Characterization of L1 ORF1p Self-Interaction and Cellular Localization Using a Mammalian Two-Hybrid System

<div><p><u>L</u>ong <u>IN</u>terspersed <u>E</u>lement<u>-1</u> (LINE-1, L1) is an active retrotransposon that mobilizes using a ribonucleoprotein particle (RNP) intermediate composed of the full-length bicistronic L1 mRNA and the two proteins (ORF1p and ORF2p) encoded by that mRNA. ORF1p and ORF2p demonstrate <i>cis</i>-preference for their encoding mRNA. Previous studies of ORF1p, purified from bacterial and insect cells demonstrated that this protein forms trimers <i>in vitro</i>. While valuable for understanding ORF1p function, these <i>in vitro</i> approaches do not provide any information on ORF1p self-interaction in the context of mammalian cells. We used a mammalian two-hybrid (M2H) system in order to study L1 ORF1p self-interaction in human and mouse cells. We demonstrate that the M2H system successfully detects human and mouse ORF1p self-interactions in transiently transfected mammalian cells. We also generated mouse and human ORF1p-specific antibodies to characterize the expression of ORF1p fusion proteins used in the M2H system. Using these antibodies, we demonstrate that ORF1p interaction <i>in trans</i> leads to the formation of heterodimers that are expected to produce a positive signal in the M2H system. Although the role for L1 ORF1p <i>cis</i>-preference in L1 mobilization is established, the impact of ability of ORF1pto interact <i>in trans</i> on the L1 replication cycle is not known. Furthermore, western blot analysis of ORF1p generated by a full-length L1, wild type ORF1, or a codon-optimized ORF1 expression vector is detected in the nucleus. In contrast, the addition of a tag to the N-terminus of the mouse and human ORF1 proteins can significantly alter the subcellular localization in a tag-specific manner. These data support that nuclear localization of ORF1p may contribute to L1 (and potentially the SINE Alu) RNP nuclear access in the host cell.</p>