CIS elements and Trans-acting factors required for minus strand DNA transfer during reverse transcription of the genomic RNA of murine leukemia virus

Abstract

During reverse transcription of the retroviral genomic RNA, two obligatory DNA strand transfers take place to synthesize the complete proviral DNA with two LTRs. We have previously shown that using an in vitro system made up of two viral RNAs mimicking the 5′ and 3′ regions of the retroviral genome, both nucleocapsid protein and the repeat (R) sequences were necessary for minus strong-stop cDNA (ss-cDNA) transfer and elongation by reverse transcriptase (RT). In this paper we show that the basic residues of nucleocapsid protein NCp10 of Moloney murine leukemia virus (MoMuLV), but not the zinc finger, are necessary for minus strand transfer. In order to examine the role of the R sequence repeated at the 5′ and 3′ ends of the genome in minus strand DNA transfer, the MoMuLV R sequence of 68 nt was replaced by either HIV-1 R of 96 nt, or RSV R of 21 nt, or by an artificial sequence of 21 nt. Analysis of MoMuLV DNA strand transfer from the 5′ RNA to the 3’ RNA and elongation in the presence of NCp10 and RT showed that it was high with control MoMuLV R, high with RSV R, reduced with HIV-1 R, and undetectable with the artificial R sequence. These results suggest that minus strand DNA transfer is a process more complex than simple hybridization of ss-cDNA to the 3′ R sequence of the genomic RNA.