Identification of Cellular Host Factors That Associate With LINE-1 ORF1p and the Effect of the Zinc Finger Antiviral Protein Zap on LINE-1 Retrotransposition.

Abstract

Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome. The human genome contains over 500,000 L1 sequences, which account for approximately 17% of human DNA. L1 sequences mobilize throughout the human genome by a copy-and-paste mechanism known as retrotransposition. Most genomic L1 sequences are incapable of mobility (i.e., retrotransposition) because they are either 5'-truncated, internally rearranged, and/or mutated; however, it is estimated that each human cell contains at least 80-100 intact L1 sequences that are retrotransposition capable. L1 retrotransposition is inherently mutagenic and on occasion can disrupt gene expression leading to diseases such as hemophilia A and cancer. Due to the mutagenic potential of L1 retrotransposition, it thus stands to reason that the host cell has evolved mechanisms to protect the cell from unabated retrotransposition. In this thesis I identified cellular host factors that associate with the first L1 open reading frame protein, ORF1p. I demonstrate that the zinc finger antiviral protein ZAP associates with L1 ORF1p and inhibits human L1 and Alu retrotransposition as well as the retrotransposition of LINE elements from mice and zebrafish. Molecular genetic, biochemical, and fluorescence microscopy data suggest that ZAP interacts with L1 RNA and reduces the expression of full-length L1 RNA and the L1-encoded proteins, thereby providing mechanistic insight into how ZAP may restrict retrotransposition. In addition to ZAP, I show that the ORF1p-associated cellular host factors MOV10, hnRNPL, and PAR-4 also inhibit L1 retrotransposition. Mechanistic data suggest that ZAP, MOV10, hnRNPL, and PAR-4 restrict L1 retrotransposition by distinct mechanisms, suggesting that each of these cellular host factors may target different post-transcriptional steps in the L1 retrotransposition cycle. Importantly, ZAP and MOV10 were first characterized as antiviral proteins due to their ability to suppress retroviral activity. Notably, several other host cell antiviral factors such as APOBEC3 proteins, TREX1, SAMHD1 and RNase L have recently been demonstrated to inhibit L1 retrotransposition. Thus, these data suggest that ZAP, MOV10 and perhaps other ORF1p-associated cellular host factors initially may have evolved to combat L1 and other endogenous retrotransposons and subsequently were co-opted as viral restriction factors.