Cellular and Viral Factors Important for Entry and Retrograde Trafficking of HPV16

Abstract

There are at least 400 types of human papillomaviruses (HPV) have been identified. HPVs infection is the most sexually transmitted disease in the US. Most of the HPVs are commensal or low-risk HPVs that cause either asymptomatic infection or benign skin and genital warts. However, about 17 types high–risk HPVs are the etiological agents responsible for 99% of cervical cancers and 5% of all human cancers worldwide. Although the vaccine is available, the protection is not full spectrum, and access to the vaccine is limited in some regions. In addition, condom use is not always effective to prevent HPV transmission. Thus, HPV is not a defeated pathogen, and it poses a significant burden on global health. HPVs are non-enveloped DNA viruses comprised of major capsid protein L1 and minor capsid protein L2. HPVs infect differentiating skin and mucosal tissues. HPV infection requires the viral particle to access the basal layer keratinocytes which sit atop the extracellular matrix-rich basement membrane. From there, virion particles will bind to the cell surface followed by endocytosis to enter the host cell. L2 is associated with viral genome (vDNA) and will act as an inducible transmembrane protein to protrude from endosome membrane and mediate the retrograde trafficking and nuclear importation of the viral genome to complete the initial infection. L2 itself and many cellular factors play an important role in these processes, but the detailed mechanisms between viral-host interaction and the L2 biology during infection are not well characterized. To address this knowledge gap, our overarching hypothesis is that after EGFR-dependent viral entry, the conserved L2 N-terminus region plays an important role in penetrating endosomal membrane while cellular SNX-BARs regulate viral retrograde trafficking. SNX-BAR proteins are likely involved with efficient entry as well. We further postulate that furin cleaved L2s may adopt a unique conformation, favorable for L2 membrane penetration and nuclear vDNA transport. In this dissertation, we studied both the host factors and L2 itself on how they can affect viral genome trafficking. First, we developed a small cell-penetrating peptide derived from the bar domain of the sorting nexin 1 (SNX1) which can significantly inhibit HPV infection. We demonstrated this peptide affects viral entry and retrograde trafficking. Second, we identified a short but well-conserved amphipathic helix (AH) located towards the N-terminus of L2. The AH is adjacent to the transmembrane domain (TMD) which is essential for viral retrograde trafficking, possibly via mediating membrane insertion of L2. Finally, we propose that furin-cleaved L2 can oligomerize together, potentially forming a double pass membrane topology. The intact L2 TMD is required for this oligomerization process. Overall, this work provides novel insight into how L2 and host factors can mediate HPV subcellular trafficking and infection.