Mechanism of nucleotide incorporation at nontemplated sites by HIV-1 reverse transcriptase

Human immunodeficiency virus type 1 (HIV-1) is highly genetically variable. This is attributed to the error-prone nature of HIV-1 replication and its proclivity for recombinaton. During replication and recombination, reverse transcriptase (RT) must polymerize DNA to the 5′ ends of multiple RNA and DNA template termini while convening HIV-1 RNA to double-stranded DNA. We have determined the fidelity of HIV-1 RT during polymerization to the 5′ ends of HIV-1 long terminal repeat DNA template sequences and to the end of a partial HIV-1 genomic RNA template that mimics a recombination intermediate. We find that HIV-1 RT binds tightly and extends blunt-end duplexes at an efficiency approaching that of recessed-end primers. These products were formed processively via a nontemplated mechanism. While these nontemplated additions are specific for purine nucleotides in incubations containing a single dNTP, highly efficient additions of both purines and pyrimidines are seen in incubations containing all four dNTPs. A molecular model of blunt-end addition is proposed from a structural analysis of HIV-1 RT complexed with primer·template. These observations suggest a potentially important mechanism for retroviral base substitution, small insertions, and small deletion mutations mediated by blunt-end additionand forced copy-choice recombination.