Resolving the intersection of HIV and F-actin in the context of cell-cell viral spread

Download files
Access & Terms of Use
open access
Embargoed until 2022-04-01
Copyright: Ospina Stella, Alberto
Abstract
HIV has evolved elaborate mechanisms to manipulate the host cell’s actin cytoskeleton. This supports specific steps of the viral life cycle but also leads to cell-type specific changes in leukocyte shape and behaviour that promote overall spread of the infection. In particular, actin manipulation is important for direct cell-cell transfer of HIV (CCTH), which is now recognized as a highly efficient mode of viral spread. This study aimed to further resolve the molecular players and mechanisms involved in this process. Through the use of CRISPR–Cas9-mediated gene editing, herein we systematically interrogated the loss-of-function phenotype of over 50 cytoskeletal regulators, in both HIV-donor and target cells during CCTH. This was then combined with a portfolio of complementary assays ranging from live cell microscopy through to the ultrastructural resolution of focused ion beam scanning electron microscopy. This approach identified several core actin regulators to be required on both sides of viral transfer, including the Rho-GTPases Cdc42/Rac1 (signalling), upstream of the Arp2/3 complex (actin nucleator), and Profilin (which indirectly supports F-actin elongation, e.g. via Formins). In infected myeloid cells, Cdc42-Arp2/3 and Formins collaborated to form distinct “HIV-Filopodia”, which were shown to be important for CCTH. The overall findings of this work support that, while HIV uses diverse strategies to influence numerous cytoskeletal targets, there is a high tolerance for the individual loss of many manipulated regulators in the context of CCTH. However, core regulators that secure the cellular ability to mediate F-actin polymerization remain essential to this process, in particular via their contribution to cortical F-actin membrane protrusions that facilitate intercellular contacts and later maturation of the virological synapse. To conclude, we have mapped a landscape of complex interactions between HIV and the actin cytoskeleton. This not only builds on our understanding of F-actin regulation in cells of our immune system but will also facilitate the continued development of host-directed therapeutics for lentiviral and/or other pathogenic infections.
Persistent link to this record
Link to Publisher Version
Additional Link
Author(s)
Ospina Stella, Alberto
Supervisor(s)
Turville, Stuart
Anupriya, Aggarwal
Creator(s)
Editor(s)
Translator(s)
Curator(s)
Designer(s)
Arranger(s)
Composer(s)
Recordist(s)
Conference Proceedings Editor(s)
Other Contributor(s)
Corporate/Industry Contributor(s)
Publication Year
2020
Resource Type
Thesis
Degree Type
PhD Doctorate
UNSW Faculty
Files
download public version.pdf 7.77 MB Adobe Portable Document Format
Related dataset(s)