Abstract
Mucosotropic, high-risk human papillomaviruses (HPV) are sexually transmitted viruses that are causally associated with the development of cervical cancer. The most common high-risk genotype, HPV16, is an obligatory intracellular virus that must gain entry into host epithelial cells and deliver its double stranded DNA to the nucleus. HPV capsid proteins play a vital role in these steps. Despite the critical nature of these capsid protein-host cell interactions, the precise cellular components necessary for HPV16 infection of epithelial cells remains unknown. Several neutralizing epitopes have been identified for the HPV16 L2 minor capsid protein that can inhibit infection after initial attachment of the virus to the cell surface, which suggests an L2-specific secondary receptor or cofactor is required for infection, but so far no specific L2-receptor has been identified. Here, we demonstrate that the annexin A2 heterotetramer (A2t) contributes to HPV16 infection and co-immunoprecipitates with HPV16 particles on the surface of epithelial cells in an L2-dependent manner. Inhibiting A2t with an endogenous annexin A2 ligand, secretory leukocyte protease inhibitor (SLPI), or with an annexin A2 antibody significantly reduces HPV16 infection. With electron paramagnetic resonance, we demonstrate that a previously identified neutralizing epitope of L2 (aa 108–120) specifically interacts with the S100A10 subunit of A2t. Additionally, mutation of this L2 region significantly reduces binding to A2t and HPV16 pseudovirus infection. Furthermore, downregulation of A2t with shRNA significantly decreases capsid internalization and infection by HPV16. Taken together, these findings indicate that A2t contributes to HPV16 internalization and infection of epithelial cells and this interaction is dependent on the presence of the L2 minor capsid protein.
Highlights
Human papillomaviruses (HPV) are one of the most common sexually transmitted viruses, and persistent high-risk HPV infections are causally associated with the development of cervical cancers, which are responsible for the deaths of approximately a quarter of a million women each year worldwide [1,2]
It was previously demonstrated that secretory leukocyte protease inhibitor (SLPI) inhibits the infection of HIV-1 through extracellular annexin A2 [34], and herpes simplex virus (HSV) causes a sustained down-regulation of SLPI [33]
The effect of SLPI and antibody inhibition of A2 heterotetramer (A2t) on HPV16 infection of epithelial cells was examined via HPV16 pseudoinfection of HaCaT cells where reporter gene transduction was used as a measure of HPV16 infectivity
Summary
Human papillomaviruses (HPV) are one of the most common sexually transmitted viruses, and persistent high-risk HPV infections are causally associated with the development of cervical cancers, which are responsible for the deaths of approximately a quarter of a million women each year worldwide [1,2]. Of the 15 different cancer-causing high-risk HPV genotypes, HPV16 is the most common, leading to approximately 50% of all cervical cancers [3] Despite these statistics and rigorous efforts in understanding the first steps in HPV16 infection, the entire mechanism of how HPV16 enters and infects human cells is yet to be defined. The timing and expression of HPV16 viral genes along with the production of infectious virions is contingent on the differentiation of basal epithelial cells into mature keratinocytes [7]. This contingency has led the majority of the field interested in papillomavirus receptors to use pseudovirions (PsV) and/or virus-like particles (VLP) to study specific aspects of viral internalization and infection. The entry of HPV16 into cells has been shown to be clathrin-, caveolin-, cholesterol-, and dynaminindependent implying a non-canonical and possibly novel ligandinduced internalization pathway related to macropinocytosis [15,16]
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