Caroe, E. | bioRxiv Science

bioRxiv2024

HIV-1 Capsid Rapidly Induces Long Lived CPSF6 Puncta in Non-dividing Cells but Similar Puncta Already Exist in Uninfected T-cells

The HIV-1 capsid (CA) protein forms the outer shell of the viral core that is released into the cytoplasm upon infection. CA binds various cellular proteins, including CPSF6, that directs HIV-1 integration into speckle associated domains in host chromatin. Upon HIV-1 infection, CPSF6 forms puncta in the nucleus. Here, we characterised these CPSF6 puncta further, in HeLa cells, T-cells and macrophages and confirm that integration and reverse transcription are not required for puncta formation. Indeed, we found that puncta formed very rapidly after infection, correlating with the time that CA entered the nucleus. In aphidicolin-treated HeLa cells and macrophages, puncta were detected for the length of the experiment, suggesting that puncta are only lost upon cell division. CA still co-localised with CPSF6 puncta at the latest time points, considerably after the peak of reverse transcription and integration. Intriguingly, the number of puncta induced in macrophages did not correlate with the MOI, or the total number of nuclear speckles present in each cell suggesting that CA/CPSF6 is only directed to a few nuclear speckles. Furthermore, we found that CPSF6 already co-localised with nuclear speckles in uninfected T-cells suggesting that HIV-1 promotes a natural behaviour of CPSF6.

microbiology↗

bioRxiv2022

Altered plasma membrane abundance of the sulfatide-binding protein NF155 links glycosphingolipid imbalances to demyelination

Myelin is a multi-layered membrane that tightly wraps neuronal axons enabling efficient, high-speed signal propagation. The axon and myelin sheath form tight contacts, mediated by specific plasma membrane proteins and lipids, and disruption of these contacts causes devastating demyelinating diseases. Using two cell-based models of demyelinating sphingolipidoses, we demonstrate that altered lipid metabolism changes the abundance of specific plasma membrane proteins. These altered membrane proteins have known roles in cell adhesion and signalling, with several implicated in neurological diseases. The cell surface abundance of the adhesion molecule Neurofascin, a protein critical for the maintenance of myelin-axon contacts, changes following disruption to sphingolipid metabolism. This provides a direct molecular link between altered lipid abundance and myelin stability. We show that the Neurofascin isoform NF155, but not NF186, interacts directly and specifically with the sphingolipid sulfatide via multiple binding sites and that this interaction requires the full-length extracellular domain of NF155. We demonstrate that NF155 adopts an S-shaped conformation and preferrentially binds sulfatide-containing membranes in cis, with important implications for protein arrangement in the tight axon-myelin space. Our work links glycosphingolipid imbalances to disturbance of membrane protein abundance and demonstrates how this may be driven by direct protein-lipid interactions, providing a mechanistic framework to understand the pathogenesis of galactosphingolipidoses.

neuroscience↗