TRIM21 –CRM-1 INTERACTION: A NOVAL MECHANISM FOR HIV RESTRICTION IN MURINE CELLS Roqaya Sabbah- Abudaabes1, Moshe Kotler1, Richard Sutton2, Hila Elinav3 1 Hebrew University, Jerusalem,Israel,2 Yale University CT USA, 3 Hadassah AIDS Center, Jerusalem, Israel Introduction Methods Results

Multiple obstacles humper the Human Immunodeficiency ’s ( HIV) replication in We transfected 293T and B79 CTy1 (murine fibroblast expressing human TRIM21 and Sept7was found to be enriched and interact significantly more mouse. While some of the reasons for this phenomenon have been identified, additional CTy1) with the following plasmids all tagged with HA at the amino terminal with murine and mutant CRM-1 in murine cells in the presence of HIV mechanisms have not been sufficiently deciphered yet. of peptides: TRIM21 One of these mechanisms is the over-splicing of viral RNA (vRNA) in mouse cells, which Human CRM-1, Murine CRM-1,Mutant CRM-1-human CRM-1 that was Spet7

leads to a reduction in the RNA transportation that is mediated by Region mutated at AA 411,412,414, Control-Empty vector encoding for HA only Cell line +/-CIV peptides Human/NC Murine/NC Mut/NC Cell line+/-CIV peptides Human/NC Murine/NC Mut/NC Maintenance-1 (CRM-1) and the viral protein REV. CRM-1 is responsible for transmitting the non-spliced, or partially spliced, vRNA molecules, which are essential for 293T 5 0.8 1.3 1.0 293T 7 0.9 0.9 0.8 Similar transfections were performed with the addition of plasmid HIV-CIY, 293T+CIY 8 1.8 1.4 1.5 293T+CIY 5 1.0 1.0 1.4 producing infectious virus, to the cytoplasm. which encode for the full genome of HIV fused to that and encode for B78CyT1 13 1.0 0.9 1.1 B78CyT1 4 0.9 1.0 0.8 A previous study in our laboratory, showed that the mouse CRM-1 protein is distinct from YFP fleur cense peptide. Each transfection was performed in triplicates B78CyT1+CIV 10 4.5 23.0 16.6 B78CyT1+CIV 4 18.4 46.1 29.9 the human protein in 21 amino acids. Three of these amino acids (411, 412, 414) are critical for the export of the viral mRNA to the cytoplasm. Interestingly, these amino acids don’t form a Co-immune precipitation: Multiple - regular binding site for viral Rev (Regulator of Expression of Virion) protein 24 hours after transfection cells were harvested, lysed and incubated overnight Plec,Actb,Msn,Actb4,Tpm1,Cltc,Rab10,Calr,Rrbp1,Hsp90aa1,Eif5a,Cct6a,CC with anti-HA antibodies that are conjugated to agarose beads (Sigma-Aldrich). t8, Cct3,Aldoa,Aco2,Mri1,Mthfd1,Rps4X,Anxa1 – playing different roles in Samples were centrifuged and washed and were analyzed by Mass- the cell-were enriched in B78CyT1 with HIV only and were more enriched spectometry in order to identify proteins that were conjugated to the different with murine CRM-1 but not with the mutant CRM-1 Structural analysis of human CRM-1 and murine CRM: HA-CRM-1’s A. general structural similarity The conjugated proteins were regarded as significant interacting if there were Conclusions B-only heat repeats 5-9 are illustrated with AA that are different between human and murine CRM-1 are indicated. identified by 2 or more peptides, the protein was enriched in a sample more than 3 fold relative to the control (HA only ) with P<0.05 Murine CRM-1 and mutant CRM-1 have a similar site at AA 411 412 414, and both have reduced ability to produce virus in `murine cells in contrast to human CRM-1. Our results implies that Trim21 and Spet7 are candidates for further Results investigation as s inhibitors of mCRM-1, since they strongly interact with both murine CRM-1 and the mutated CRM-1 and much less to human In human cells, (293T) 1800 proteins were identified to significantly interact CRM-1. Hypothesis at least with one of the proteins: human CRM-1 Murine CRM-1 or mutant More research is needed to explore the phenomenon of multiple proteins Another protein binds to the site 411-414 , resulting in mouse CRM-1 inhibition, or human CRM-1. When HIV-CIV was added 1680 proteins were found to be that interact mainly with murine CRM-1, in B78Cyt1 only in the presence A B of HIV. CRM-1 activation. C enriched. In murine cells (B78CyT1) 993 different proteins were identified to Further understanding of the role of proteins interacting with CRM-1 significantly interact at least with one of the proteins: human CRM-1 might provide better understanding of the pathogenesis of HIV and will ,Murine CRM-1 or mutant CRM-1. When HIV-CIV was added 990 proteins significantly promote the use of mice models in studying HIV replication, were found to be enriched. pathogenesis, and development of new effective treatments.

A. A protein that can interact with the murine CRM-1 binding site (AA 411,412,414) but not with the human binding site, blocks the binding site of CRM-1 and REV. B. Protein that can interact with the human CRM-1 binding site (AA 411,412,414) but not with the murine binding site, facilitate the binding of CRM-1 and REV. C. Rev interacts with CRM-1 in a few binding sites and the one of binding sites is incompatible with the murine AA 411,412,414 Poster#200 Supported by the Israel science foundation