The interaction of nucleocapsid protein, a reverse transcription accessory factor, with replication primer transfer RNA

Abstract

In the initiation of reverse transcription in animal retroviruses, an obligate step is the formation of RNA duplex between the transfer RNA primer and a complementary genomic RNA sequence, termed the primer binding site (PBS). A virally encoded protein, nucleocapsid protein (NC), has been shown to be required for this step in vitro and in vivo. Aimed at understanding the initiation of reverse transcription, this work describes the purification of recombinant nucleocapsid proteins, the determination of the structural changes induced in a model TRNA as a result of binding by NC and the identification of the functional domains of NC required for productive binding to TRNA. A particularly efficient and relatively easy purification protocol for NC proteins which avoids denaturation steps of previous methodology is outlined and gives high purity and high yield of protein. The Mason-Pfizer monkey virus nucleocapsid protein (MPMV NC) and 15N-labelled MPMV NC are currently being used to determine the molecular details of the protein structure. An RNase Tl digestion of 3,-32p labelled Mg2+-tRNAHis in the presence and absence of human immunodeficiency virus type 1 nucleocapsid protein (HIV-1 NC) shows that the 3'-end acceptor stem of the NC-TRNA complex appears to have a far greater reactivity toward the single strand-specific RNase than that of the uncomplexed TRNA. Investigation of the lead-catalyzed auto-cleavage of the 5'-32p labelled yeast tRNAPhe shows that HIV-1 NC changes the conformation of the tRNAPhe molecule such that the lead cleavage is completely inhibited. From examination of an HIV-1 NC-concentration dependence of this reaction, it is found that HIV-1 NC and tRNAPhe mixed at a 0.5 to 1:1 ratio is sufficient to completely suppress the observable cleavage activity, consistent with a 1:1 stoichiometry. A number of substitution and domain deletion mutants of HIV-1 NC which have one or both zinc-fingers destroyed by mutagenesis or chemical modification and/or have either or both N-or C-terminal flanking region deleted have been examined for their abilities to inhibit the cleavage. The data taken together suggest the possibility that the N-terminal domain region (residues 1 1 1) and the zinc-finger cooperate to alter the conformation of TRNA.