The mature HIV-1 capsid core has emerged as a key antiviral target because of its critical role in HIV infectivity and the discovery of capsid-specific host restriction factors, such as TRIM5? and MX2. Recent work has demonstrated that capsid does much more than simply house the viral genetic material and required replication enzymes. It also participates in and may mediate several critical replication events, including uncoating, initiation of reverse transcription, nuclear import, integration, and evasion of host immune responses. For each of these processes to occur, a delicate balance between capsid stability and dissociation must be maintained, demonstrating an intricate link between capsid and viral infectivity. The mature capsid lattice is formed following protease-mediated cleavage of the Gag polyprotein. The basic structural element of the mature lattice is a capsid protein (CA) hexamer, comprising a trimer of CA dimers. The fullerene cone structure contains ~250 hexamers, along with 12 pentamers to facilitate closing. Mutations that alter the relative stability of capsid protein (CA) assembly states (dimers, pentamers, hexamers, or the assembled lattice) result in severe infectivity defects due to disruption of one or more replication events. Replication can also be impacted through alteration of host factor binding. Notably, the specific roles of CA in these events is not well understood, and it is unknown how the various CA assembly states contribute to capsid function or interactions with host factors involved in replication. There are currently no tools available to differentiate CA assembly states in vivo to assess their role during replication events. This project will identify and characterize RNA aptamers that bind sites specific to the assembled hexamer lattice and differentiate among CA assembly states by binding to unique solvent-exposed crevices that define each independent CA assembly state. The proposed experiments capitalize on the research team's expertise in poly-target aptamer selection, advances in post-selection bioinformatics analysis, intracellular aptamer expression, HIV biology, and innate immunity. Importantly, the proposed approach could be widely applicable to other viruses of importance to public health.
Health Relevance This study may lead to better understanding the role of HIV-1 capsid protein in replication and the interactions of capsid with cellular factors through the proposed work to develop important tools (aptamers) that differentiate among independent capsid assembly states, allowing investigation of replication events that are not well understood. Of particular significance, these aptamers could identify important, previously unidentified interaction sites for both viral and host proteins. Future work beyond this proposal could inform targeting of newly identified, critical sites on HIV capsid by small molecule drugs, and notably, the proposed approach could be further applied to other viruses of public health importance.
