EFFECT OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 (HIV-1) NUCLEOCAPSID PROTEIN ON HIV-1 REVERSE-TRANSCRIPTASE ACTIVITY IN-VITRO

Conversion of human immunodeficiency virus type 1 (HIV-1) genomic RNA to viral DNA is a requisite step in the virus life cycle. This convers ion is catalyzed by reverse transcriptase (RT) associated with a large nucleoprotein complex composed of several viral proteins including nu cleocapsid (NC). To better characterize the biochemical mechanisms of viral DNA synthesis, we overexpressed and purified recombinant HIV-1 N C and studied its effect on the activity and processivity of HIV-1 RT during polymerization of HIV-1 template sequences in vitro. The effect of NC on steady-state RT activity was dependent on the order of addit ion of reaction components. Addition of NC prior to formation of RT-pr imer . template-dNTP ternary complexes inhibited primer extension and reduced total product yields by slowing steady-state RT turnover. In c ontrast, addition of NC to preformed ternary complexes resulted in eff icient primer extension and increased RT processivity at specific DNA template sites. NC stimulated polymerization (2-4 times) through eight of 13 sites examined in the cRRE region of HIV-1 env and increased th e rate of polymerization through the D3/CTS region of HIV-1 pol 10 tim es. The data suggest that NC affects RT processivity by facilitating p olymerization through regions of template secondary structure. Thus, N C functions as a single-strand binding (SSB)-like accessory replicatio n factor for RT in vitro and may be part of a multicomponent retrovira l replication complex.