The evolving field of human papillomavirus receptor research: a review of binding and entry.
Journal: 2013/July - Journal of Virology
ISSN: 1098-5514
Abstract:
Human papillomaviruses (HPVs) infect epithelia and can lead to the development of lesions, some of which have malignant potential. HPV type 16 (HPV16) is the most oncogenic genotype and causes various types of cancer, including cervical, anal, and head and neck cancers. However, despite significant research, our understanding of the mechanism by which HPV16 binds to and enters host cells remains fragmented. Over several decades, many HPV receptors and entry pathways have been described. This review puts those studies into context and offers a model of HPV16 binding and entry as a framework for future research. Our model suggests that HPV16 binds to heparin sulfate proteoglycans (HSPGs) on either the epithelial cell surface or basement membrane through interactions with the L1 major capsid protein. Growth factor receptors may also become activated through HSPG/growth factor/HPV16 complexes that initiate signaling cascades during early virion-host cell interactions. After binding to HSPGs, the virion undergoes conformational changes, leading to isomerization by cyclophilin B and proprotein convertase-mediated L2 minor capsid protein cleavage that increases L2 N terminus exposure. Along with binding to HSPGs, HPV16 binds to α6 integrins, which initiate further intracellular signaling events. Following these primary binding events, HPV16 binds to a newly identified L2-specific receptor, the annexin A2 heterotetramer. Subsequently, clathrin-, caveolin-, lipid raft-, flotillin-, cholesterol-, and dynamin-independent endocytosis of HPV16 occurs.
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J Virol 87(11): 6062-6072

The Evolving Field of Human Papillomavirus Receptor Research: a Review of Binding and Entry

INTRODUCTION

Since the discovery of human papillomaviruses (HPVs), researchers in the field have sought to identify the mechanism by which these viruses enter host cells. Although much work has been done to date and many possible receptors have been identified, a clearly defined description of the entry of HPVs has remained controversial. In many cases of viral infection, our understanding of simple binding and uptake through a singular mechanism has given way to a model of a more complex interaction between several specific receptors, coreceptors, and cofactors (13). The purpose of this review is to synthesize the known data regarding HPV type 16 (HPV16) binding proteins at the cell surface, and their associated molecules, and attempt to connect them, if possible, into a testable framework of binding and entry. Due to the fact that HPVs are a diverse group of over 150 viruses, this review focuses primarily on the most common of the cancer-causing genotypes, HPV16, while making it clear when non-HPV16 genotypes are reviewed. The development of several HPV particle production systems has allowed researchers to begin to delineate the mechanisms behind viral entry; however, important differences between particles developed in vitro made direct comparisons challenging. Therefore, not only HPV16 structure but also the multiple forms the virus structure takes in the laboratory are discussed below. Finally, due to the tropism of HPV16 for human epithelial cells during a natural infection, this review focuses on studies that employ human epithelial cell lines and makes specific mention when nonhuman or nonepithelial cells are discussed.

HPV16 STRUCTURE

The HPV16 virion is approximately 55 nm in diameter and consists of a T=7 icosahedral capsid composed of two structural proteins, the L1 major capsid protein and the L2 minor capsid protein (4, 5). The capsid is formed by 360 molecules of L1 organized into 72 pentamers (6). In different particle production systems, between 12 and 72 molecules of L2 which appear to be located in the central internal cavity of the L1 pentamer are incorporated into the capsid, while naturally produced virions average approximately 30 L2 molecules (5). The majority of L2 is hidden from the capsid surface, except for some residues within the N terminus, which are exposed on the viral capsid surface (7). The proline-rich C terminus of L2 binds directly to L1 primarily through hydrophobic interactions (8), and it has been suggested that this may facilitate bending of L2 to loop through the central cavity of the L1 pentamer, though convincing structural and biochemical data for this are lacking. Although not strictly required for capsid formation, L2 was first shown to be essential for infection in HPV33 (9) and later in HPV16 (10). L2 is needed for efficient DNA encapsidation in some papillomaviruses, such as bovine papillomavirus type 1 (BPV-1) and HPV18, and, although not essential, L2 appears to play an important role in DNA encapsidation in other papillomaviruses, such as HPV16 and HPV31 (1114). Further functions of L2 include but are not limited to the following: facilitation of endosomal escape of the viral genome after infection (15), possibly assisted through the L2 GxxxG transmembrane domain (16) and/or interaction with sorting nexin 17 (17); binding of the virion to the cytoskeleton and transport within the cytoplasm (18); interaction with dynein light chain for transport to the nucleus (19); chaperoning of packaged DNA to the host cell nucleus (20); and assistance in capsomere assembly (21).

Because the life cycle of HPVs is dependent on the differentiation of basal cells into keratinocytes, it can be challenging to study this in vitro. Therefore, as an alternative to native virions, virus-like particles (VLP) were developed. There are two types of VLP that are currently being utilized by researchers in the field—L1 VLP and L1L2 VLP. When the L1 major capsid protein is expressed alone, it can self-assemble into an L1 VLP with an icosahedral structure, and if L1 and the L2 minor capsid protein are simultaneously expressed, the proteins assemble into L1L2 VLP (2225). These VLPs can be made in insect cells, mammalian cells, and yeast (e.g., Saccharomyces cerevisiae) (22, 23, 26). While the general structure similarity of VLP to native virions allows the presentation of conformational epitopes, they do not contain the viral genome, rendering them noninfectious. The subsequent development of particles containing reporter DNA plasmids, termed pseudovirions (PsV), allowed verification and quantification of host cell infection through reporter plasmid gene transduction and the investigation of possible DNA-induced capsid structural changes (12, 2731). Finally, the development of organotypic (raft) culture systems has allowed epithelial cell differentiation in vitro and yielded virions such as HPV31, HPV18, and HPV16, which have been shown to have infectious potential (3236).

While these systems provide invaluable resources for the study of papillomavirus binding and entry, the resulting particles have subtle structural and functional differences. Studies show that L1 VLP, L1L2 VLP, and PsV interact differently with cells, indicating they cannot be utilized interchangeably without proper discussion and interpretation. For example, O-linked heparan sulfates (HS) were shown to be sufficient for HPV33 L1L2 VLP binding to COS-7 cells (monkey kidney fibroblast cell line) whereas HPV33 PsV infection also required N-linked heparan sulfates, indicating that there may be differences in VLP versus PsV interactions with heparin sulfate proteoglycans (HSPGs) (37). Those authors suggest that the observed differences between VLP and PsV in HSPG sulfation requirements are due to structural changes from DNA encapsidation. In the same study, internalization of HPV33 L1 and L1L2 VLP occurred significantly faster (half-time [the time it takes for half of the particles to be internalized] of 3.5 h) than HPV33 PsV uptake (half-time of 7.5 h) in COS-7 cells (37). Additionally, HPV16 L1 VLP binding to the basement membrane in an in vivo mouse vaginal challenge model was unaffected by the presence of a proprotein convertase (PC) inhibitor, while HPV16 PsV demonstrated a reduction in the length of basement membrane binding in the presence of a PC inhibitor (38). Further, the internalization of HPV16 L1 VLP into HeLa cells (HPV18-transformed cervical cancer cell line) was unaffected by a reduction in annexin A2 levels, while internalization of HPV16 L1L2 VLP and infection with HPV16 PsV were significantly reduced (39). The presence of L2 within the particle affects L1 structure, as demonstrated by virions from raft cultures with chimeric HPV16 L2 proteins that sufficiently alter the structure of wild-type HPV16 L1 proteins such that HPV16 L1-specific conformational antibodies (H16.7E, H16.V5) cannot neutralize the virions (40). Even within HPV16 PsV preparations, there are differences between “immature” and “mature” conformations in the number of intercapsomeric disulfide bonds (13). HPV33 VLP contain fewer disulfide bonds between L1 molecules than do tissue-derived HPV33 virions (31). Additionally, it has been demonstrated that the encapsidation of DNA into HPV33 PsV leads to an increase in disulfide bond formation and an increased resistance to trypsin degradation, indicating an alteration in capsid structure when DNA is present (31). These are just some of the known caveats to consider when evaluating studies that use different capsid systems which make integration of studies more challenging. Despite these differences, research has shown that there are occasions when events in binding and entry are consistent between different particle types, such as HPV16 L1L2 VLP competing with BPV1 virions for binding to C127 cells (murine mammary tumor line) or HPV16 L1 VLP colocalizing with BPV1 virions in C127 cells (41, 42). Therefore, this review attempts to designate clearly the type of particles used in each study discussed and offer interpretation when appropriate.

HPV16 BINDING AND ENTRY

Initial HPV16 binding occurs via heparan sulfate proteoglycans (HSPGs) either located on the epithelial cell surface, as shown using HPV16 PsV on HaCaT cells (HPV-negative keratinocyte cell line derived from adult skin [43]), or residing on the basement membrane, as shown using HPV16 PsV in an in vivo mouse vaginal challenge model (38, 44, 45). HSPGs were first described as HPV binding receptors using HPV11 L1 VLP on HaCaT cells (46) and were later shown to be essential for infection using HPV16 PsV and HPV33 PsV on COS-7 cells (28). Additionally, it is possible that initial HPV16 binding occurs via the extracellular matrix (ECM) through laminin-332 (formerly named laminin-5), as shown using HPV11 virions from human xenografts on HaCaT cells (47). However, this finding was not supported in an in vivo mouse vaginal challenge model where heparinase treatment was shown to reduce HPV16 PsV basement membrane binding, despite the observation that laminin-332 levels were unchanged (45).

HSPGs consist of a cell surface or matrix protein with covalently attached heparan sulfate (HS) glycosaminoglycans, and they regulate a wide variety of biological activities as well as functioning as binding receptors for a multitude of viral and bacterial pathogens (4850). Among the HSPGs, syndecan-1, which consists of a single transmembrane (type 1) core domain with an ectodomain that carries three to five HS chains (51), is the most prevalent in keratinocytes and its expression is increased during wound healing (52). Wound healing facilitates HPV16 PsV infection of epithelial cells, as shown in the in vivo mouse vaginal challenge model (53). Although syndecan-1 may be the most prevalent, several HSPGs can potentially serve as binding receptors (54, 55). At least in K562 cells (erythroleukemia cell line), which lack common HSPGs, exogenous expression of syndecan-1, syndecan-4, and glypican 1 enhanced HPV16 L1 VLP binding in correlation with the level of HSPG expression (54). Early studies demonstrated that HPV16 L1L2 VLPs bind to the keratinocyte cell surface primarily through interaction with the L1 protein (41). It was later shown that the HPV16 L1 lysine residues Lys278 and Lys361 on the top of the pentamer appear to be the primary attachment points to HS (56, 57), indicating a direct interaction between HSPGs and the HPV16 capsid. Interestingly, it has been demonstrated that, compared to epithelial cell lines such as HeLa and HaCaT, primary keratinocytes cultured in vitro are difficult to infect with multiple different HPV PsV genotypes (33, 58, 59). One hypothesis for this is that the HSPGs on the keratinocyte cell surface in vitro may become modified, such as through O and N sulfation, due to culture conditions and are therefore not receptive for infection (59). It has been speculated that specific HSPG modifications may be preferentially present on basal keratinocytes during wound healing, which may make them more receptive to HPV infection (reviewed in reference 60). As described previously, differences in HS sulfation affect HPV33 PsV binding and infection of COS-7 cells (37), and similar results were subsequently confirmed for HPV16 PsV transduction of HeLa cells (61). Furthermore, pgsF-17 cells (mutant hamster ovarian cell line) deficient in 2-O and 3-O sulfation showed reduced HPV16 PsV transduction, further indicating that specific HS modifications facilitate HPV16 infection (61). The initial binding of the capsid to HSPGs was also shown to cause changes in the L1 major capsid proteins, as demonstrated through differences in pre- and postattachment monoclonal antibody neutralization (H33.J3) of HPV33 (37), which seem to be required for transfer to secondary receptors for viral entry.

After the binding of HPV16 PsV to HSPGs and initial capsid changes, cyclophilin B (CyPB) mediates a conformational change in the virus capsid that increases the exposure of the N terminus of the L2 protein and facilitates efficient infection (59, 6264). CyPs are a family of peptidyl-prolyl cis-/trans-isomerases of which CyPB is one of the most abundant (65). CyPB is located predominantly in the endoplasmic reticulum, from which it can be secreted to the extracellular space. There, CyPB associates with HSPGs, in particular, syndecan-1 (66). When the putative CyPB binding site on HPV16 L2 (97-PVGPLDP-103) is mutated, the L2 N terminus becomes permanently exposed and CyPB is no longer required for HPV16 PsV conformational changes that facilitate internalization into HaCaT cells (64). However, this mutant still required the presence of CyPs for effective infection, suggesting the postinternalization involvement of CyPs. A subsequent study demonstrated that CyPs actually facilitate the dissociation of HPV16 L1 from the L2/DNA complex following HPV16 PsV internalization in HaCaT cells and acidification of endocytic vesicles (67). Interestingly, CyPB was found in viral genome-containing vesicles, suggesting that it may be cointernalized with HPV16 PsV particles (67).

The HSPG/CyPB-mediated conformational change in the HPV16 PsV capsid exposes a highly conserved consensus furin convertase site (9-RTKR-12) on the HPV16 L2 N terminus, which can lead to proprotein convertase (PC)-mediated cleavage of L2 via furin or PC 5/6 (59, 6264). Furin cleavage of L2 in HPV16 PsV exposes the region of amino acids (aa) 17 to 36 of L2 (62). HPV16 PsV were able to bind to and infect HSPG-negative pgsA-745 cells (a Chinese hamster ovary mutant cell line deficient in xyloslytransferase) when they were first furin precleaved (59). However, there was a significant reduction in infection of furin-precleaved HPV16 PsV seen in a murine model after heparinase treatment, indicating that HSPG binding may cause conformational changes in addition to exposing the furin cleavage site in vivo (38). Interestingly, furin/PC cleavage-resistant HPV16 PsV mutants are still able to enter and traffic normally in HPSG-positive cells, but the particles accumulate in late endosomes, supporting a model in which furin/PC cleavage is required for L2-mediated endosome escape rather than being a prerequisite for binding and entry (62). Furin is membrane bound, while PC 5/6 either can be membrane bound or can be secreted in a soluble form in which it binds to HSPGs, depending on alternative splicing of mRNA (reviewed in reference 68). Whether cleavage occurs exclusively on the target cell surface by furin or from PC 5/6 bound to the BM or whether cleavage can occur prior to viral release from infected epithelium is not clear.

Since binding of HPV16 to HSPGs/CyPB does not mediate infectious HPV16 endocytosis, a separate secondary receptor or coreceptor may be involved in the infectious internalization of HPV16 into epithelial cells (59, 69). One of the secondary receptors speculated to be involved in HPV16 binding and entry is the cell adhesion molecule α6 integrin (44, 70). α6 integrin was first identified as a candidate receptor using HPV6 L1 VLP on HaCaT cells (71). It has been demonstrated that the level of HPV16 PsV binding correlates with the levels of α64 integrin, as decreasing α6 integrin expression through small interfering RNA (siRNA) significantly reduces HPV16 PsV infection (44), and that HPV16 PsV reporter gene transduction in β4 integrin knockout mice was significantly reduced (55). Further, activation of focal adhesion kinase (FAK) by HPV16 PsV is important for infection of HaCaT cells, and since this activation can be blocked through an α6 integrin blocking antibody in mouse embryonic fibroblasts, it may suggest that HPV16 PsV signals through α6 integrin to activate FAK (44). Until a direct interaction between integrins and HPV16 can be demonstrated, these signaling events indicate that, at the very least, integrins act as coreceptors for HPV16 binding and entry. Interestingly, there are several interactions between α6 integrins, tetraspanins, HSPGs, CyPB, and intracellular signaling pathways. Specifically, there is a close association between HSPGs and integrins as matrix components (reviewed in reference 72). Further, CyPB has been shown to interact with and mediate cell signaling through integrins (73). Signaling events by HPV16 L1L2 VLP have been shown to activate the phosphoinositide 3-kinase (PI3K) pathway via α6 integrin binding on A431 cells (epidermoid carcinoma cell line) (74), and PI3K signaling is required for HPV16 PsV infection of HeLa and HaCaT cells (75, 76).

In a recent study, the role of epidermal growth factor receptor (EGFR) and keratinocyte growth factor receptor (KGFR) in HPV16 PsV cell surface binding, signaling, and infection was explored (77). These growth factor receptors (GFRs) are highly expressed on human keratinocytes and are important in wound-healing processes (78). It was demonstrated that HPV16 PsV can bind to HSPG/growth factor (GF) complexes and subsequently activate signaling through these GFRs on HaCaT cells (77). Those authors propose that HPV16 particles become decorated with HSPG/GF complexes at the cell surface or in the mucosal layer, where they might be released from cells. Such predecorated particles could then bind to any cell via an interaction between the GFRs and the GF attached to virions via HSPGs. Though no direct evidence was provided showing an interaction between HPV16/HSPG/GF complexes and GFRs at the cell surface, GFR activation was suggested by an observed activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) (77). A recent study by the same group showed that early binding of HPV16 PsV to HaCaT cells leads to activation of the PI3K/Akt/mTOR pathway through EGFR stimulation and that this signaling plays an important role in infection (76). Interestingly, the early activation of PI3K has been shown to be mediated by both integrins and GFR, suggesting a possible additive effect; however, this attractive hypothesis may need to be further substantiated. Additionally, these early signaling events leave a number of open issues such as whether the observed activation is directly connected to entry or internalization. An important consideration may be that the kinetics of signaling activation and internalization do not coincide; i.e., signal activation appears to be early whereas internalization occurs at a later event. However, blocking EGFR activation blocks infection and internalization. Hence, there may be two (or more) unrelated waves of signal transduction activation. It is interesting that EGFR signaling is modulated by integrins, further associating these known HPV16 coreceptors (80, 81).

In addition to HSPGs, integrins, and GFRs, the HPV16 PsV entry pathway was shown to utilize tetraspanins, specifically, CD63 and CD151 (82). Tetraspanins are widely expressed transmembrane domain-containing proteins that interact with other tetraspanins and with other proteins to form tetraspanin-enriched microdomains (TEMs) (83, 84). The expression of CD151 was recently shown to be localized to the basal cells of human cervical epithelium matching the tropism of HPV16 infection, and depletion of CD151 reduces HPV16 PsV endocytosis in HeLa cells but does not affect cell surface binding (85). Of note, tetraspanins have been shown to interact with molecules identified in HPV16 infection such as HSPGs, integrins, and GFRs (84). For example, there is a tight association between tetraspanin CD151 and α6 integrin, and this association is thought to facilitate the coupling of signaling pathways (86). Knockdown of the alpha subunits of CD151-associated integrins (α3 and α6) decreased HPV16 PsV infectivity, and mutations in CD151 that ablate integrin binding did not restore HPV16 PsV internalization in CD151-depleted HeLa cells, indicating that CD151 may mediate HPV16 infection via integrins (85).

Until recently, there was no direct evidence that the HPV16 L2 minor capsid protein possessed any function at the cell membrane. Previously, several studies suggested that L2 was not involved in cell surface binding, such as studies by Volpers et al. which demonstrated that HPV33 L1 VLP and L1L2 VLP bind equally well to HeLa cells (87) and studies by Roden et al. which showed that BPV1 virions are competitively inhibited from binding to C127 cells by either BPV1 L1 VLP or L1L2 VLP (41). However, in the context of subsequent HSPG research, it appears that those studies may have simply highlighted the significant role of HSPGs in binding the L1 protein. The L2 protein has been indirectly linked to HPV16 infectious entry, as demonstrated by PsV with low average levels of L2 being less infectious that those with higher average levels (5). Further, HPV16 L1L2 VLP are twice as efficient as HPV16 L1 VLP in entry into HeLa cells (39), and preincubation of COS-1 cells (monkey kidney fibroblast cell line) with the HPV16 L2 peptide aa 108 to 126 decreased infectivity of HPV16 PsV (88). Additionally, this L2 peptide was shown to interact directly with the epithelial cell surface in the absence of L1 on human cervical cancer cell lines (HeLa, SiHa, and CaSki), indicating the presence of an unidentified L2-specific binding molecule on host cells (88). It was demonstrated in a recent study that the annexin A2 heterotetramer (A2t) is an L2-specific receptor and contributes to HPV16 internalization and infection of epithelial cells in an L2-dependent manner (39). The L2 peptide 108–126 was shown to specifically interact with the S100A10 subunit of A2t, with an equilibrium dissociation constant (KD) of 6.7 × 10 M. This binding strength is physiological and within the same order of magnitude as that of other viruses (89, 90). It was further shown that reduction of annexin A2 expression by short hairpin RNA (shRNA) in HeLa cells led to a 75% decrease in HPV16 L1L2 VLP internalization and significantly reduced infection with HPV16 PsV (39). While our laboratory recently found that HPV16 PsV transduction of the annexin A2-deficient HepG2 cell line (human hepatocellular carcinoma) can occur (W. M. Kast, unpublished data), it is unclear what significance this result has for the nonphysiological cell type for HPV16 infection. However, this cell line, as well as Alexander cells (annexin A2-deficient human hepatoma), was previously shown to bind to the L2 108–126 peptide approximately 80% less than cell lines that contain annexin A2 (88). Taken together, these results may suggest that in the absence of annexin A2, an alternative HPV16 internalization pathway exists which could utilize different cell surface components. However, no clear evidence for a multiple-pathway concept exists to date. Since A2t was shown to be involved in HPV16 PsV infectious entry into relevant epithelial cells (39), we suggest that A2t should be included conceptually as a component of HPV16 binding and entry. Annexin A2 is a member of a family of Ca and phospholipid binding proteins and is found at the cell surface as a heterotetramer, consisting of two annexin A2 monomers noncovalently linked by a S100A10 dimer (91). Annexin A2 has been shown to function in exocytosis, endocytosis, cell adhesion, membrane fusion, and membrane trafficking, along with the binding and uptake of multiple viruses (9298). In addition, the annexin A2 C terminus has been shown to bind heparin while the N terminus is the site for S100A10 interaction (91). Interestingly, cytomegalovirus (CMV), which uses annexin A2 for infection, also uses a multitude of other molecules similar to those mentioned for HPV16 such as HSPGs, EGFR, and integrins (99).

The dynamic binding of HPV16 to the cell surface also coincides with movement of the viral particle. By exploring the lateral mobility of HPV16 PsV on the epithelial cell surface during the initial phases of infection, it has been shown that the extracellular directed motion of HPV16 PsV occurs much more frequently on actin-containing filopodia or retraction fibers than on the cell body (100). It was previously shown that both HPV16 PsV and HPV31 PsV highly increase the formation of filopodia on HaCaT cells, a process that appears to be dependent on FAK phosphorylation, tyrosine kinases, and PI3K (44, 101). While attached to these cell protrusions, HPV16 PsV are juxtaposed to the plasma membrane with a space of 12 nm ± 4 nm, suggesting a membrane receptor with a large ectodomain (100). In contrast to the diffusive motion of other viruses, movement of HPV16 PsV along filopodia was directed toward the cell body at an average rate similar to that of actin retrograde transport, which suggests a specific receptor-mediated process (100). This active transport of HPV16 PsV, while not required for infection, facilitated infection in subconfluent cells, as demonstrated by an ∼50% reduction in infection when virus transport was blocked in epithelial cells (100). Interestingly, the HPV16 PsV movement on actin filopodia resembles the movement of EGFR (102). Taking those data together with the results of the previous study linking EGFR and HPV16 PsV (77), it is tempting to speculate that these HSPG/GF/HPV16 PsV complexes may be stimulating active filopodial transport when they bind to the EGFR, particularly as disruption of active transport and EGFR signaling reduces levels of HPV16 PsV infectivity by similar amounts. However, this hypothesis will need to be explored in future studies.

Subsequent to cell surface binding, HPV16 must enter the cell in order to induce a productive infection. Although many studies have been conducted, the complete details of endocytosis are yet to be fully elucidated. A recent study showed that the infectious process of HPV16 PsV occurs very slowly and asynchronously in epithelial cells, with an average half-time of 12 h (75), which coincides with previous studies done with HPV16 PsV and HPV18 PsV (28, 69). Most viruses are internalized rapidly, whereas the overall kinetics for HPV16 internalization is among the slowest of those of the viral entry pathways described. It is interesting that HSPGs have been shown to also have a low rate of internalization after ligand binding (103). It may be that the required conformational changes, furin cleavage, and interaction with multiple disparate cell surface molecules create the rate limiting step(s) in HPV16 internalization (82, 104). Although occurring asynchronously over many hours, when HPV16 PsV endocytic events do occur on epithelial cells, they occur quickly, on average within 120 s after movement has stopped (75).

Nonenveloped viruses that enter by endocytosis typically use one of two canonical pathways, the clathrin- and caveolae-mediated pathways. Early studies on HPV endocytosis relied on biochemical inhibitors to block components of these known internalization pathways; however, these small-molecule inhibitors often exhibit pleiotrophic effects on cell function. These studies were also complicated by the usage of different types of particles (L1 VLP, L1L2 VLP, and PsV) and different HPV genotypes, making conflicting results difficult to interpret. However, recent research confirms that HPV16 PsV endocytosis is independent of clathrin and caveolae in epithelial cells (75, 82). It was also demonstrated that inhibition of clathrin- and caveolin/raft-dependent endocytosis by either siRNA-mediated downregulation or dominant-negative mutants, along with inhibition of dynamin function, did not impair HPV16 PsV infection. HPV16 PsV endocytosis in HeLa and HaCaT cells was further described as a clathrin-, caveolin-, lipid raft-, flotillin-, cholesterol-, and dynamin-independent mechanism distinct from macropinocytosis (75). HPV16 PsV infection required actin polymerization but was independent of Rho-like GTPases. The pathway may indicate a novel ligand-induced endocytosis pathway whose closest relative appears to be macropinocytosis in terms of required host cell factors. Internalization was shown to be dependent on several signaling factors, namely, EGFR, protein kinase C (PKC), p21-activated kinase 1 (PAK-1), and PI3K and possibly other tyrosine and serine/threonine kinases. Specifically, for the endocytic internalization of HPV16 PsV, the particles required actin polymerization, Na/H exchanger, PAK-1, PI3K, and tyrosine and serine/threonine kinases. Once bound, the PsV caused slight indentations in the plasma membrane that were 65 to 120 nm in diameter, with about 5 to 10 nm of distance between the particle and the membrane, and actin polymerization was involved in scission of the HPV16 PsV-containing vesicles after the particle had sunk into the plasma membrane (75).

MODEL OF HPV16 BINDING AND UPTAKE

In the context of the current research reviewed here, we propose a model of HPV16 uptake in epithelial cells (Fig. 1). This model of HPV16 uptake synergistically incorporates the varied observations into a cohesive concept while fitting within the current body of HPV16 receptor knowledge. For reference, we have included a table of HPV16-associated cell surface molecules discussed in this review, noting the HPV genotype, model particle system, and cell type used (Table 1).

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HPV16 binds to heparin sulfate proteoglycans (HSPGs) on either the epithelial cell surface or the basement membrane or through laminin-332 on the ECM. Epidermal growth factor receptor (EGFR) and/or keratinocyte growth factor receptor (KGFR) may also become activated at this time through HSPG/growth factor HPV16 complexes and may initiate intracellular signaling cascades that include the activation of the phosphoinositide 3-kinase (PI3K) pathway. After binding to HSPGs, the virion undergoes a conformational change that is facilitated by cyclophilin B (CyPB), which exposes more amino acids of the N terminus of L2. Subsequently, HPV16 binds to α6 integrin, which initiates a second intracellular signaling cascade. After undergoing conformational changes and signaling, the HPV16 capsid binds to A2t. Upon HPV binding to the A2t, clathrin-, caveolin-, lipid raft-, flotillin-, cholesterol-, and dynamin-independent endocytosis of HPV16 is triggered. HPV16-induced PI3K activation may then be involved in vesicle closure and actin scission. Tetraspanins are hypothesized to be involved in forming an enriched membrane domain that stabilizes connections between HPV16-associated molecules. Therefore, as opposed to a sequential handoff of the virion from one receptor to another, we hypothesize that a receptor complex coalesces and includes HSPGs, CyPB, α6 integrin, tetraspanins, EGFR, and A2t.

Table 1

HPV16-associated cell surface molecules

Binding/uptake component(s)ReferenceHPV type(s)Cell type(s)
Heparin sulfate proteoglycan28HPV16 PsV, HPV33 PsVHaCaT
37HPV33 PsV, HPV33 VLPCOS7
41HPV16 L1L2 VLP, HPV11 L1L2 VLP, BPV-1C127, human foreskin keratinocytes, other transformed epithelial and fibroblast cell lines
44HPV16 PsVHaCaT
45HPV16 PsV, HPV31 PsV, HPV5 PsVIn vivo BALB/c murine genital tract, HaCaT
46HPV11 L1 VLPHaCaT, CHO-K1, mutant CHO (pgsA-745)
54HPV16 L1 VLP, HPV11 virionsK562, KH-SV, BOUA-SV
56HPV16 PsV, HPV16 L1L2 VLPCOS-7
57HPV16 PsV, HPV16 L1 VLP, HPV18 L1 VLPHaCaT
61HPV16 PsVA549, CHO-K1, mutant CHO (pgsF-17)
Furin/PC cleavage38HPV16 PsV, HPV16 L1 VLPIn vivo BALB/c murine genital tract
59HPV16 PsVFD11, mutant CHO (pgsA-745), human foreskin keratinocytes
62HPV16 PsVHeLa, C127, FD11
63HPV16 PsVHaCaT
Laminin-33247HPV16 PsV, HPV18 PsV, HPV 11 PsV, HPV11 virions, BPV-1, CRPVaHaCaT, KH-SV, BOUA-SV, human vaginal keratinocytes
Cyclophilin B64HPV16 PsV, HPV18 PsV, HPV5 PsV, HPV6 PsV, HPV31 PsV, HPV45 PsV, HPV52 PsV, HPV58 PsV, BPV-1HaCaT, 293TT, HeLa
67HPV16 PsVHaCaT, 293TT
α6 integrins44HPV16 PsVHaCaT
55HPV16 PsV, HPV31 PsV, HPV45 PsVIn vivo BALB/c murine genital tract, C127
70HPV16 L1 VLPHaCaT, HeLa, C33A, Caski, T98G, SK-N-SH, OVCAR-4, K562, T47D, COS-7
71HPV6b L1 VLPCV-1, HaCaT, DG75
74HPV16 L1L2 VLP, HPV31 L1L2 VLP, HPV35 L1L2 VLP, HPV18 L1 VLP, HPV6b L1 VLP, BPV1 L1L2 VLPA431
Annexin A2 heterotetramer39HPV16 PsV, HPV16L1L2 VLP, HPV16L1 VLPHaCaT, HeLa
Epidermal growth factor receptor76HPV16 PsVHaCaT
77HPV16 PsV, HPV31 PsVHaCaT, CHO-K1, mutant CHO (pgsd-677)
Tetraspanins82HPV16 PsVHeLa, 293TT
85HPV16 PsVHaCaT, HeLa, NHEK
CRPV, cottontail rabbit papillomavirus.

In our proposed model, HPV16 initially binds to HSPGs through L1 on either the epithelial cell surface or the basement membrane. HPV16 may also initially bind to laminin-332 on the ECM and subsequently transfer to the cell surface. Included in this model is the presence of GFRs, which become activated by HSPG/GF/HPV16 complexes and initiate an early intracellular signaling cascade most likely by lateral association of HSPG/GF/HPV16 and GFRs without the need to be released from cells. This activation of EGFR may also be related to the active transport of HPV16 along filopodia; however, this remains to be further explored. This model does not exclude the possibility of an occasional complex release and renewed binding for infection. After binding to HSPGs and initial changes in L1, the virion undergoes a conformational change to further expose the N terminus of L2 that is facilitated by CyPB, which has been shown to bind to HSPGs. After primary binding to HSPGs, HPV16 may initiate an additional signaling cascade through interactions with α6 integrins; however, a direct biochemical interaction between HPV16 and α6 integrins remains to be shown. After initial attachment to the cell surface, and possibly after initial signaling events and conformational changes, the HPV16 capsid binds to A2t. We propose that, upon HPV16 binding to A2t, clathrin-, caveolin-, lipid raft-, flotillin-, cholesterol-, and dynamin-independent endocytosis of HPV16 is triggered. Binding of HPV16 leads to PI3K activation through EGFR and/or integrin signaling, and PI3K signaling in macrophages has been shown to be required for closure of macropinosomes (105). This leads us to hypothesize that HPV16-induced PI3K activation, occurring late after binding and asynchronously, is involved in vesicle closure and perhaps actin scission. Tetraspanins are hypothesized to be involved in forming an enriched membrane domain containing HPV16-associated receptor molecules, such as HSPGs, integrins, GFRs, and others that have been shown to interact directly with tetraspanins (85). Therefore, as opposed to a sequential handoff of the virion from one receptor to another, we hypothesize that a receptor complex coalesces and may incorporate some or all the mentioned components, including HSPGs, CyPB, α6 integrin, tetraspanins, GFRs, and A2t. While this proposed model may at first seem complex, it may be a simple evolutionary result of targeting a preexisting cellular complex and subsequent entry mechanism for viral invasion. In fact, in similarity to HPV16, other viruses, such as CMV, which utilizes annexin A2 for infection, HSPGs for tethering, EGFR for signaling, and integrins for internalization, have been previously shown to use a variety of molecules for different purposes (99).

CONCLUSION

While research to date has brought the field significantly closer to understanding the complex HPV16 infectious process, questions still remain. Our model proposes that many of the HPV16-associated receptors form a complex that eventually leads to internalization; however, it is possible that an alternative sequence occurs, such as a sequential handoff of the virion at one or more steps in the pathway. It may be that not one but multiple HPV16 internalization and/or infectious entry pathways exist and that these pathways may use different cell surface molecules. Further, while it is clear that signaling plays a critical role in HPV16 entry, the connection between these signals and downstream events is not completely clear. Additionally, although there is evidence in the literature to support the notion of an interaction between the different identified HPV16 cell surface receptors and coreceptors, these dynamic connections have not been well studied specifically for HPV16 uptake. An important requirement for future consensus on the mechanism of HPV16 entry is confirmation of individual findings by multiple laboratories. Finally, whether this model holds for other (both high-risk and low-risk) HPV types besides HPV16, along with other cell types besides epithelial cells, remains to be seen. These future studies will be needed to explore the interactions of all the molecules discussed in this review and to further elucidate the binding and uptake of HPV16. Although the model we propose remains speculative, it provides a firm framework that can guide future research on HPV16 cell entry.

Norris Comprehensive Cancer Centera
Department of Molecular Microbiology & Immunologyb
Department of Obstetrics & Gynecology, University of Southern California, Los Angeles, Los Angeles, California, USA
Emmy-Noether Group Virus Endocytosis, Institutes of Molecular Virology and Medical Biochemistry, University of Münster, Münster, Germanyd
Corresponding author.
Address correspondence to W. Martin Kast, ude.csu@tsakm.
Present address: Adam B. Raff, Department of Internal Medicine, Kaiser Permanente Los Angeles Medical Center, Los Angeles, California, USA.
Address correspondence to W. Martin Kast, ude.csu@tsakm.

Abstract

Human papillomaviruses (HPVs) infect epithelia and can lead to the development of lesions, some of which have malignant potential. HPV type 16 (HPV16) is the most oncogenic genotype and causes various types of cancer, including cervical, anal, and head and neck cancers. However, despite significant research, our understanding of the mechanism by which HPV16 binds to and enters host cells remains fragmented. Over several decades, many HPV receptors and entry pathways have been described. This review puts those studies into context and offers a model of HPV16 binding and entry as a framework for future research. Our model suggests that HPV16 binds to heparin sulfate proteoglycans (HSPGs) on either the epithelial cell surface or basement membrane through interactions with the L1 major capsid protein. Growth factor receptors may also become activated through HSPG/growth factor/HPV16 complexes that initiate signaling cascades during early virion-host cell interactions. After binding to HSPGs, the virion undergoes conformational changes, leading to isomerization by cyclophilin B and proprotein convertase-mediated L2 minor capsid protein cleavage that increases L2 N terminus exposure. Along with binding to HSPGs, HPV16 binds to α6 integrins, which initiate further intracellular signaling events. Following these primary binding events, HPV16 binds to a newly identified L2-specific receptor, the annexin A2 heterotetramer. Subsequently, clathrin-, caveolin-, lipid raft-, flotillin-, cholesterol-, and dynamin-independent endocytosis of HPV16 occurs.

Abstract

ACKNOWLEDGMENTS

This review contains data generated with support from Public Health Service grant R01 CA074397 from the National Cancer Institute to W.M.K., who also holds the Walter A. Richter Cancer Research Chair. A.W.W. and L.Y. are both TL1 Scholars and are supported by SC CTSI (NIH/NCRR/NCATS) grant TL1TR000132;. A. W.W. is supported in part by award number P30CA014089 from the National Cancer Institute through the Norris Comprehensive Cancer Center Heidelberger Award. M.S. was supported by the German Science Foundation (DFG; grants SCHE 1552/2-1;, SFB629A16;, and GRK1409C2).

The content is solely our responsibility and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.

ACKNOWLEDGMENTS

Footnotes

Published ahead of print 27 March 2013

Footnotes

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