Roles of HIV-1 capsid-binding FG-motif containing cellular cofactors in infection

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Abstract Early events in HIV-1 lifecycle, such as post-fusion trafficking of viral cores across the cytoplasm, through the nuclear pore complex (NPC) and into the nucleus, remain poorly understood due to limited information about virus-host interactions. Interactions of the core’s surface, which is composed of the capsid protein (CA) arranged into large hexameric lattices and exactly 12 pentamers, with a variety of host cell proteins that aid infection (dependency factors) are crucial for this journey. However, their full identity and molecular mechanisms of action remain largely unknown. Our preliminary studies resulted in the following two principal discoveries. 1) We have identified SEC24C as a new, crucial HIV-1 host dependency factor. SEC24C is a predominantly cytoplasmic protein that employs a phenylalanine-glycine (FG)-motif to specifically interact with the hexameric CA lattice at the hydrophobic pocket comprised of two adjoining subunits. These novel findings, coupled with the known roles of other CA-binding FG-motif containing cellular factors NUP153 and CPSF6 in nuclear import and integration site selection of HIV-1, suggest that these proteins collectively provide a dependable platform for continuous HIV-1 journey throughout different cellular compartments during the virus ingress to ensure productive infection. 2) We discovered a prion- like domain (PrLD)-mediated mechanism for avid binding of SEC24C, NUP153 and CPSF6 to hexameric CA lattices. Our preliminary cryo-EM and HDX-MS studies provide novel structural information indicating that, in addition to known CPSF6 FG peptide binding to the cognate CA hydrophobic pocket, PrLD-PrLD interactions enable polyvalent assembly of CPSF6 molecules along the extended lattices of adjoining CA hexamers. Our virology experiments further support an essential role of CPSF6 PrLD in functional virus- host interactions in infected cells. To extend these exciting, paradigm-shifting preliminary studies we propose the following three specific aims: Aim 1 will define a role of SEC24C in HIV-1 infection; Aim 2 will elucidate interplay between SEC24C and other CA-binding host factors; Aim 3 will determine the structural basis for avid recognition of hexameric HIV-1 CA lattices by FG-motif and PrLD containing cellular factors SEC24C, NUP153 and CPSF6. To accomplish these aims we have assembled a highly collaborative team with complementary expertise in virology, proteomics, biochemistry and X-ray crystallography (Kvaratskhelia), cryo-EM (Asturias), live cell microscopy (Melikian) and HDX-MS (Griffin). These studies are expected to uncover novel virus-host interaction mechanisms crucial for HIV-1 infection. Furthermore, elucidating structural determinants for functionally relevant interactions of SEC24C, NUP153 and CPSF6 to hexameric CA lattices will provide a new frontier in HIV-1 structural biology and improve our understanding of these virus-host interactions as an important therapeutic target.