Human immunodeficiency virus nucleocapsid protein stimulates strand transfer from internal regions of heteropolymeric RNA templates

Abstract

We have examined the influence of HIV nucleocapsid protein (NCp) on strand transfer from internal regions of a heteropolymeric RNA template. The system consisted of a DNA-primed 225 nucleotide RNA donor template, on which reverse transcriptase initiated primer extension, and a 189 nucleotide RNA acceptor template, to which extended primers could transfer. The last 133 nucleotides on the 3′ end of the acceptor were homologous to an internal region on the donor, limiting homologous strand transfer to this region. Primers extended to the end of the donor were 212 nucleotides while those transferred to, and extended on the acceptor were 259 nucleotides in length. The rate of strand transfer and the level of transfer products increased several-fold when nucleocapsid was included in the reactions. The increase was due, at least in part, to the transfer to, and extension on the acceptor, of incompletely elongated primer-extension products that were “chased” into transfer products in the presence of nucleocapsid. Nucleocapsid did not directly influence reverse transcriptase elongation as the enzyme processivity (number of nucleotides incorporated before the enzyme dissociates from the primer-template) was approximately the same in the presence and absence of nucleocapsid.