+TIPs as novel host capsid-binding co-factors in early HIV-1 infection

Upon entry into a host cell, the unusual cone-shaped capsid core of HIV-1 must traverse the cytoplasm to deliver its genome to the nucleus. The viral core consists of pentamers and hexamers of capsid (CA) protein and is known to be metastable, undergoing restructuring and CA loss during early infection. 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 the known binding strategies of these proteins center around hexamers. Notably, HIV-1 exploits host microtubules and microtubule motors to regulate both capsid stability and transport to the nucleus, yet unlike many other viruses HIV-1 does not engage motors directly. We recently discovered that HIV-1 exploits Fasiculation and Elongation Factor Zeta-1 (FEZ1) as its Kinesin-1 adaptor and determined that it uses negatively charged amino acids in one of its coiled-coil domains to bind to the positively charged central pore of CA hexamers. While the structural basis of its interactions have yet to be determined, others have shown that another coiled-coil domain protein, Bicaudal D Homolog 2 (BICD2) acts as the Dynein adaptor for HIV-1. Distinct from these motor adaptors, we also recently discovered that Cytoplasmic Linker Protein 170 (CLIP170), a prototypical member of the highly specialized family of microtubule regulatory proteins known as +TIPs, binds HIV-1 cores and capsid structures in a unique manner that centers around pentameric organizations, and functions to regulate both capsid stability and transport. This previously unrecognized capsid-recognition strategy involves binding to the Major Homology Region (MHR) of CA, which is structurally oriented inward and predicted to be inaccessible to cytosolic co-factors. However, in new preliminary data, cryoEM imaging of capsid structures reveals that specific pentamer organizations create a pore that makes the MHR domain accessible from outside the capsid, a previously unrecognized state predicted by our prior CLIP170 binding and functional studies. Furthermore, our data shows that Dynactin 1 (DCTN1), a key component of the primary Dynein adaptor complex, Dynactin, also “moonlights” as a +TIP and negatively regulates the pro-viral functions of CLIP170. We hypothesize that this dual functionality of DCTN1 made it incompatible for HIV-1 to separately engage motors while using +TIPs such as CLIP170 to regulate core metastability, and that this was an evolutionary driver for HIV-1 to use the coiled-coil-based motor adaptors, FEZ1 and BICD2. We further hypothesize that studies of CLIP170’s unusual pentamer-based recognition strategy will enable us to capture and study metastable states of the HIV-1 core during early infection. In this proposal, we employ cutting-edge cryoEM, biochemical and functional approaches to test these hypotheses. We anticipate that findings from the proposed studies will provide fundamental new insights into host co-factors that control both capsid structure and metastability during early stages of infection, along with a better understanding of why HIV-1 exploits less conventional motor adaptors.