Filoviruses Ebola (EBOV) and Marburg (MARV) are emerging pathogens capable ofcausing explosive outbreaks with high mortality rates. Host proteins play critical roles in regulating (positively or negatively) EBOV and MARV entry and exit, and their identification can provide biological and mechanistic insight critical for development of new therapeutics and countermeasures. The overall goal of my thesis project was to identify and characterize novel host regulators of the filovirus lifecycle, with a specific focus on filovirus egress. The VP40 matrix proteins of EBOV (eVP40) and MARV (mVP40) are the key drivers of virus assembly and egress, and VP40 can bud independently from cells in the form of virus-like particles (VLPs). We identified E3 ubiquitin ligase Smad Ubiquitin Regulatory Factor 2 (SMURF 2) as a novel interactor with VP40 that positively regulates VP40 VLP release. Our results show that eVP40 and mVP40 directly interact with SMURF2 and provide evidence that the eVP40-SMURF2 interaction is functional. Notably, expression of SMURF2 positively regulates VLP egress, while siRNA knockdown of endogenous SMURF2 decreases VLP egress. Additionally, we identified actin-binding cytoskeletal crosslinking proteins filamin A (FLNa) and B (FLNb) as important regulators of both EBOV and MARV entry, as well as EBOV exit. EBOV and MARV entry is mediated by the EBOV glycoprotein (eGP) or MARV glycoprotein (mGP), respectively, and our results show that entry of VSV-eGP pseudotypes, infectious VSV-eGP-mCherry recombinant virus, and live authentic EBOV and MARV was inhibited in filamin A knockdown (FLNaKD) cells, but was surprisingly enhanced in filamin B knockdown (FLNbKD) cells. Mechanistically, our findings suggest that differential regulation of macropinocytosis by FLNa and FLNb likely contributes to their distinct effects on filovirus entry. Interestingly, we found that budding of eVP40 VLPs, eVP40-expressing lentivirus particles, as well as authentic EBOV was impaired significantly in both FLNaKD and FLNbKD cells. In sum, we identified and characterized novel host regulators of the filovirus lifecycle, which will provide new insights into the development of antivirals to prevent and/or treat filovirus infections.