WAVE1 and WAVE2 facilitate human papillomavirus-driven actin polymerization during cellular entry

Abstract

Human Papillomavirus Type 16 (HPV16) is an etiological agent of human cancers that requires endocytosis to initiate infection. HPV16 entry into epithelial cells occurs through a non-canonical endocytic pathway that is actin-driven, but it is not well understood how HPV16-cell surface interactions trigger actin reorganization in a way that facilitates entry. This study provides evidence that Wiskott-Aldrich syndrome protein family verprolin-homologous proteins 1 and 2 (WAVE1 and WAVE2) are molecular mediators of the actin polymerization that facilitates HPV endocytosis and intracellular trafficking. We demonstrate through post-transcriptional gene silencing and genome editing that WAVE1 and WAVE2 are critical for efficient HPV16 infection, and that restoration of each in knockout cells rescues HPV16 infection. Cells lacking WAVE1, WAVE2, or both, internalize HPV16 at a significantly reduced rate. Analysis of fluorescently labeled cells exposed to HPV16 and acquired by confocal fluorescence microscopy revealed that HPV16, WAVE1, WAVE2, and actin are all colocalized at the cellular dorsal surface. We also found that HPV16 stimulates WAVE1 and WAVE2-mediated cellular dorsal surface filopodia formation during the viral endocytic process. Taken together, this study provides evidence that the HPV endocytic process needed for infection is controlled by actin reorganization into filopodial protrusions and that this process is mediated by WAVE1 and WAVE2. Author SummaryHuman Papillomavirus (HPV) is the most common sexually transmitted infection in the United States. While its mode of entry into cells has yet to be fully described, extensive studies indicate HPV entry occurs via a macropinocytosis-like pathway. Interestingly, more than 10 viruses enter cells via macropinocytosis-like entry, with no two viruses utilizing identical factors for entry. It is unclear whether these viruses are entering cells via the same pathway, or if the term "macropinocytosis" describes a subset of endocytic pathways. One unifying feature of entry for each of these viruses is their requirement of actin polymerization. In this study, we identify the cellular factors necessary for actin polymerization to participate in HPV endocytosis. The findings of this study are of importance to the field of virology as they may extend to the infection of other viruses. It is also of interest in cancer studies as macropinocytosis has been associated with the scavenging of nutrients and methuosis, a form of cell death in cancer cells that occurs from over- scavenging. Nanoparticle delivery can also occur via macropinocytosis. Therefore, the contribution of WAVE proteins to macropinocytosis and macropinocytosis-like endocytic events is informative to a broad audience.