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> </div

Analysis of subcellular localization of hORF1p expressed from vectors containing wild type sequences.

<p><b>A</b>. (top) Western blot analysis of subcellular localization of ORF1p (blue rectangle labeled as hORF1) expressed from human full-length L1 (hL1wt, 40 kDa), human ORF1 wt (hORF1wt, 41 kDa) or hORF1co (40 kDa) vectors transiently expressed in HeLa cells. Human ORF1p is detected in nuclear (N) and cytoplasmic (C) fractions with human-specific ORF1p polyclonal antibodies. GAPDH (cytoplasmic marker) and Lamin A (nuclear marker) are used as loading and cell fractionation controls. 37 kDa is a molecular marker. Control lanes indicate cells transfected with empty vector. (bottom) Quantitation of western blot results: each construct expressing hORF1p was normalized to its respective GAPDH and Lamin A loading controls. The relative amounts of nuclear and cytoplasmic ORF1p were calculated as a fraction of the relative total ORF1p detected. No statistical significance was found between the subcellular localization of ORF1p expressed by the above-described constructs. <b>B</b>. The same experiment and analysis as in A was performed in NIH-3T3 cells. No statistical significance was found between the subcellular localization of ORF1p expressed by the above-described constructs. <b>C</b>. Subcellular localization of mORF1p (green rectangle labeled as mORF1) transiently expressed from the full-length mouse L1 (mL1wt) or codon-optimized mORF1 (mORF1co) in HeLa or NIH-3T3 cells using mouse specific polyclonal antibodies. 50 kDa is a molecular marker. </p

Analysis of ORF1p interaction using the M2H system.

<p><b>A</b>. (left) Schematic of the M2H constructs, arrows denote promoters. One ORF1p is fused to a GAL4 binding protein, derived from the GAL4 yeast transcription factor which binds a specific sequence of DNA. The other ORF1 protein is fused to the VP16 viral transcriptional transactivator protein sequence, with an added SV40-derived internal nuclear localization signal. Tandemly-arrayed GAL4 binding sites are present upstream of the firefly luciferase gene. (top right) Interaction between proteins of interest expressed from the M2H expression plasmids leads to firefly luciferase expression. (bottom right) If the proteins do not interact with one another, no firefly luciferase is produced. <b>B</b>. M2H results for codon-optimized and wild type hGORF1p:hVORF1p interaction in HeLa and NIH-3T3 cells normalized to the positive control. Asterisk denotes statistical significance between the relative luciferase signals detected for hG:VORF1p interaction (wt or co) in HeLa or NIH-3T3 cells compared to their respective negative control (p-value <0.05). Double asterisk denotes statistical significance between relative signal reflecting interaction between hORF1co and VORF1co detected in HeLa versus NIH-3T3 cells (p-value<0.05). <b>C</b>. M2H results for codon-optimized and wild type mGORF1p:mVORF1p interactions in HeLa and NIH-3T3 cells normalized to their respective positive controls. Asterisk denotes statistical significance between the relative luciferase signals detected for mG:VORF1p (wt or co) when compared to their respective negative controls (p-value<0.05).</p

Analysis of ORF1p interactions <i>in</i><i>trans</i>.

<p><b>A</b>. Schematic of predicted ORF1p species expected to be detected if ORF1p heterodimerization takes place when cells are transiently cotransfected with tagged and untagged ORF1p expression plasmids. Blue rectangle represents ORF1, grey rectangle represents a GAL4 tag, with expected molecular weights expressed in kilodaltons (kDa). Red asterisk denotes heterodimers. <b>B</b>. Western blot analysis of ORF1p species expressed by hORF1co (blue rectangle labeled hORF1co) and hGORF1co (grey/blue rectangle labeled hGORF1co) plasmids individually transfected (hORF1co lane and hGORF1co lane) or cotransfected (hORF1co+hGORF1co lane) in HeLa cells. Western blot analysis of ORF1 protein species in HeLa cells under non-reducing (left panel) and reducing (right panel) conditions. Proteins are detected with human specific polyclonal antibodies. GAPDH used as loading control. A molecular weight ladder in kilodaltons (kDa) is presented on right side. Red asterisk indicates an ORF1p heterodimer. <b>C</b>. The same experiment as in B is performed in NIH-3T3 cells. Non-reducing western blot conditions are shown. Red asterisk denotes ORF1p heterodimers. <b>D</b>. Quantitation of western blot results from B and C using heterodimer/ORF1p monomer ratio. Statistical significance is indicated by dashed lines. An asterisk indicates statistically significant difference (T-test, p-value<0.05).</p

Analysis of M2H ORF1 fusion protein expression in human and mouse cells.

<p><b>A</b>. (top) Western blot analysis of codon-optimized human ORF1 (hORF1co, 40 kDa, blue rectangle labeled as ORF1) and human ORF1co N-terminally fused to GAL4 or VP16, (hGORF1co, 57 kDa, and hVORF1co, 49 kDa, blue and grey rectangles labeled as ORF1 fusion) transiently transfected in HeLa or NIH-3T3 cells. Human ORF1 protein was detected with custom-made human-specific ORF1 polyclonal antibodies. GAPDH is used as a loading control. 37 and 50 kDa are molecular markers. Control lanes indicate cells transfected with an empty vector. (bottom) Quantitation of western blot results. Signals obtained for hORF1p and its fusions were normalized to their respective GAPDH loading controls and expressed as a percentage of the relative signal detected for each protein in NIH-3T3 cells. <b>B</b>. (top) Western blot analysis of codon-optimized mouse ORF1 (mORF1co, 47 kDa, T7-Myc-His tag, green rectangle labeled as ORF1) and mouse ORF1co N-terminally fused to GAL4 and VP16 (mGORF1co, 60 kDa, and mVORF1co, 52 kDa, green and grey rectangles labeled as ORF1 fusion) transiently expressed in HeLa or NIH-3T3 cells. 50 kDa is a molecular marker (bottom) Quantitation was performed as described in A. </p

Analysis of subcellular localization of ORF1 protein fusions in human and mouse cells.

<p><b>A</b>. (top) Western blot analysis of subcellular localization of hORF1co (blue rectangle labeled as hORF1), hGORF1co and hVORF1co (grey/blue rectangle labeled as hORF1 fusion) transiently expressed in HeLa cells. Subcellular localization of the human ORF1 protein detected in nuclear (N) and cytoplasmic (C) fractions with human-specific ORF1p polyclonal antibodies. GAPDH (cytoplasmic marker), Lamin A (nuclear marker), and Calregulin (endoplasmic reticulum marker) proteins are used as loading and cell fractionation controls. Control lanes indicate cells transfected with empty vector. (bottom) Quantitation of western blot results. hORF1 and its fusions were normalized to their respective GAPDH and Lamin A loading controls. The relative nuclear and cytoplasmic ORF1p was calculated as a fraction of the relative total ORF1p detected in respective cell types. <b>B</b>. The same experiment and analysis as in A, but using NIH-3T3 cells. <b>C</b>. Western blot analysis as above using mORF1co (green rectangle labeled as mORF1), mGORF1co or mVORF1co (grey/green rectangle labeled as mORF1 fusion) in HeLa cells. Mouse specific ORF1p polyclonal antibodies were used for protein detection. <b>D</b>. The same experiment and analysis as in C, using NIH-3T3 cells. For all panels, statistically significant data points are indicated by dashed lines. An asterisk represents a significant difference between the subcellular localization of ORF1p and VP16-fused ORF1p (T-test, p-value <0.05).</p