Mutations in Human Immunodeficiency Virus Type 1 RNase H Primer Grip Enhance 3′-Azido-3′-Deoxythymidine Resistance

Abstract

We recently observed that mutations in the human immunodeficiency type 1 (HIV-1) reverse transcriptase (RT) connection domain significantly increase 3-azido-3-deoxythymidine (AZT) resistance up to 536 times over wildtype (WT) RT in the presence of thymidine analog resistance mutations (TAMs). These mutations also decreased RT template switching, suggesting that they altered the balance between nucleotide excision and template RNA degradation, which in turn increased AZT resistance. Several residues in the HIV-1 connection domain contact the primer strand and form an RNase H primer grip structure that helps to position the primer-template at the RNase H and polymerase active sites. To test the hypothesis that connection domain mutations enhanced AZT resistance by influencing the RNase H primer grip, we determined the effects of alanine substitutions in RNase H primer grip residues on nucleoside RT inhibitor resistance in the context of a WT, TAM-containing, or K65R-containing polymerase domain. Ten of the 11 RNase H primer grip mutations increased AZT resistance 20 to 243 times above WT levels in the context of a TAM-containing polymerase domain. Furthermore, all mutations in the RNase H primer grip decreased template switching, suggesting that they reduced RNase H activity. These results demonstrate that mutations in the RNase H primer grip region can significantly enhance AZT resistance and support the hypothesis that mutations in the connection and RNase H domains can increase resistance by altering the RNase H primer grip region, changing interactions between RT and the template-primer complex and/or shifting the balance between the polymerase and RNase H activities.Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is responsible for converting the singlestranded RNA viral genome into double-stranded DNA (3, 27). To accomplish this, HIV-1 RT contains two enzymatic properties: a DNA polymerase, which can incorporate nucleotides using RNA or DNA as a template, and an RNase H, which degrades the RNA template after it has been copied into DNA (9, 26). The process of reverse transcription requires that RNase H make both specific and nonspecific cleavages. Nonspecific cleavages are required to degrade the viral RNA genome, and specific cleavages are required to remove the tRNA primer, cleave the polypurine tract (PPT) to make it available for use as a plus-strand primer, and finally degrade and remove the PPT primer.For reverse transcription to be completed accurately, the nucleic acid hybrid must be properly aligned at both the polymerase and RNase H active sites (2, 13, 22). Crystal structure studies of HIV-1 RT show the presence of several amino acids that contact the DNA primer strand and the RNA template strand near the RNase H active site (6,7,11,22). These amino acids (G359, A360, H361, K390, K395, E396, T473, Q475, K476, Y501, and I505) are collectively referred to as the RNase H primer grip and lie either in the p51 or p66 subunit of RT. In vivo and in vitro studies of the RNase H prim...

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

“…We have previously shown that increases in AZT resistance associated with CN subdomain mutations occur in the presence of TAMs (4,5,19). To first determine if any NRTI resistance was associated with the CN subdomain of AE T1 in the context of its own TAMs-containing POL domain, the POL plus CN subdomain from pHL(AE T1 ) was subcloned into pHL(B-WT) to create AE T1 -AE T1 -B, which was then assayed for AZT resistance (Fig.…”

Amino Acid Mutation N348I in the Connection Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Multiclass Resistance to Nucleoside and Nonnucleoside Reverse Transcriptase Inhibitors

“…Several studies, including recent work by Delviks-Frankenberry et al and Brehm et al (4,11), highlighted the necessity to expand sequencing analysis to include the connection and RNase H subdomains. This contention is further supported by results in this work and by others (16,28,42,43) showing that mutations at the connection subdomain influence susceptibility to some antiretroviral drugs.…”

N348I in HIV-1 Reverse Transcriptase Can Counteract the Nevirapine-mediated Bias toward RNase H Cleavage during Plus-strand Initiation

“…Mutations in HIV-1 RT that confer resistance to the NRTI, 3Ј-azidothymidine, increase rates of excision (15)(16)(17)(18)(19)(20)(21). More recently, it has been shown that mutations in the connection and RNase H domains of HIV-1 RT confer resistance to NRTIs and NNRTIs (9,10,(22)(23)(24)(25)(26). These mutations appear to exert their effects on susceptibility to 3Ј-azidothymidine more indirectly.…”