Interactions between HIV-1 reverse transcriptase and chain-terminated primer/template: Discovery of a novel primer unblocking activity involved in resistance to AZT

HIV-1 reverse transcriptase is the key enzyme responsible for replication of the HIV-1 genome. It acts as an RNA-dependent DNA polymerase, an RNaseH, and a DNA-dependent DNA polymerase to convert the HIV-1 positive single stranded RNA into double stranded DNA that is incorporated by HIV-1 integrase into the host call genome. DNA polymerization by HIV-1 RT is a sequential process in which the rate limiting step is a conformational change that occurs after binding of the dNTP complementary to the next nucleotide position on the template, but before phosphodiester bond formation.;Due to its vital role in the HIV-1 life cycle, HIV-1 RT has been one of the main targets of pharmaceutical intervention. Inhibitors of HIV-1 RT include analogs of deoxynucleosides which lack a functional 3' -OH and are phosphorylated by host-cell kinases to their active triphosphorylated form. Once the nucleotide analog is incorporated, the kick of a functional 3'-OH on the primer terminus blocks any further elongation of the primer (chain-terminators). Resistance to 3'-azido,3 '-deoxythymidine (AZT) does not seem to involve discrimination by mutant RT against AZTTP. This suggests that perhaps resistance instead is due to increased removal of AZTMP from blocked primers.;The main goal of my research was to better understand the interactions that occur between HIV-1 RT and chain-terminated primer/template in the absence and presence of the dNTP complementary to the nod nucleotide position (+1) on the template. I used DNAse-1 protection assays to probe the binding of HIV-1 RT to primer/template. The pattern of protection suggests that RT slides forward by one base relative to the primer/template in the presence of the dNTP complementary to the nucleotide at template position +1, and by two bases in the presence of dNTP complementary to the nucleotide at template position +2. The forward movement by RT suggests that the translocation stop of polymerization occurs before chemical bond formation, not after, which is the commonly held belief.;While doing DNase-1 protection assays I uncovered a novel, nucleotide-dependent activity of HIV-1 RT that can unblock chain-terminated primers. The enzyme is capable of transferring the 3'-terminal nucleotide from the primer terminus to a free acceptor nucleotide, such as ATP. The mechanism of the reaction is related to pyrophosphorolysis, but instead of using PPi as a substrate, HIV-1 RT uses the distal two phosphate groups of the acceptor nucleotide as a PPi analog. Importantly, the presence of the dNTP complementary to the nucleotide at the +1 position on the template strongly inhibits the novel unblocking reaction. This indicates that chain-terminated primer/template is not a substrate for the unblocking reaction while present in the stable DEC.;Experiments will be presented that show that the mechanism of AZT-resistance involves both increased unblocking of AZTMP-terminated primers through the novel nucleotide-dependent mechanism, but not through pyrophosphorolysis, as well as decreased sensitivity to inhibition by the complementary dNTP.;Although mutant RT can remove either 2',3 '-dideoxyadenosine monophosphate (ddAMP) or AZTMP through nucleotide-dependent primer unblocking more efficiently than WT RT, removal of ddAMP is strongly inhibited by the presence of complementary dNTP, while removal of AZTMP is insensitive to the presence of dNTPs. (Abstract shortened by UMI.)...