PROJECT SUMMARY/ABSTRACT Several aspects of early HIV-1 infection are both unusual and still poorly understood. In particular, HIV-1's cone-shaped viral core consists of pentamers and hexamers of capsid (CA) protein and is known to be metastable, undergoing restructuring and CA loss that is driven by and facilitates reverse transcription of the viral genome. Yet only a relatively small number of host proteins have been both structurally and functionally well-characterized in terms of how they bind to and influence core stability, and to date their binding strategies all center around recognition of hexamers. This includes microtubule motor adaptor proteins, which HIV-1 exploits to indirectly engage motor proteins to regulate both capsid stability and transport to the nucleus. Our recent work was at the forefront in identifying the Kinesin-1 adaptor for HIV-1 as Fasiculation and Elongation Factor Zeta-1 (FEZ1) and determining that negatively charged amino acids in one of FEZ1's coiled-coil domains mediate binding to the positively charged central pore of CA hexamers. While the structural basis of its interactions have yet to be determined, others subsequently found that another coiled-coil domain protein, Bicaudal D Homolog 2 (BICD2) acts as HIV-1's Dynein adaptor. Independently, we discovered that the specialized microtubule regulatory protein, Cytoplasmic Linker Protein 170 (CLIP170) binds to HIV-1 cores and in vitro assembled CA structures in a unique manner that is distinct from currently known co-factors. Specifically, unlike hexamer-binding co-factors, CLIP170 binds to the extreme ends of wildtype CA assemblies and also has a unique ability to bind and stabilize CA-R18L mutant assemblies, which form pentamer-rich rather than hexamer-rich structures. Moreover, CLIP170 binds to the Major Homology Region (MHR) of CA, which is structurally oriented inward and predicted to be inaccessible to cytosolic co-factors. However, cryoEM imaging reveals that native HIV-1 cores contain breaks while we reveal unusual pentamer organizations in R18-derived CA assemblies that create a pore which makes the MHR domain accessible from outside the capsid. From this, we hypothesize that CLIP170 recognizes the MHR upon exposure by natural breaks in the CA lattice of native cores or WT CA assemblies, or through previously unrecognized pores that form in R18- derived assemblies, and functions to then control the HIV-1 metastable state. Furthermore, our data shows that Dynactin 1 (DCTN1), a key component of the primary Dynein adaptor complex, Dynactin, also functions independently to negatively regulate the pro-viral functions of CLIP170. We hypothesize that this makes DCTN1 incompatible with HIV-1's goal of separately engaging motors through its hexamers while using CLIP170 to regulate core metastability, and that this was an evolutionary driver for HIV-1 to instead use BICD2 to engage Dynein. In this proposal, we employ cutting-edge cryoEM, biochemical and functional...