Essential Roles for Soluble Virion-Associated Heparan Sulfonated Proteoglycans and Growth Factors in Human Papillomavirus Infections

Ozbun MA (2012) Essential Roles for Soluble Virion-Associated Heparan Sulfonated Proteoglycans and Growth Factors in Human Papillomavirus Infections. PLoS Pathog 8(2): e1002519. doi:10.1371/journal.ppat.1002519 Essential Roles for Soluble Virion-Associated Heparan Sulfonated Proteoglycans and Growth Factors in Human Papillomavirus Infections Zurab Surviladze 0 Agnieszka Dziduszko 0 Michelle A. Ozbun 0 Michael Imperiale, University of Michigan, United States of America 0 Department of Molecular Genetics & Microbiology, University of New Mexico School of Medicine , Albuquerque, New Mexico , United States of America A subset of human papillomavirus (HPV) infections is causally related to the development of human epithelial tumors and cancers. Like a number of pathogens, HPV entry into target cells is initiated by first binding to heparan sulfonated proteoglycan (HSPG) cell surface attachment factors. The virus must then move to distinct secondary receptors, which are responsible for particle internalization. Despite intensive investigation, the mechanism of HPV movement to and the nature of the secondary receptors have been unclear. We report that HPV16 particles are not liberated from bound HSPG attachment factors by dissociation, but rather are released by a process previously unreported for pathogen-host cell interactions. Virus particles reside in infectious soluble high molecular weight complexes with HSPG, including syndecan-1 and bioactive compounds, like growth factors. Matrix mellatoproteinase inhibitors that block HSPG and virus release from cells interfere with virus infection. Employing a co-culture assay, we demonstrate HPV associated with soluble HSPG-growth factor complexes can infect cells lacking HSPG. Interaction of HPV-HSPG-growth factor complexes with growth factor receptors leads to rapid activation of signaling pathways important for infection, whereas a variety of growth factor receptor inhibitors impede virus-induced signaling and infection. Depletion of syndecan-1 or epidermal growth factor and removal of serum factors reduce infection, while replenishment of growth factors restores infection. Our findings support an infection model whereby HPV usurps normal host mechanisms for presenting growth factors to cells via soluble HSPG complexes as a novel method for interacting with entry receptors independent of direct virus-cell receptor interactions. - Funding: This work was supported by NIH Grants R01CA132136 and U19AI084081. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Human papillomaviruses (HPVs) are small, DNA-containing viruses that infect mucosal and cutaneous epithelium to cause benign and malignant tumors, including many anogenital, oropharyngeal and some skin cancers [1,2]. HPVs demonstrate remarkable host restrictions and have strict tropism for stratifying squamous epithelium. HPV virions consist of 360 copies of the L1 capsid protein, 1272 copies of the L2 protein and the circular viral genome (<8 kb) condensed by cellular histones. Like a number of other pathogens, HPV entry into target cells is a multistep process initiated by binding to cell surface attachment factors, the most common of which are glycosaminoglycan chains, especially heparan sulfate in proteoglycans (HSPGs) [3,4]. Binding to these negatively charged polysaccharides is usually electrostatic and relatively nonspecific. Many microbes like HPVs must transfer from HSPG to a distinct secondary receptor responsible for active pathogen internalization [5]. For HPVs this entry receptor has been elusive. Despite intensive investigation, the mechanism of HPV movement from primary HSPG attachment receptors to secondary high-affinity receptors has been unclear. Several studies suggest a role for HPV L2 protein in facilitating infection via interaction with a secondary receptor (reviewed in ref. [6]). In this model, initial virus attachment to HSPG causes a conformational change in L1 that facilitates a critical proteolytic cleavage of L2 by furin, a proprotein convertase [68]. L2 cleavage is thought to expose the L2 binding site for the secondary cell receptor, lowering the affinity of L1 for HSPG binding and resulting in transfer to the entry receptor [8]. Many, but not all, of the accumulating experimental data support this attractive hypothesis. Although antibodies raised to L2 can neutralize infection [9] and in vitro synthesized L2 peptides and proteins can interact with the cell surface [10,11], there is no direct evidence that L2 in the context of the virion has a function at the cell plasma membrane. Scatchard plot analyses indicate high affinity binding of HPV33 VLP to HeLa cells, with a Kd of ,85610212 M [12]. This strong binding affinity of L1 VLP for cells makes it difficult to conceive how cleavage of L2, which is not involved in primary binding, could change the affinity of L1 so dramatically as to cause particle dissociation from the HS chain. Moreover, a recent report shows that heparin binding does not induce obvious conformational changes in the HPV16 capsid structure in vitro, except for slight movements of the surface loops and the residues directly involved in oligosaccharide binding [13]. Additional observations that call into question a function for L2 in early entry steps include the fact that L1-only containing virus-like A subset of the .120 different types of human papillomaviruses (HPVs) are the most common cause of sexually transmitted infections. Certain HPVs are also associated with approximately 5% of all cancers worldwide. Like many pathogens, HPVs bind first to heparan sulfate proteoglycans (HSPGs) on cells before moving to more specific uptake receptors. However, relatively little is known about the mechanism(s) that triggers the translocation of HPV from HSPGs to the receptors that facilitate entry. As obligate parasites, viruses have evolved numerous means to hijack host cell functions to cause infection. We report two novel mechanisms of pathogen-host interactions. First, bound HPV particles are liberated from cells in an active complex with HSPGs and growth factors rather than dissociating from the sugars to engage secondary receptors. Second, HPV uses the specificity of the associated growth factors to bridge to their cognate receptors as opposed to direct binding to a cell internalization receptor. Signals transduced during these interactions are important for HPV infection. Our study provides new insights into the transmission of a significant viral pathogen and reveals novel means whereby microbes may repurpose normal cell functions during infection of their hosts. Likewise, this work uncovers new targets for HPV prophylaxis. particles (VLP) are capable of normal internalization in cells [14 16] and PsVs containing a furin-resistant L2 mutant bind, enter, and uncoa (...truncated)