Kinetic analysis of the effect of HIV nucleocapsid protein (NCp) on internal strand transfer reactions

The mechanism of HIV reverse transcriptase (RT) catalyzed strand transfer s ynthesis (i.e., switching of the primer to a new template) from internal re gions on RNA templates in the presence and absence of HIV nucleocapsid prot ein (NCp) was investigated. Two different systems each consisting of DNA-pr imed RNA donor (on which primer extension initiated) and acceptor (to which DNAs initiated on the donor could transfer) templates were used to determi ne kinetic parameters of strand transfer. The donor and acceptor shared an internal region of homology where homologous strand transfer could occur. T he rate of strand transfer at various acceptor concentrations was determine d by monitoring the production of transfer products over time. These rates were used to construct Lineweaver-Burk plots. In each system, NCp increased the V-max about 3-fold while the K-m for acceptor template was decreased s everalfold. NCp's effects on RT extension ranged from no effect to inhibiti on depending on the primer-template used. The lowered K-m shows that NCp in creases the affinity of the acceptor template for the transferring DNA. V-m ax increases despite the inhibition of RT extension. The increased V-max im plies a stimulatory mechanism that cannot be mimicked by high acceptor conc entrations. Therefore, NCp does not act by merely increasing the effective concentration of nucleic acids.