A. Raja et Jj. Destefano, Kinetic analysis of the effect of HIV nucleocapsid protein (NCp) on internal strand transfer reactions, BIOCHEM, 38(16), 1999, pp. 5178-5184
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.