Cryoelectron Microscopy Maps of Human Papillomavirus 16 Reveal L2 Densities and Heparin Binding Site

Guan, Jian; Bywaters, Stephanie M.; Brendle, Sarah A.; Ashley, Robert E.; Makhov, Alexander M.; Conway, James F.; Christensen, Neil D.; Hafenstein, Susan

doi:10.1016/j.str.2016.12.001

Cited by 56 publications

(84 citation statements)

References 71 publications

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“…However, these have not been clearly mapped, and structural data are not yet available. Recent high-resolution reconstruction of cryoelectron microscopic images of HPV16 pseudovirions provided evidence for L2 density present on the outer capsid surface is suggestive of interaction between the L1 and L2 proteins (62). This supports previous findings based on the accessibility of L2 epitopes, which have suggested that L2 protein penetrates the capsid barrier allowing binding of L2 specific antibodies to N-terminal segments (residues 60 to 120) (4).…”

Section: Discussionsupporting

confidence: 82%

Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

DiGiuseppe

Bienkowska-Haba

Guion

et al. 2017

J Virol

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During infectious entry, acidification within the endosome triggers uncoating of the human papillomavirus (HPV) capsid, whereupon host cyclophilins facilitate the release of most of the major capsid protein, L1, from the minor capsid protein L2 and the viral genome. The L2/DNA complex traffics to the trans-Golgi network (TGN). After the onset of mitosis, HPV-harboring transport vesicles bud from the TGN, followed by association with mitotic chromosomes. During this time, the HPV genome remains in a vesicular compartment until the nucleus has completely reformed. Recent data suggest that while most of L1 protein dissociates and is degraded in the endosome, some L1 protein remains associated with the viral genome. The L1 protein has DNA binding activity, and the L2 protein has multiple domains capable of interacting with L1 capsomeres. In this study, we report that some L1 protein traffics with L2 and viral genome to the nucleus. The accompanying L1 protein is mostly full length and retains conformation-dependent epitopes, which are recognized by neutralizing antibodies. Since more than one L1 molecule contributes to these epitopes and requires assembly into capsomeres, we propose that L1 protein is present in the form of pentamers. Furthermore, we provide evidence that the L1 protein interacts directly with viral DNA within the capsid. Based on our findings, we propose that the L1 protein, likely arranged as capsomeres, stabilizes the viral genome within the subviral complex during intracellular trafficking.IMPORTANCE After internalization, the nonenveloped human papillomavirus virion uncoats in the endosome, whereupon conformational changes result in a dissociation of a subset of the major capsid protein L1 from the minor capsid protein L2, which remains in complex with the viral DNA. Recent data suggest that some L1 protein may accompany the viral genome beyond the endosomal compartment. We demonstrate that conformationally intact L1 protein, likely still arranged as capsomeres, remains associated with the incoming viral genome throughout mitosis and transiently resides in the nucleus until after the viral DNA is released from the transport vesicle.KEYWORDS HPV entry, HPV16, L1 protein, L2 protein, mitosis, nuclear transport, virus trafficking P apillomaviruses are a family of nonenveloped DNA viruses that infect a wide range of hosts with a preference for epithelial cells. The papillomaviruses that infect humans (HPVs) are a particular health burden. While most infections with HPVs are cleared by the immune system, a persistent infection with the high-risk types is associated with increased risk of carcinomas. The high-risk type 16 accounts for

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Section: Discussionsupporting

confidence: 82%

Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

DiGiuseppe

Bienkowska-Haba

Guion

et al. 2017

J Virol

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“…These molecular envelopes demonstrate that heparin asymmetrically decorates rather than symmetrically decorates the surface of the capsid, the latter of which has been demonstrated for a number of icosahedral viruses. This may be because prior structural studies of icosahedral viruses in complex with heparin utilized averaging techniques that may have eliminated the asymmetric distribution of heparin on the capsid surface (53,54,(57)(58)(59)(60). The packing of capsids into a crystal for X-ray crystallography inherently imposes the averaging of structures (61).…”

Section: Discussionmentioning

confidence: 99%

Porcine Circovirus 2 Uses a Multitude of Weak Binding Sites To Interact with Heparan Sulfate, and the Interactions Do Not Follow the Symmetry of the Capsid

Dhindwal

Avila

Feng

et al. 2019

J Virol

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Porcine circovirus 2 (PCV2) is the smallest pathogenic virus capable of autonomous replication within its host. Infections result in immunosuppression and subsequent death of the host and are initiated via the attachment of the PCV2 icosahedral capsid to heparan sulfate (HS) and chondroitin sulfate B (CSB) glycosaminoglycans on the cell surface. However, the underlying mechanism of structural recognition remains to be explored. Using heparin, a routinely used analog of heparan sulfate, we demonstrate that increasing lengths of heparin exhibit a greater affinity toward PCV2. Our competition assays indicate that dextran sulfate (8 kDa) has a higher affinity for PCV2 than heparin (12 kDa), chondroitin sulfate B (41 kDa), hyaluronic acid (1.6 MDa), and dextran (6 kDa). This suggests that polymers high in sulfate content are capable of competing with the PCV2-heparan sulfate interaction and, thus, have the potential to inhibit PCV2 infection. Finally, we visualized the interaction between heparin and the PCV2 capsid using cryo-electron microscopy single-particle analysis, symmetry expansion, and focused classification. The image reconstructions provide the first example of an asymmetric distribution of heparin on the surface of an icosahedral virus capsid. We demonstrate that each of the 60 capsid subunits that generate the T=1 capsid can bind heparin via one of five binding sites. However, not all of the binding sites were occupied by heparin, and only one-third to two-thirds of the binding sites were occupied. The binding sites are defined by arginine, lysine, and polar amino acids. Mutating the arginine, lysine, and polar amino acids to alanine diminished the binding capacity of PCV2 to heparin. IMPORTANCE It has been demonstrated that porcine circovirus 2 (PCV2) attaches to cells via heparan sulfate (HS) and chondroitin sulfate B (CSB) glycosaminoglycans; however, the underlying structural mechanism describing the HS/CSB recognition by PCV2 remains to be explored. We used cryo-electron microscopy with single-particle analysis, symmetry expansion, and focused classification to visualize the interaction between the PCV2 capsid and heparin, an analog of heparan sulfate, to better than 3.6-Å resolution. We observed that the interaction between PCV2 and heparin does not adhere to the icosahedral symmetry of the capsid. To the best of our knowledge, this is the first example where the interaction between heparin and an icosahedral capsid does not follow the symmetry elements of the capsid. Our findings also suggest that anionic polymers, such as dextran sulfate, may act to inhibit PCV2 infection.

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“…This could be explained by the location of the insertion and whether the whole peptide is readily displayed on the surface of the VLP and to the immune system. Modelled cryo-electron microscopy images of MS2 [79] and HPV16 L1 [80] seem to show that the N-termini of MS2 coat proteins and the DE loops of HPV16 L1 coat proteins (where the peptides are inserted) are readily exposed on the surface of their icosahedral structures compared with the H4 helix of HPV16 L1 (Figure 4). In addition to this, insertions at these different locations can affect the assembly of the chimeric coat proteins into VLPs and ultimately, their immunogenicity.…”

Section: Expert Commentary and Perspectives For The Futurementioning

confidence: 99%

Virus-like Particle-Based L2 Vaccines against HPVs: Where Are We Today?

Yadav

Zhai

Tumban

2019

Viruses

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Human papillomaviruses (HPVs) are the most common sexually transmitted infections worldwide. Ninety percent of infected individuals clear the infection within two years; however, in the remaining 10% of infected individuals, the infection(s) persists and ultimately leads to cancers (anogenital cancers and head and neck cancers) and genital warts. Fortunately, three prophylactic vaccines have been approved to protect against HPV infections. The most recent HPV vaccine, Gardasil-9 (a nonavalent vaccine), protects against seven HPV types associated with ~90% of cervical cancer and against two HPV types associated with ~90% genital warts with little cross-protection against non-vaccine HPV types. The current vaccines are based on virus-like particles (VLPs) derived from the major capsid protein, L1. The L1 protein is not conserved among HPV types. The minor capsid protein, L2, on the other hand, is highly conserved among HPV types and has been an alternative target antigen, for over two decades, to develop a broadly protective HPV vaccine. The L2 protein, unlike the L1, cannot form VLPs and as such, it is less immunogenic. This review summarizes current studies aimed at developing HPV L2 vaccines by multivalently displaying L2 peptides on VLPs derived from bacteriophages and eukaryotic viruses. Recent data show that a monovalent HPV L1 VLP as well as bivalent MS2 VLPs displaying HPV L2 peptides (representing amino acids 17–36 and/or consensus amino acids 69–86) elicit robust broadly protective antibodies against diverse HPV types (6/11/16/18/26/31/33/34/35/39/43/44/45/51/52/53/56/58/59/66/68/73) associated with cancers and genital warts. Thus, VLP-based L2 vaccines look promising and may be favorable, in the near future, over current L1-based HPV vaccines and should be explored further.

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Cryoelectron Microscopy Maps of Human Papillomavirus 16 Reveal L2 Densities and Heparin Binding Site

Cited by 56 publications

References 71 publications

Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

Porcine Circovirus 2 Uses a Multitude of Weak Binding Sites To Interact with Heparan Sulfate, and the Interactions Do Not Follow the Symmetry of the Capsid

Virus-like Particle-Based L2 Vaccines against HPVs: Where Are We Today?

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