Mutations within the Primer Grip Region of HIV-1 Reverse Transcriptase Result in Loss of RNase H Function

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...

Section: Effects Of Connection Domain Mutations On Rnase H-competentmentioning

confidence: 99%

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

Ehteshami

Beilhartz

Scarth

et al. 2008

“…Surprisingly, mutations in the primer grip residues also affect the distant RNase H domain. Residues 226 and 227 have been shown to be involved in positioning RT correctly for 5Ј-end-directed cleavage of RNA fragments remaining after minus strand synthesis (39). Mutations P226A, F227A, L228A, M230A, G231A, Y232A, E233A, and H235A had reduced RNase H activity for the specific cleavage involved in the removal of the polypurine tract (38,40).…”

mentioning

confidence: 99%

“…The W229A mutation reduces RNA and DNA-primed synthesis (37)(38)(39)(40)(41)(42). Since the binding affinity of this mutant is 27-fold lower than that of wild type RT (41), we believe that this mutation results in lower processivity of the enzyme.…”

mentioning

confidence: 99%

Mutations in the Primer Grip Region of HIV Reverse Transcriptase Can Increase Replication Fidelity

Wisniewski¹,

Palaniappan²,

Fu³

et al. 1999

Self Cite

Mutations in the primer grip region of human immunodeficiency virus reverse transcriptase (HIV-RT) affect its replication fidelity. The primer grip region (residues 227-235) correctly positions the 3-ends of primers. Point mutations were created by alanine substitution at positions 224 -235. Error frequencies were measured by extension of a dG:dA primer-template mismatch. Mutants E224A, P225A, P226A, L228A, and E233A were approximately equal to the wild type in their ability to extend the mismatch. Mutants F227A, W229A, M230A, G231A, and Y232A extended 40, 66, 54, 72, and 76% less efficiently past a dG:dA mismatch compared with the wild type. We also examined the misinsertion rates of dG, dC, or dA across from a DNA template dA using RT mutants F227A and W229A. Mutant W229A exhibited high fidelity and did not produce a dG:dA or dC:dA mismatch. Interestingly, mutant F227A displayed high fidelity for dG:dA and dC:dA mismatches but low fidelity for dA:dA misinsertions. This indicates that F227A discriminates against particular base substitutions. However, a primer extension assay with three dNTPs showed that F227A generally displays higher fidelity than the wild type RT. Clearly, primer grip mutations can improve or worsen either the overall or base-specific fidelity of HIV-RT. We hypothesize that wild type RT has evolved to a fidelity that allows genetic variation without compromising yield of viable viruses. Human immunodeficiency virus (HIV-1) reverse transcriptase (HIV-RT)1 is the enzyme that converts the RNA genome of the virus to a double-stranded DNA, which is ultimately integrated into the host chromosome (1). This enzyme is multifunctional, possessing RNA-and DNA-dependent DNA polymerase, RNase H, strand transfer, and strand displacement activities (2, 3). Studies in vivo and in vitro have addressed the role of RT in the variability of the genome (4 -14). RT contributes to the generation of sequence diversity, partly because it produces frequent replication errors. One reason for its low fidelity is that RT lacks a 3Ј to 5Ј exonuclease activity (15). DNA polymerases in other organisms generally have this activity, which removes incorrectly added nucleotides by recognizing the mismatched base with the template sequence. RT misincorporates nucleotides at an estimated frequency of 1 per 5000 polymerized and extends mismatched termini at varying efficiencies (4, 6, 11, 14 -20). Additionally, RT alters the sequence of viral progeny by participating in recombination between the two copies of the viral genome (13, 21-28). Polymerization errors and recombination produce a high frequency of frameshift, deletion, and deletion with insertion mutations observed both in vivo and in vitro (6,7,11,13,21,29). This proclivity for mutations by RT helps HIV to evade immune responses and drug treatment.HIV-1 RT is a heterodimer composed of p66 and p51 subunits. The p66 subunit contains both the polymerase and RNase H active sites. The p51 subunit is a proteolytic product of p66 lacking the 15-kDa carboxyl-terminal (RNase H) d...

“…Differences in RNA and DNA primer usage by RT are likely to result from differences in the helical structures of hybrids having RNA or DNA in the primer strand (20,38,39). Mutations in the primer grip region can also affect RNase H function (37,38,40); for example, mutant Y232A is defective in utilization of an RNA primer and also exhibits altered cleavage specificity at the PPT (38).…”

mentioning

confidence: 99%

Residues in the αH and αI Helices of the HIV-1 Reverse Transcriptase Thumb Subdomain Required for the Specificity of RNase H-catalyzed Removal of the Polypurine Tract Primer

Powell¹,

Beard²,

Bębenek³

et al. 1999

During retrovirus replication, reverse transcriptase (RT) must specifically interact with the polypurine tract (PPT) to generate and subsequently remove the RNA primer for plus-strand DNA synthesis. We have investigated the role that human immunodeficiency virus-1 RT residues in the ␣H and ␣I helices in the thumb subdomain play in specific RNase H cleavage at the 3-end of the PPT; an in vitro assay modeling the primer removal step was used. Analysis of alanine-scanning mutants revealed that a subgroup exhibits an unusual phenotype in which the PPT is cleaved up to seven bases from its 3-end. Further analysis of ␣H mutants (G262A, K263A, N265A, and W266A) with changes in residues in or near a structural motif known as the minor groove binding track showed that the RNase H activity of these mutants is more dramatically affected with PPT substrates than with non-PPT substrates. Vertical scan mutants at position 266 were all defective in specific RNase H cleavage, consistent with conservation of tryptophan at this position among lentiviral RTs. Our results indicate that residues in the thumb subdomain and the minor groove binding track in particular, are crucial for unique interactions between RT and the PPT required for correct positioning and precise RNase H cleavage.The virus-encoded enzyme reverse transcriptase (RT) 1 of human immunodeficiency virus type 1 (HIV-1) and other retroviruses catalyzes the conversion of genomic RNA to a doublestranded DNA replicative intermediate. As minus-strand DNA is synthesized, the RNA template is degraded by the RNase H activity of RT, which cleaves the RNA strand in an RNA-DNA hybrid (Refs. 1 and 2; for reviews, see . This results in the production of many small RNA fragments, any one of which could potentially serve as a primer to initiate synthesis of plus-strand DNA (6). However, a short, purine-rich sequence known as the polypurine tract (PPT) is almost exclusively used as the primer for plus-strand initiation (Refs. 7-12, 61; for a review, see Ref. 6). Why the PPT sequence is selected from all of the other available primers has been the subject of much speculation.One possibility is that the PPT sequence is intrinsically resistant to RNase H degradation and therefore survives as the sole RNA primer available for plus-strand initiation. However, experimental evidence from HIV-1 (13-15) 2 and murine leukemia virus (MuLV) (9, 16, 17) model systems demonstrates that cleavages within the PPT can occur. The MuLV PPT can also be internally cleaved by the isolated RNase H domain from MuLV RT; furthermore, the specificity of cleavage at the 3Ј-end of the PPT is lost when the polymerase domain is removed (18,19). Finally, Escherichia coli RNase H catalyzes cleavages within the MuLV PPT (9,16,17,19) as well as within the HIV-1 PPT. 3A second possibility to explain selection of the PPT primer is related to its unique helical structure (12,20) and the shape and width of the major groove, which is wider than that of other RNA-DNA hybrids (20, 21). These structural factors could cause bi...