Y586F mutation in murine leukemia virus reverse transcriptase decreases fidelity of DNA synthesis in regions associated with adenine–thymine tracts

Abstract

Using in vivo fidelity assays in which bacterial ␤-galactosidase or green fluorescent protein genes served as reporters of mutations, we have identified a murine leukemia virus (MLV) RNase H mutant (Y586F) that exhibited an increase in the retroviral mutation rate Ϸ5-fold in a single replication cycle. DNA-sequencing analysis indicated that the Y586F mutation increased the frequency of substitution mutations 17-fold within 18 nt of adenine-thymine tracts (AAAA, TTTT, or AATT), which are known to induce DNA bending. Sequence alignments indicate that MLV Y586 is equivalent to HIV-1 Y501, a component of the recently described RNase H primer grip domain, which contacts and positions the DNA primer strand near the RNase H active site. The results suggest that wild-type reverse transcriptase (RT) facilitates a specific conformation of the template-primer duplex at the polymerase active site that is important for accuracy of DNA synthesis; when an adeninethymine tract is within 18 nt of the polymerase active site, the Y586F mutant RT cannot facilitate this specific template-primer conformation, leading to an increase in the frequency of substitution mutations. These findings indicate that the RNase H primer grip can affect the template-primer conformation at the polymerase active site and that the MLV Y586 residue and template-primer conformation are important determinants of RT fidelity. R everse transcriptase (RT), which copies retroviral genomic RNA into double-stranded DNA during reverse transcription, contains two major enzymatic activities: a DNA polymerase activity that uses either RNA or DNA as a template and an RNase H activity that can degrade RNA in an RNA⅐DNA hybrid (1). The crystal structure of HIV-1 RT in complex with a template-primer and a dNTP substrate has shown that the polymerase active site and the RNase H cleavage site are separated by 18 nt of the template-primer (2). In the case of murine leukemia virus (MLV) RT, the DNA polymerase and RNase H activities reside in physically separable domains of a single monomeric protein (3).The RNase H activity of all retroviral RTs performs several functions that are essential for the viral life cycle (4), which include degradation of the RNA template after synthesis of the minusstrand DNA, generation of a specific polypurine primer from which plus-strand DNA synthesis initiates, subsequent removal of the polypurine primer to complete synthesis of the viral doublestranded DNA, and removal of the minus-strand tRNA primer (5). During minus-strand DNA synthesis, RT positions the RNase H active site and cleaves the RNA Ϸ18 nt 3Ј of the site of DNA synthesis (6).All retroviral RTs carry out error-prone DNA synthesis, in part because they lack a proofreading activity and possess a low affinity for the template (7). The low fidelity of retroviral replication is a major force for generation of high levels of variation in retroviral populations (8), which allows them to adapt quickly to changes in their environment. One important outcome of this high adaptability is t...

Interaction of the Ty3 Reverse Transcriptase Thumb Subdomain with Template-Primer

“…Contacts with the template are mediated via elements of the p66 fingers and palm subdomains collectively designated the "template grip" (8 -10). Participation of the RNase H primer grip in imposing the correct trajectory on the RNA strand of RNA/DNA hybrid for hydrolysis at the RNase H catalytic center has been suggested (11)(12)(13). Finally, a network of contacts between residues of helices ␣H and ␣I at the base of the p66 thumb with the primer backbone 3-6 bp behind the polymerase active center may compose a "translocation track" and also assist the transition in nucleic acid geometry from the A to B form downstream of the DNA polymerase catalytic center, an event accompanied by considerable duplex bending (14 -20).…”

Mutations in the RNase H Primer Grip Domain of Murine Leukemia Virus Reverse Transcriptase Decrease Efficiency and Accuracy of Plus-Strand DNA Transfer

“…D17 dog osteosarcoma cells and 293T cells were obtained from the American Type Culture Collection. The D17-based cell lines ANGIE P or A3GFP11, which contain a single GA-1 or MP-1 provirus, respectively, also express an amphotropic MLV envelope (13,44). The GN-MLV-GFFP cell line contains a single MLV-based provirus derived from the vector pGN-MLV-GFFP-IHy.…”

“…The culture medium from pooled cells containing either GA-1 or MP-1 virus was used to infect D17 target cells plated at a density of 2 ϫ 10 5 cells per 60-mm-diameter dish as previously described (13). After selection for resistance to G418, GA-1-infected D17 cells were stained with X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside), and lacZ inactivation was determined by counting blue and white colonies, whereas MP-1-infected cells were observed under a fluorescence microscope to determine the frequency of GFP inactivation (8,13,44). Isolation of single nonfluorescent cell clones by FACS.…”

“…We recently reported that the Y586F substitution in the MLV RNase H domain resulted in an approximately fivefold increase in the MLV mutation rate in vivo, which is the highest reported to date (44). Residue Y586 of MLV is equivalent to HIV-1 residue Y501, a constituent of the recently described RNase H primer grip domain, which contacts the DNA primer strand and positions the template strand near the RNase H active site, influencing RNase H cleavage efficiency and specificity (31,34).…”

“…Unlike most high-fidelity cellular DNA polymerases, RT lacks a classical 3Ј-5Ј exonuclease proofreading activity. Other structural features also known to affect RT fidelity include positioning of the template-primer complex at the polymerase active site that is dictated by contacts with RT residues and the local geometry of the polymerase active site (3,15,44). In addition, reverse transcription of the retroviral RNA genome requires two template-switching events, namely, minus-strand DNA transfer and plus-strand DNA transfer.…”