Structural and functional characterization of the HPV16 entry platform on the cell surface

Abstract

Human papillomaviruses (HPV) are small, non-enveloped DNA viruses that infect skin and mucosa tissues. Among the family, HPV16 is one of the most oncogenic subtypes responsible for a variety of cancers, including cervical, anal and head and neck cancers. HPV internalization is a slow and asynchronous process involving many steps and host cell factors. Among these factors is the tetraspanin CD151, which is essential for HPV infection. In microscopy, it colocalizes with viral particles on the cell surface. Moreover, CD151 associates with other host cell factors, notably laminin-binding integrins. While integrin α6 is accepted to have a role during infection, the involvement of integrin α3 remains controversial. <br /> In this study, HPV16 pseudoviruses (PsVs) associated with large assemblies of CD151 at the plasma membrane were studied. Superresolution microscopy revealed that those assemblies are crowds of closely arranged CD151 nanodomains. It could also be observed that integrin α3 and integrin α6 clusters both associate with these nanodomains. CD151 and integrin nanodomains segregate with only their margins being in contact with each other rather than concentrating in the same cluster to form a homogenous protein mixture. In line with this, integrin clusters were similarly densely packed in the PsV associated CD151 assemblies. <br /> Moreover, the functionality of integrin α3 and integrin α6 for HPV infection was verified in a keratinocyte cell model. As in accordance with literature, integrin α6 is a proviral factor mediating host cell binding. More importantly, a role in HPV entry was demonstrated for integrin α3 as well. Integrin α3 mediates internalization rather than virus binding, as knockdown of the protein hardly affected PsV binding but all analysed post-binding steps were reduced. The CD151 assemblies were sites of actin accumulation. Overexpression of tetraspanins seemed to enhance the occurrence and size of the assemblies, possibly by enhancing the availability of TEM components at the cell surface. <br /> Viral entry platforms were shown to be enriched with another tetraspanin, CD63, which is required for intracellular trafficking of the virus. These platforms seem to be very variable in size, invaginating to the cell interior and reaching up to a few micrometres under overexpressing conditions. Moreover, the cytoskeletal adaptor protein OBSL1 was identified as a putative platform component possibly acting as a linker of the platform to cytoskeletal dynamics needed for internalization. Preliminary data indicate that OBSL1 may be recruited to early endocytic structures at the plasma membrane. <br /> Thus, it was concluded that CD151 organizes viral entry platforms containing integrin α3 and α6, forming cluster crowds that associate with viral particles, while both integrins have different functions in viral entry. The crowds also contain additional components like CD63 and actin and may be linked to the cytoskeleton via OBSL1. Since numerous viruses utilize TEMs for viral entry, the concept of a tetraspanin scaffolding several host cell factors to build up an entry platform could be a general model for cell surface architectures utilized by viral particles.