Determining the role of microtubules and motor proteins during early HIV-1 replication SUMMARY During HIV-1 infection, viral membrane fuses with the host cell membrane to deliver the HIV-1 virus to the host cell cytoplasm. Once within the cell, HIV-1 must translocate to the nucleus for the reverse transcribed DNA to be incorporated into the host genome. Despite 40+ years of research devoted to HIV-1 infection, surprisingly little is known about how HIV-1 exploits the host cell cytoskeleton to facilitate transport to the nucleus and accelerate infection. The goal of this proposal is to establish and validate an assay to measure HIV-1 microtubule trafficking in vitro via kinesin and dynein microtubule motor proteins. Recent work has led to the identification of two host factors - FEZ1 and BicD2 - that serve as cargo adaptors for transport via kinesin-1 and cytoplasmic dynein-1, respectively. To study the role of FEZ1 and BicD2 in the transport of HIV-1, we will utilize single-molecule total internal reflection fluorescence (TIRF) microscopy to monitor the transport of fluorescently-labeled virus-motor complexes on reconstituted microtubules (Aim 1). We will use a novel synthetic icosahedral scaffold derived from encapsulins to validate the assay, which we will use to measure the motility of motor protein teams bound to viral cargo via cargo adaptors. We will then determine how HIV-1 determines directionality on microtubules (kinesin vs. dynein) by measuring the combined influences of FEZ1, BicD2, kinesin-1, and dynein (Aim 2). By establishing that microtubule motors are capable of transporting HIV-1 in vitro, this proposal will provide a new avenue in the study of HIV-1 and provide new targets to block HIV-1 infection.