In vitro reconstitution of HIV-1 capsid transport

Abstract

The HIV-1 capsid interacts with mutiple host factors during its early life cycle to achieve succesfful infection. In this thesis we aim to expand our understanding of how interactions between the capsid and host cell factors regulate viral activities, with chapter 4 and 5 specifically focusing on the cytoplasmic transport process. Based on cellular and biochemical evidence, recent studies suggest that HIV-1 capsid hijacks both dynein and kinesin-1, via the adaptor proteins BICD2 and FEZ1 respectively, for active transport along microtubule network. We will present our work to reconstitute these complexes and demonstrate their motilities along microtubles in vitro, providing more direct evidence to support these models.

To investigate the interactions between HIV-1 capsid and the motor adaptors, we applied a fluorescence fluctuation spectroscopy based binding assay and a total internal reflection microscopy (TIRFm) based binding assay. These approaches allowed us to generate quantitative descriptions of the interactions between HIV-1 capsid and the adaptor proteins.

Subsequently, we reconstituted the motor-adaptor-cargo complexes (DDBC and KFC) using recombinant proteins as well as components isolated from native tissues. We have successfully demonstrated both the dynein- and kinesin-dependent transport of HIV-1 capsid along microtubules in vitro using a TIRFm based single molecule motility assay. We also further charaterized the motile behaviors and properties of the KFC complex.

Our work validated the proposed models for the cytoplasmic transport of HIV-1 capsid and demonstrated the minimum requirement for this process. In general, this work has made solid contributions towards the understanding both HIV viology and motor-driven cargo transport, as well as opened oppotutnities to a range of exciting research questions in both fields.