Single Molecule Study of HIV-1 Reverse Transcriptase Polymerization Activity in the Presence of NC

Abstract

HIV-1 reverse transcriptase (RT) is a multifunctional polymerase, which synthesizes double-stranded proviral DNA from single-stranded viral RNA by catalyzing RNA- and DNA-dependent DNA polymerization and degrading RNA via its RNase H activity. Reverse transcription is an essential step in HIV-1 infection, and HIV-1 RT is the target of many anti-AIDs therapeutic drugs. HIV-1 nucleocapsid (NC) protein is a nucleic acid chaperone, which facilitates DNA duplex melting and re-annealing, and shows rapid protein-nucleic acid interaction kinetics. The effect of NC on the reverse transcription process is not fully understood. To gain insights into the polymerase activity of RT in the presence of NC protein, we use single molecule force spectroscopy to examine DNA polymerization activity of HIV-RT along long single-stranded DNA (ssDNA) templates with and without NC. Our preliminary observations show the polymerization activity of RT is dependent on the force on ssDNA templates; an increase in the force on ssDNA templates reduces the polymerization activity of HIV-RT. The observed exponential dependence of polymerization activity of RT with force on long ssDNA templates is consistent with previous single molecule studies, and NC appears to enhance the polymerization rate. Our studies will test the polymerization activity of RT in the presence of NC, allowing us to determine the biophysical mechanism by which NC enhances this activity.