Characterizing the physicochemical properties of membraneless condensates and its regulation by delta-9-tetrahydrocannabinol in HIV/SIV infection.

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Abstract The `central dogma' of molecular biology is that in cells, the functions of proteins and nucleic acids are primarily determined by their sequence. However, this dogma has been challenged by studies of secreted molecules including the membrane encased extracellular vesicles (EVs) and the membraneless structures known as BMCs, which indicate that cells create discrete chemical environments where they organize complex biomolecules. BMCs form in many compartments of the body, including the nucleus (nucleolus, centrosome, Cajal body, stress granules), the cytoplasm (P-bodies, stress granules), and even the neuronal cytoplasm (neuronal transport granules). While the details of BMC biology is still evolving, it is known that RNA and proteins with intrinsically disordered regions (IDRs) are key factors that influence intracellular composition, size, and morphology of BMCs. The size and composition of BMCs are also thought to be different. BMCs participate in the regulation of various physiological processes, such as modulation of host transcription and stress response. BMCs also play important roles in pathophysiological conditions, including cancer, neurodegenerative diseases, and viral pathogenesis, including modulation of HIV replication. Indeed, it is known that HIV infection results in peripheral and neuro inflammation, and that chronic exposure to THC in the context of HIV in humans or SIV infection in rhesus macaques results in amelioration of disease or symptoms. However, the regulators of HIV disease and THC response is not completely understood. Despite the reported role of BMCs in various conditions, whether or not BMCs regulate the pathogenesis of HIV infection and the intersection of HIV and THC use is unknown. It is also unknown whether BMCs assemble in vivo, where they may play relevant biological roles. There is paucity of information on the presence of BMCs in body fluids (in vivo) and their role in HIV pathogenesis. The reason for this scarcity of information is partly due to lack of tools for isolation of BMCs from body fluids. In preliminary studies, we developed a novel size-guided Particle Purification Liquid Chromatography (PPLC). We used PPLC to isolate and retrieve dye-free BMCs from human semen, rhesus macaque blood plasma and cerebrospinal fluid (CSF). Using standard electron microscopy and Energy Dispersive X-ray equipped transmission electron microscope (TEM-EDX) analyses, we obtained structural and elemental information of the BMCs. Proteomics analysis and RNA bioanalyzer study were used to show that the BMCs contain protein and RNA, while cell biology assay indicate that BMCs are cytotoxic to cells. These data provide compelling evidence that bioactive BMCs are present in vivo. Thus, in this proposal, we will use PPLC to isolate BMCs from rhesus macaque blood, CSF, saliva, and intestinal contents. We will then use various structural, biochemical, and cell biology assays to provide structural, molecular, and functional classification of BMCs from SIV infected and THC treated macaques, which will provide a framework to dissect BMC functions in HIV/AIDS and THC use.