Abstract
DNA and RNA polymerases share a core architecture composed of three structurally conserved motifs: A, B, and C. Although the amino acid sequences of these motifs are highly conserved between closely related organisms, variation across broader evolutionary distances suggests that only a few residues in each motif are indispensable for polymerase function. To test this, we constructed libraries of human immunodeficiency virus type-1 (HIV-1) containing random single amino acid replacements in motif B of reverse transcriptase (RT), and we used selection in culture to assess RT function. Despite the nearly absolute constancy of motif B in vivo, virus replicating in culture tolerated a range of conservative and nonconservative substitutions at 10 of the 11 amino acid positions examined. These included residues that are invariant across all retroviral subfamilies and highly conversed in diverse retroelements. Several mutants retained wild type infectivity, and serial passage experiments revealed replacements that were neutral or even beneficial to viral fitness. In addition, a number of the selected variants exhibited altered susceptibility to the nucleoside analog inhibitors AZT and 3TC. Taken together, these data indicate that HIV-1 tolerates a range of substitutions at conserved RT residues and that selection against slightly deleterious mutations (purifying selection) in vivo masks a large repertoire of viable phenotypic variants. This mutational flexibility likely contributes to HIV-1 evolution in response to changing selection pressures in infected individuals.
Highlights
Despite these biochemical differences, viral polymerases are structurally very similar [2,3,4,5,6]
To minimize wild type sequences in the mutant pools, we started with reverse transcriptase (RT) subclones containing either a stop codon at position 154 or a unique ClaI restriction site insertion at position 152 (Fig. 2A, bottom left) and used mutagenesis primers that corrected these lethal mutations while simultaneously introducing random nucleotides at the target codon sites
Each library of RT subclones was ligated en masse into an RTdeleted human immunodeficiency virus type-1 (HIV-1) construct to generate the corresponding full-length HIV-1 pR9 libraries (Fig. 2A, right). These full-length mutant libraries were separately transfected into 293T cells to produce mutant HIV-1 populations, each randomized at a single codon site in motif B (Fig. 2B). 293T cells do not express the HIV-1 CD4 receptor and cannot be reinfected by progeny virions, enabling us to examine the infectious potential of the virus pools prior to biologic selection
Summary
Viral polymerases are structurally very similar [2,3,4,5,6]. We constructed libraries of human immunodeficiency virus type-1 (HIV-1) containing random single amino acid replacements in motif B of reverse transcriptase (RT), and we used selection in culture to assess RT function.
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