Human papillomavirus type 16 (HPV16) binding, entry and signaling

Human papillomaviruses (HPVs) are non-enveloped 8 kb DNA viruses. There are over one hundred HPV genotypes. Several of these genotypes have been recognized as the etiologic agent of genital, skin and largyngopharyngeal warts as well as certain human cancers including cervical, anal, head, neck and upper airway cancers. The HPV16 genotype is the primary causative agent of cervical cancer, the second leading cause of death in women worldwide.For a successful infection, HPV16 binds to receptors and enters the host cell via clathrin-mediated endocytosis. Several HPV16 entry studies have suggested a role of cell surface heparan sulfate (HS), a ubiquitous cellular attachment factor, in virus binding, entry and infection. In recent years, it has been speculated that a secondary receptor putatively an a6 integrin receptor may mediate HPV16 infection. Controversies for the role of HS and the a6 integrin receptor in mediating HPV16 infection are still debatable, because some of these studies were done with non-human keratinocyte cell lines including COS-7 cells. However, because a natural target for HPV16 is the human keratinocyte cell lines, the role of HS and the a6 integrin receptor in HPV16 infection needs to be studied in the appropriate cell target. Integrin interaction with a virus activates a variety of cellular signaling molecules. The focal adhesion kinase (FAK), non-receptor cytoplasmic molecule, is the earliest activating molecule reported. Activated FAK mediates several downstream effector molecules that may play a role in endocytosis. Currently, an understanding of the early signaling events that HPV16 induces is not known.My initiative in this doctoral dissertation was to determine and understand the role of the attachment factor and the secondary receptor that HPV16 utilizes in infection of human keratinocytes as well as the signaling events induced by the virus. Although HPV16 has been shown to enter target cells via a clathrin-mediated endocytosis, the binding events and signaling molecules mediating internalization of the virus into the cytoplasm have not been defined. We hypothesize that HPV16 binds to a cellular heparan sulfate attachment factor which induces a conformational change that enables the virus to bind to an integrin receptor. Integrin binding activates an early signaling molecule FAK which directly or indirectly phosphorylates and recruits other cellular molecules including dynamin GTPases to the plasma membrane leading to a dynamin-dependent, clathrin-mediated endocytosis.My studies have confirmed the usage of HS and the a6 integrin receptor for HPV16 infection of human keratinocytes. I have demonstrated that HPV16 induces phosphorylation and hence activation of the FAK molecule which is essential for infection. I have shown the role of dynamin in the release of formed vesicles from the plasma membrane to the cytoplasm using a dynamin GTPase inhibitor, dynasore. This work has provided potential targets of inhibiting the spread of HPV16 infection, however, more studies need to be done to decipher the role of other cellular signaling molecules in infection.I believe that the contribution of my proposed work will define the specific receptor profile that the virus utilizes to mediate a successful infection. Findings from this study will enable us determine for the first time the early cellular signaling molecules that is utilized by the virus for an infectious entry.