Nucleocapsid Protein Annealing of a Primer–Template Enhances (+)-Strand DNA Synthesis and Fidelity by HIV-1 Reverse Transcriptase

Abstract

Human immunodeficiency virus type-1 (HIV-1) requires reverse transcriptase (RT) and HIV-1 nucleocapsid protein (NCp7) for proper viral replication. HIV-1 NCp7 has been shown to enhance various steps in reverse transcription including tRNA initiation and strand transfer which may be mediated through interactions with RT as well as RNA and DNA oligonucleotides. With the use of DNA oligonucleotides, we have examined the interaction of NCp7 with RT and the kinetics of reverse transcription during (+)-strand synthesis with an NCp7-facilitated annealed primer-template. Using a pre-steady state kinetics approach, the NCp7-annealed primer-template has a substantial increase (3-7 fold) in the rate of incorporation (kpol) by RT as compared to heat annealed primer-template with single nucleotide incorporation. There was also a 2-fold increase in the binding affinity constant (Kd) of the nucleotide. These differences in kpol and Kd were not through direct interactions between HIV-1 RT and NCp7. When examining extension by RT, the data suggests that the NCp7-annealed primer-template facilitates the formation of a longer product more quickly compared to the heat annealed primer-template. This enhancement in rate is mediated through interactions with NCp7’s zinc fingers and N-terminal domain and nucleic acids. The NCp7-annealed primer-template also enhances the fidelity of RT (3-fold) by slowing the rate of incorporation of an incorrect nucleotide. Taken together, this study elucidates a new role of NCp7 by facilitating DNA-directed DNA synthesis during reverse transcription by HIV-1 RT that may translate into enhanced viral fitness and offers an avenue to exploit for targeted therapeutic intervention against HIV.

Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ

“…The RT R72I substitution was introduced to both monomers of the recombinant RT p66/p51 heterodimer using QuikChange II (Agilent). The C-terminal His-tagged R72I RT was made in E. coli and purified as described previously (42,44). Purity of all proteins was estimated to be >95% after SDS-polyacrylamide gel electrophoresis.…”

Balancing Antiviral Potency and Host Toxicity: Identifying a Nucleotide Inhibitor with an Optimal Kinetic Phenotype for HIV-1 Reverse Transcriptase

“…Stephen Hughes and Andrea Ferris (Frederick Cancer Research and Development Center, Frederick, MD). The C-terminal hexa-histidine-tagged RT was purified as described previously (Kerr and Anderson, 1997;Kim et al, 2012). The purity of all proteins, as judged through SDS-polyacrylamide gel electrophoresis with Coomassie staining, was Ͼ90%.…”

Human Immunodeficiency Virus Reverse Transcriptase

“…The tRNA may also be hybridized to the template by thermal denaturation and still be competent for reverse transcription . Previous reports found no difference in the structure of the template between heat-annealed and NC-annealed RNAs (Brule et al 2002 ;Kim et al 2012 ). Alternatively, vRNA and tRNA Lys3 gently extracted from virions can serve as substrates for in vitro extension assays in which extension via radiolabel incorporation is a measure of the amount of annealed complex (Cen et al 2009 ;.…”