The triphosphate forms of NRTIs and cellular dNTPs serve as substrates for HIV-1 RT. In contrast to natural dNTPs, NRTIs lack the 3Ј-hydroxyl group of the sugar moiety. The incorporated monophosphate (MP) therefore acts as a chain terminator, which prevents phosphodiester bond formation with the next complementary nucleotide (6). NNRTIs are structurally diverse, and members of this family of compounds bind to a hydrophobic pocket (NNRTI-binding pocket) near the polymeras...

The N348I Mutation at the Connection Subdomain of HIV-1 Reverse Transcriptase Decreases Binding to Nevirapine

“…Moreover, both Ile-348 p66 and Ile-348 p51 slightly enhanced processivity. These results were consistent with trap-based experiments performed in our laboratory (data not shown) and by others (39).…”

“…Reaction products were resolved in denaturing polyacrylamide-urea DNA gels and quantitated as described above. The primary RNase H cleavage product is mainly 18 nucleotides from the 3Ј-end of the DNA primer (Ϫ18 nucleotides), and the secondary cleavage product is mainly 12 nucleotides from the 3Ј-end of the primer (Ϫ12 nucleotides) as reported previously (39,43).…”

“…40) annealed to the Pd 18 primer was used. For the RNase H assays, a 22-base pair duplex (Cy3-Tr 35 /Pd 22 ) consisting of a 5Ј Cy3-labeled RNA template (Cy3-Tr 35 ) of the sequence 5Ј-Cy3-GGAAAUCUCUAGCAGUGGCGCCCGA-ACAGGGACCU-3Ј annealed to a DNA primer (Pd 22 ) of the sequence 5Ј-AGGTCCCTGTTCGGGCGCCACT-3Ј was used (39). Primers were annealed to templates at concentrations corresponding to a 1:3 molar ratio.…”

“…Yap et al (31) showed that N348I reduces the rate of RNA template degradation by RT in either a WT background or in the presence of excision-enhancing AZT resistance mutations. Ehteshami et al (39) proposed that CSMs N348I and A360V enhance resistance to AZT through both RNase H-dependent and -independent mechanisms. Specifically, addition of the CSMs N348I and A360V to the AZTresistant M41L/D67N/T215Y RT enhanced AZT resistance by causing preservation of AZT-terminated RNA/DNA that can be unblocked by RT with CSMs (39).…”

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

“…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.…”