PROJECT SUMMARY: The placenta forms a physical barrier to HIV transmission by separating maternal and fetal blood and a functional barrier by expressing a plethora of restriction factors to inhibit viral replication. However, vertical transmission of HIV does occur in utero and shows that these defenses are imperfect. This is consistent with the recently described viral restriction factor, Serinc, which is highly expressed in the placenta and can incorporate into budding viral particles to inhibit their ability to infect. However, Serinc is an imperfect restriction factor as it is inactivated by the viral accessory protein, Nef, and some sequences of the HIV surface protein, Env. We plan to study the mechanism of how Serinc restricts HIV infection to gain a better understanding of host-pathogen interactions occurring in the placenta and with the hope of enabling the development of novel anti- virals that can exploit the same viral weakness as Serinc but are not susceptible to the same pitfalls. Serincs are a family of 5 human plasma membrane proteins expressed in select tissues. Isoforms Serinc3 and Serinc5 restrict HIV proliferation while Serinc2 incorporates into viral particles but does not restrict. The exact mechanism by which Serinc3 and 5 reduce infectivity is incompletely understood but is known to block infection at a step before cell entry. Two major theories have emerged: 1) Serinc inactivates Env by causing changes to the conformation and distribution of Env trimers in the viral particle or 2) Serinc slows or disrupts membrane fusion of the virus with the host cell plasma membrane. In this study, we will take advantage of the development of plasma membrane “blebs” as a model for viral membrane fusion to test the relative merits of two proposed mechanisms of Serinc. We will assess Serinc- containing or -lacking HIV pseudoviruses for changes in structure and distribution of Env by cryo- electron tomography and subtomogram averaging (aim 1). We will also assess whether Serincs block membrane fusion by observing HIV pseudoviruses containing or lacking Serincs as they fuse to bleb model membranes with high resolution cryo-electron tomography snapshots (aim 2.1) and higher throughput fluorescence microscopy single-particle viral fusion assays (aim 2.2). Preliminary results show Serinc3 and Serinc5 block membrane fusion by disrupting fusion pore opening but do not affect Env distribution or the speed at which viral particles undergo membrane fusion. With these two aims, we will be able to discriminate between competing hypotheses about the mechanism of Serinc restriction of HIV infection, gain a better understanding of how HIV transmits in the placenta, and potentially enable the development of new anti-virals.