Sunday, May 5, 2024
5/5/2024
Project Summary HIV remains a significant problem worldwide. The CA protein of HIV is involved in several critical replication events, including Gag oligomerization and viral assembly, maturation, reverse transcription (RT), trafficking to the nucleus via interaction with host factors, nuclear import, integration, and evasion of host immune responses. Despite significant advances in our understanding of the role of CA in replication, there are many unresolved questions regarding CA structural dynamics during viral assembly and post-entry replication steps, CA-host factor interactions, and the impact of these interactions on virus biology. The genetic fragility of CA and difficulty of examining CA-host interactions in cells represent significant barriers to the resolution of these questions. To address these challenges and knowledge gaps, we are working to identify and develop novel tools capable of discriminating among different CA assembly forms. To date, we have identified RNA aptamers that bind specifically to the CA lattice, but not other assembly forms, and those that bind both the CA lattice and CA hexamer assembly forms. Aptamers represent unique tools particularly well-suited to the study of CA assembly forms and interaction sites, as they bind targets with high specificity, discriminate among different conformations of the same protein, can be expressed in or delivered to cells, can be used to outcompete other interacting partners, and are amenable to a variety of different modifications. Interestingly, our aptamers identified to date display different biological phenotypes, suggesting that they may target unique sites on CA or influence CA function in distinct ways. Here, we propose two aims in support of our goal to develop aptamers as useful tools for the field. The focus of Aim 1 is to identify new aptamers that target additional CA assembly forms, building upon our prior work supporting feasibility of our approach. Our ultimate goal is to develop a panel of aptamers with specificities to all relevant CA assembly states. The focus of Aim 2 is to develop an aptamer-mediated, CA assembly state-specific affinity purification method paired with crosslinking mass spectrometry to identify novel CA assembly state-associated host factors. Collectively, these tools will tremendously benefit the field, as there are currently no tools available for the differentiation of CA assembly states. Importantly, aptamers are amenable to a variety of modifications that will facilitate applications including affinity purification, microscopy-based tracking of CA during replication, expression in cells for evaluation of replication effects and mechanistic studies, and delivery to cells to compete for binding to specific CA sites. Furthermore, future determination of sites of aptamer-CA interaction will help identify novel accessible sites on CA for potential therapeutic targeting.
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