The role of the ORF1 protein leucine zipper domain and cellular host factors in LINE-1 retrotransposition.

Abstract

The non-LTR retrotransposon L1 comprises &sim;17% of the human genome and L1-mediated retrotransposition events have created one-third of human DNA. Therefore, L1s are a driving force in genome evolution and diversification. L1s also may be important tools for gene therapy vectors and transposon mutagenesis mouse models, yet a better understanding of the mechanism of L1 retrotransposition and the cellular processes that inhibit retrotransposition is required. I have examined the role of ORF1p and cellular host factors in L1 retrotransposition. A retrotransposition-competent L1 encodes two proteins (ORF1p and ORF2p) that localize with L1 RNA to form a cytoplasmic ribonucleoprotein particle (RNP). ORF2p contains endonuclease (EN) and reverse transcriptase (RT) activities. ORF1p can bind RNA and participate in ORF1p-ORF1p interaction. In humans, a leucine zipper (LZ) domain lies within the region required for ORF1p-ORF1p interaction. We determined that the LZ domain is required for L1 retrotransposition, but is dispensable for ORF1p-ORF1p interaction. Mutations in the LZ domain result in the formation of mutant RNPs and block ORF2p RT activity at initiation or early steps of reverse transcription. Therefore, we conclude that mutation of the LZ domain behaves in a <italic>cis</italic>-dominant manner. Furthermore, these results suggest there is an interplay between ORF1p and ORF2p that is required for retrotransposition. We hypothesize that cellular proteins are required to promote and restrict L1 retrotransposition. We performed a yeast two-hybrid screen for proteins that interact with ORF1p as this protein has few known functions and shows evidence of positive selection. We also examined members of the APOBEC3 family for their effect on retrotransposition as these proteins restrict retroviral infection and inhibit LTR retrotransposons. We determined APOBEC3A and APOBEC3B can inhibit L1 and Alu retrotransposition, while APOBEC3G inhibits only Alu retrotransposition. Inhibition was independent of hypermutation, and in the case of Alu retrotransposition was ORF1p-independent. These results suggest that L1-mediated retrotransposition is inhibited at multiple stages. Finally, it is tempting to speculate that the APOBEC3 family may have evolved, in part, to restrict retrotransposition. In conclusion, these experiments have further elucidated a process that is responsible for one billion of the three billion bases in human DNA.