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.
