Mechanism of Dynein-mediated Early Intracellular Trafficking of Human Papillomavirus - PROJECT SUMMARY/ABSTRACT Human papillomavirus (HPV) infections are responsible for almost all cases of cervical cancer, as well as most anogenital and oropharyngeal cancers. HPV, a non-enveloped DNA virus, is transported from the cell surface to the nucleus for genome replication. Recent studies have shown that, rather than exiting directly from the endosome into the cytosol after endocytosis, incoming HPV remains within a vesicular compartment and moves along microtubules (MTs) toward the nucleus. Previous research demonstrated that the C-terminus of the HPV L2 minor capsid protein contains a novel cell-penetrating peptide. This peptide facilitates L2 protrusion through the endosomal membrane, enabling its interaction with the endosomal sorting complex, retromer, which directs the virus into a retrograde transport vesicle en route to the Golgi. These processes promote the exposure of L2 protein to the cytosol, thereby facilitating the recruitment of host factors necessary for HPV retrograde transport. These findings raise important questions about how and when, after endocytosis, the HPV-containing endosomal compartment interacts with the dynein transport machinery for intracellular movement along MTs across the host cytoplasm and reach the nucleus. The discovery of dynein adaptors involved in retrograde transport highlights their roles in the precise recruitment and interaction of dynein with specific membranous compartments. This has led us to investigate the mechanisms by which dynein is recruited to HPV-carrying endosomal vesicles to mediate viral transport after endocytosis. Our recent research has uncovered a novel molecular basis for dynein recruitment to the HPV-carrying endosome. In this process, HPV utilizes the early endosomal small GTPase Rab5 and its effector Rabankyrin-5 to form a complex that links the HPV-carrying endosome to dynein, facilitating viral transport along MTs. We propose that HPV repurposes Rabankyrin-5 as a new dynein adaptor, enabling precise dynein recruitment and physical interactions with HPV-carrying endosomes. This process is linked to the exposure of HPV L2 protein to the cytosol, which facilitates Rabankyrin-5 recruitment. In Aim 1, we will determine how HPV infection triggers Rabankyrin-5-mediated virus-dynein association at the endosome, specifically investigating whether L2 protrusion is required for Rabankyrin-5 recruitment and examining the mechanism of L2-Rabankyrin-5 interaction. Using live-cell imaging, we will examine the timing of dynein recruitment relative to L2 protrusion during HPV movement along MTs. In Aim 2, we will investigate whether Rabankyrin-5 acts as a novel dynein adaptor, directly interacting with dynein and activating its function to promote HPV transport. This research has the potential to uncover a novel mechanism for HPV intracellular transport during the early stages of viral entry, involving the spatial and temporal coordination of viral membrane protrusion, endosomal coat complex formation, and precise dynein recruitment and activation. By advancing our understanding of the cell biology and molecular virology of HPV infections, this research offers molecular insights that could lead to the identification of new therapeutic targets and the development of innovative antiviral approaches.
