Role of the tetraspanin CD151 in the early human papillomavirus type 16 infection cascade

Abstract

Human papillomaviruses (HPV) are a diverse group of non-enveloped, double-stranded DNA viruses with profound significance in human health. An estimated 5% of all human cancer cases are attributed to HPVs. For infection to occur, virions must reach dividing basal cells of the lower epithelium through a break in the epithelial barrier. On the basement membrane, viral particles bind to their primary attachment site, heparan sulfate (HS) within the extracellular matrix (ECM). This binding triggers a series of conformational changes which promote viral cell entry, with virions migrating along actin-rich protrusions to engage a yet uncharacterized secondary receptor complex on keratinocytes. Current evidence suggests that this secondary receptor is likely a multimeric complex rather than a single molecular entity. Several proteins have been implicated as essential for cell entry, including the tetraspanin CD151, integrin-α6 and growth factor receptors. Among these, CD151 plays a crucial role in facilitating viral access and organizing the cellular components necessary for successful infection. However, this multistep entry process is slow and asynchronous, and involvement of CD151 in HPV entry prior to endocytosis remains unclear. <br/> In this study, HPV16 pseudovirions (PsVs) were used to investigate early infection stages and their association with CD151. PsV/CD151 assemblies were analyzed, revealing that PsVs preferentially bind to regions with increased CD151 density. Overexpression of CD151-GFP induced the formation of large CD151 aggregates, although these aggregates also form independently of PsV binding, indicating that aggregate formation depends on CD151 expression levels. <br/> Furthermore, the actin inhibitor cytochalasin D was employed to synchronize PsV uptake, revealing that actin dynamics within filopodia are crucial for releasing virions from the ECM and transferring them to the cell body. The human keratinocyte cell line HaCaT was validated as an ideal model system for studying early HPV infection events. Inhibition of actin-dependent processes resulted in entrapment of PsVs in the ECM. Upon removal of this blockade, HS-coated PsVs rapidly translocated to the cell body and to CD151 assemblies. After reaching the cell body, the viral capsid sheds its HS coat, followed by PsV/CD151 endocytosis. These findings demonstrate an early involvement of CD151 in HPV16 cell entry. <br/> Moreover, the HSPG-binding peptide 19-2.5 was evaluated for its potential to inhibit PsV infection. While peptide 19-2.5 did not affect PsV/CD151 association, it caused the formation of large viral aggregates several micrometers away from the plasma membrane, indicating a block in the translocation from the primary HSPG attachment site to the secondary receptor complex. This suggests that peptide 19-2.5 may serve as a promising alternative or complement to current HPV prevention strategies.