Article

Cryoelectron Microscopy Maps of Human

Papillomavirus 16 Reveal L2 Densities and Heparin
Binding Site
Highlights Authors
d 4.3-A resolution 3D reconstructions based on gold- Jian Guan, Stephanie M. Bywaters,
standard FSC Sarah A. Brendle, ..., James F. Conway,
Neil D. Christensen, Susan Hafenstein
d Classification of HPV overcomes heterogeneity that limits the
resolution Correspondence
d L1 protein was built and used to reveal L2 minor protein shafenstein@hmc.psu.edu

d Heparin binding elicits conformational changes to L2 In Brief

Guan et al. solved near-atomic resolution
cryo-EM maps of HPV16 and HPV16-
heparin, building the L1 protein structure
including C-terminal arm. The structural
analysis revealed the heterogeneity of
HPV16, the location and features of L2
protein, the heparin binding site, and
suggested L2 conformational changes
during cell entry.

Accession Numbers
5KEP
5KEQ

Guan et al., 2017, Structure 25, 253263

February 7, 2017 2016 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.str.2016.12.001
Structure
Article
Cryoelectron Microscopy Maps of Human
Papillomavirus 16 Reveal L2 Densities
and Heparin Binding Site
Jian Guan,1 Stephanie M. Bywaters,2 Sarah A. Brendle,2 Robert E. Ashley,1 Alexander M. Makhov,3 James F. Conway,3
Neil D. Christensen,2 and Susan Hafenstein1,4,*
1Division of Infectious Diseases, Department of Medicine, Penn State College of Medicine, The Pennsylvania State University College of
Medicine, Mail Code H036, 500 University Drive, P.O. Box 850, Hershey, PA 17033-0850, USA
2Department of Pathology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA
3Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 5th Avenue, Pittsburgh, PA 15260, USA
4Lead Contact
*Correspondence: shafenstein@hmc.psu.edu
http://dx.doi.org/10.1016/j.str.2016.12.001
SUMMARY
Human papillomavirus (HPV) is a significant health
burden and leading cause of virus-induced cancers.
The current commercial vaccines are genotype spe-
cific and provide little therapeutic benefit to patients
with existing HPV infections. Host entry mechanisms
represent an excellent target for alternative thera-
peutics, but HPV receptor use, the details of cell
attachment, and host entry are inadequately under-
stood. Here we present near-atomic resolution struc-
tures of the HPV16 capsid and HPV16 in complex
with heparin, both determined from cryoelectron
micrographs collected with direct electron detection
technology. The structures clarify details of capsid
architecture for the first time, including variation in
L1 major capsid protein conformation and putative
location of L2 minor protein. Heparin binds spe-
cifically around the capsid icosahedral vertices and
may recapitulate the earliest stage of infection,
providing a framework for continuing biochemical,
genetic, and biophysical studies.
INTRODUCTION
Human papillomavirus 16 (HPV16) is the most prevalent onco-
genic genotype in HPV-associated anogenital and oral cancers.
Having co-evolved with the human host for more than a million
years, HPV develops chronic infections by activating the cell
cycle as epithelial cells differentiate in order to create a replica-
tion competent environment that allows viral genome expression
and packaging into infectious particles (Doorbar et al., 2015).
Since the first reported association with cancer, extensive HPV
research has focused on the prevention of cervical cancer,
including vaccine development to prevent virus infection (Malik
et al., 2014; Nicol et al., 2015; Toh et al., 2015). However, the
current commercial vaccines are genotype specific and provide
little therapeutic benefit to patients with existing HPV infections
Structure 25, 253263, February 7, 2017 2016 Published by Elsevier Ltd. 253
(Dickson et al., 2015; Gilmer, 2015; McKee et al., 2015). Target-
ing host entry mechanisms may be a successful alternative
approach because virus entry is a key step of HPV infection
regardless of genotype. However, receptor use for HPV, the de-
tails of cell attachment, and capsid conformational changes
required for host entry are poorly understood.
HPV is epitheliotropic and its replication is tightly associated
with the terminal differentiation of basal cells into keratinocytes.
This restricted tropism makes production and purification of
high-titer virus preparations for research problematic. Therefore,
alternative HPV production methods have been developed
within the HPV community for molecular biology and structural
studies. Virus-like particles can be composed of only the major
structural protein (L1) or of the major and minor capsid proteins
(L1/L2) and are not infectious since they are devoid of viral
genome (Hernandez et al., 2012; Kirnbauer et al., 1992, 1993).
Pseudovirus is composed of both structural proteins, which
are expressed along with a plasmid so that a copy of the plasmid
DNA can be packaged as a mock genome (Buck et al., 2005a,
2005b). Quasiviruses assemble a structurally complete capsid
and are infectious since they package a copy of the cottontail
rabbit papillomavirus genome (CRPV) (Christensen, 2005). All
of these types of HPV capsids preserve the main attributes of
the native capsid structure and have been used successfully
for vaccine development and for studies of antigenicity, receptor
usage, entry mechanisms, and capsid structure. Quasiviruses
have been used for the HPV16 work presented here.
Structures of sub-viral particles have been solved by X-ray
crystallography, including the HPV16 L1 pentamer and a T = 1
particle (Chen et al., 2000; Dasgupta et al., 2011); however, the
complete HPV T = 7d capsid has only been visualized by cryoe-
lectron microscopy (cryo-EM) reconstructions (Baker et al.,
1991; Buck et al., 2008; Cardone et al., 2014; Guan et al.,
2015a; Lee et al., 2014). Currently, the highest-resolution struc-
ture of the intact T = 7d HPV16 capsid is a 9-A resolution cryo-
EM structure, from which a pseudoatomic model was developed
using the major structural protein, L1 (Cardone et al., 2014). The
5560 nm diameter HPV capsid is composed of 360 copies of
L1 arranged into 72 capsomers, each composed of five copies
of L1. Twelve capsomers lay on the icosahedral 5-fold vertices
and are referred to as pentavalent capsomers, whereas the
remaining 60 capsomers are each surrounded by six other
capsomers and referred to as hexavalent capsomers. An exten-
sion of the L1 C terminus, or C-terminal arm, links capsomers
together and provides interactions leading to the formation of
disulfide bonds between Cys175 and Cys428 from two neigh-
boring L1 proteins (Buck et al., 2005a, 2005b; Cardone et al.,
2014; Sapp et al., 1998). Although the invading arm structure
of bovine papilloma structure is known (Wolf et al., 2010), the
stabilizing C-terminal arm structure has not been solved yet
for HPV.
Besides the L1 major capsid protein, there is an uncertain
number of L2 minor structural proteins that co-assemble with
L1 to form infectious virus capsids. The L2 minor protein has a
number of functions including encapsidation of DNA, involve-
ment in the conformational changes of the capsid during cell
entry, disruption of the endosomal membrane, and subcellular
trafficking of the incoming viral genome (Bronnimann et al.,
2013; Buck et al., 2008; Raff et al., 2013). Specifically, the N-ter-
minal residues of L2 (188) are exposed on the surface of the
capsid although the anti-L2 antibody, RG-1 recognition of L2
(1736) is dependent on maturation status or cell attachment
(Day et al., 2008a, 2008b; Finnen et al., 2003; Lowe et al.,
2008; Okun et al., 2001). An N-terminal L2 motif (RXXR, furin
cleavage site) is conserved across HPV genotypes and thus
has promise as a new target for vaccine development (Bronni-
mann et al., 2013; Conway et al., 2011; Roden et al., 2001). A
previous structural study using pseudovirus maps (23 and
25 A) interpreted small densities beneath each capsomer as
belonging to L2 (Buck et al., 2008). Based on analysis of the
late gene products, L2 protein has been predicted to bind within
the center of the L1 capsomer with the antigenic region facing
outward (Lowe et al., 2008). Stoichiometric studies have indi-
cated that there are about 1236 L2 molecules per HPV capsid
(Okun et al., 2001); however, other works have suggested the
amount of L2 per capsid is variable, and may include up to 72
L2 molecules per capsid, perhaps one L2 at each capsomer
(Buck et al., 2008; Doorbar and Gallimore, 1987; Trus et al.,
1997). Naturally produced virions average approximately 2436
L2 molecules per capsid (Bronnimann et al., 2013; Buck et al.,
2008; Campos and Ozbun, 2009). Despite these variable reports,
there is general acceptance that between 12 and 72 L2 proteins
per capsid are incorporated, each into a central internal cavity of
the capsomer via L2 C-terminal interactions.
During host entry and endocytosis, HPV conformational
changes are triggered by binding to a cell receptor (Abban and
Meneses, 2010; Kines et al., 2009; Selinka et al., 2003). Cell
surface heparan sulfates (HSs) serve as a primary attachment
receptor (Buck et al., 2006; Rommel et al., 2005; Schowalter
et al., 2011; Shafti-Keramat et al., 2003) and it is not the protein
backbone but the glycan moieties, specifically the sulfation
pattern, that are required for virus infection (Cerqueira et al.,
2013). HS proteoglycans are expressed and secreted by nearly
all mammalian cells, and are located on both the cell surface
and extracellular matrix (Bernfield et al., 1992; Buck et al.,
2006; Schowalter et al., 2011; Selinka et al., 2003). However,
because HS polysaccharides are expensive and difficult to
obtain, heparin has been used in lieu of HS in most biological
and structure studies (Cerqueira et al., 2013; Selinka et al.,
2007). Both heparin and HS binding have been shown to induce
254 Structure 25, 253263, February 7, 2017
conformational changes of virus during entry (Florin et al., 2012;
Knappe et al., 2007; Levy et al., 2009). Co-crystallization of a
truncated L1 capsomer together with heparin oligosaccharides
(8 and 10 monosaccharide units) suggested four distinct heparin
binding sites that mapped to the L1 protein (Dasgupta et al.,
2011). Three of these HS binding sites were verified by subse-
quent mutational analysis (Dasgupta et al., 2011; Richards
et al., 2013). Nonetheless, the molecular mechanisms regarding
the binding of heparin during entry remain fragmented and not
completely understood.
With recent technological advances in cryo-EM image detec-
tion and the introduction of new software classification schemes,
we can now investigate HPV at near-atomic resolution. Here, we
present the near-atomic resolution structures of HPV16 alone
and HPV16 interacting with heparin molecules at 4-A resolutions.
Three-dimensional (3D) classification of the data revealed that
HPV capsids are heterogeneous in nature but contain sub-pop-
ulations of capsids with consistent diameters. The classification
of particles improved the high-resolution maps, which facilitated
building de novo the L1 protein (residues 3485), revealing the
structure of the C-terminal arm. Solving the L1 asymmetric unit
allowed us to calculate an L1-only capsid that was used to iden-
tify the location of L2 densities in our maps. Binding of heparin
induced no conformational changes in the capsid that include
local changes in L1, but there are alterations to putative L2 den-
sities. Besides the identification of the heparin binding site in the
context of the intact capsid, the changes induced by heparin
binding provide the first glimpse of an HPV entry intermediate.
We propose an entry mechanism model that may include isola-
tion of specific 5-fold capsomers due to potential interaction
between heparin and L2 protein.
RESULTS
Near-Atomic Resolution Maps Were Reconstructed by
Selecting Homogeneous Sub-populations
HPV16 capsids and capsids incubated with heparin in excess
were vitrified for cryo-EM data collection using new direct elec-
tron detector technology. The drift-corrected micrographs of
HPV and HPV-heparin displayed bumpy spherical shapes
with evident capsomer protrusions (Figures 1A and 1B). Cap-
sids contained internal density corresponding to packaged
DNA and appeared to have diameters of about 60 nm. Howev-
er, 3D classification using REgularized LIkelihood OptimizatioN
(RELION) showed that the populations of HPV and HPV-hepa-
rin capsids were heterogeneous in size (Figures S1 and S2).
Both capsid types included a large group of approximately
50% of the total number of particles that had the same diam-
eter of 569570 A. Final reconstructions using AUTO3DEM
with the gold-standard approach used 50% of the HPV parti-
cles and 50% of the HPV-heparin complex particles to recon-
struct a homogeneous population in each case to near-atomic
resolution (Figures 1C and 1D). The 4.3-A resolution of the
maps was estimated where the Fourier shell correlation (FSC)
dropped below 0.143 (Figure 2) (Table 1). Central sections
through the density maps illustrate the modest quality of the re-
constructions (Figures 1E and 1F). At this resolution, we can
see strand separation, the secondary structures of a helices
and b strands (Figures 3AI, 3AII, 3BI, and 3BII), and side chains

Figure 1. Cryo-EM Reconstruction of HPV16 and HPV16-Heparin
(A and B) Representative regions of cryo-EM micrographs of the HPV16
particle capsids (A) and HPV16 capsids complexed with heparin (B).
(C and D) The surface rendered 3D maps of HPV16 (C) and capsid-heparin
complex (D) were radially colored according to the distance from the center
(color key in angstroms) of the capsid and surface rendered at 1s. The heparin
difference density (black, difference map rendered at 4.2 s) was found at
positions surrounding only the 5-fold vertices of the capsid.
(E and F) The central sections through the cryo-EM density maps show the
quality of the reconstructions. Capsids were cut vertically through the 2-, 3-,
and 5-fold icosahedral symmetry axes (black lines), with the central 2-fold axis
appearing at the 12 oclock position.
(G) The zoom-in view of the HPV16-heparin complex shows the detailed fea-
tures of bound heparin molecules. The 5-fold symmetric axis was represented
by black pentagon.
Figure 2. Plot Showing the FSC versus Spatial Frequency of the
Icosahedrally Averaged Reconstructions for HPV16 and HPV16-
Heparin Complex
The resolution of the reconstructions was assessed where the FSC curve
crossed a correlation value of 0.143 (dashed line).
of some residues can be identified (Figures 3A13A3 and
3B13B3).
Building L1 into the Cryo-EM Density Solved the
Structure of the C-Terminal Invading Arm and Revealed
the Location of L2
Strong continuous density facilitated the modeling of the L1
protein structure from residues 3 to 485, which included the
C-terminal arm that links capsomers. The structure building
was initiated with a combination of the previously solved pseu-
doatomic L1 model (PDB: 3J6R) and the crystal structure of
the truncated L1 (PDB: 3OAE). During the iterative modeling
and refinement, six full-length L1 molecules were built separately
to make an asymmetric unit of the capsid (Figure 4A). After
applying icosahedral symmetry to this asymmetric unit an L1-
only density map was generated (see Experimental Procedures).
A difference map was calculated by subtracting the L1-only
density from the L1/L2-containing HPV16 experimental map. Dif-
ference density mapped to both pentavalent and hexavalent
capsomers (Figures 4B and 4C). Putative L2 density began at
the inside base of the capsomer (Figure 4C), rose along the
outside of the capsomer to the shoulder, ending at the outside
center of the capsomer (Figure 4B). Corresponding densities in
the pentavalent capsomer were significantly smaller than the
densities in hexavalent capsomers, both at the surface and the
internal part at the base.
Distinct Structural Differences Were Observed between
Pentavalent and Hexavalent Capsomers
Our near-atomic structure of the HPV16 capsid supplied more
detailed information on the capsid surface topology than the
maps we produced before (Guan et al., 2015a, 2015b; Lee
et al., 2014). Besides the entire C-terminal arm, surface loops
could be distinguished separately, especially the BC and EF
Structure 25, 253263, February 7, 2017 255
Table 1. Cryo-EM Image Reconstruction Data
HPV HPV-Heparin
No. of micrographs 9,137 6,175
Defocus level range (mm) 1.045.19 0.784.85
No. of particles selected from 115,112 102,829
micrograph
No. of particles selected for 57,556 51,422
reconstruction
Final resolution 4.3 4.3
loops that were seen as merged densities in the previous lower-
resolution maps (Figure 4A). To further visualize the differences
between L1 conformations in the two different environments,
the newly built structures of the pentavalent and hexavalent
capsomers were superimposed (Figure 5A). The conformational
differences were obvious on both surface loops and C-ter-
minal arms, with a root-mean-square deviation (RMSD) value
of 4.77 A between the entire sets of L1 atoms of the two cap-
somers. Since the C-terminal arm conformations were notice-
ably different, the RMSD was recalculated with this part of the
structure truncated, and found to be 1.49 A, indicating sig-
nificantly different loop structures throughout. Specifically the
hexavalent C-terminal arm extended further away from the
hexavalent capsomer, whereas the pentavalent arms were
arranged closer in a curved arc at the site of interaction with
the neighboring hexavalent capsomer. The individual BC, DE,
FG, HI, and EF loops are in decidedly different conformations
throughout; however, overall the hexavalent capsomers formed
a more open crown-like structure compared with the pentavalent
capsomers.
Differences between the pentavalent and hexavalent cap-
somers were also apparent in the local resolution map (Figures
5B and 5C). The pentavalent capsomer surface had lower local
resolution compared with the hexavalent capsomer, especially
near the center, suggesting the pentavalent capsomer loops
had more flexibility. Overall the very center and the outer edges
of the capsomers had lower resolution than the mid-regions of
the capsomers. Specifically, the BC and EF loops, which were
Figure 3. Secondary Structure and Residue Side Chain of the L1 Protein Were Identified in the Maps
Secondary structure of a helix and b sheet (blue) in the L1 protein was shown in the corresponding density in the map (green) of HPV16 (AI) and (AII) and
HPV-heparin complex (BI) and (BII). The side chain of the residues were also identified in each of maps. For example, (A1) 311.Tyr, (A2) 447.Trp, (A3) 467.Lys in
HPV16 map; and (B1) 311.Tyr, (B2) 447.Trp, (B3) 467.Lys in the HPV16-heparin map.
256 Structure 25, 253263, February 7, 2017
located at the tip of the star-shaped capsomer, had the lowest
resolution (6.5 A) in both pentavalent and hexavalent capsomers.
The HPV-Heparin Complex Showed that Heparin
Receptors Only Bound at the 5-fold Vertex of the
HPV16 Virus
Significant extra densities were found at each 5-fold vertex of the
HPV-heparin complex map compared with the HPV map. This
putative heparin density was comparable with the magnitude
of the average capsid density, which suggested nearly full occu-
pancy of bound heparin. The regions of strongest density were
located between neighboring pairs of BC and EF loops; however,
there was noisy discontinuous density stretching out and away
from the bound heparin (Figure 1E). The mixture of polysaccha-
ride chain lengths (1030) in the heparin incubated with the virus
in our study likely caused the lesser densities extending away
from the bound heparin, which corresponded to longer polysac-
charide chains of bound heparin that were averaged with shorter
chains during the reconstruction process.
Heparin Binding Triggers Conformational Changes of L2
Protein without Altering the Capsid Diameter
No significant difference in diameter between HPV16 and
HPV16-heparin complex maps was seen in the radial density
plots (Figure 6A), indicating that heparin binding did not trigger
a change in the overall size of the capsid. The structure of L1
was built into the HPV-heparin complex map to make an asym-
metric unit and an L1-only capsid map was calculated as
described above for the capsid. The mass axis of L1 proteins
in pentavalent and hexavalent capsomers in the HPV and the
HPV-heparin maps were calculated (Figure 6B) to track both
rotational and translational movements of L1 proteins. L1 pro-
teins rotated no more than 0.4 and shifted less than 0.1 A (Fig-
ure S3). Thus, no significant movement of L1 protein was found
after binding with heparin.
To check the surface loop rearrangement of the L1 pro-
tein, the HPV and HPV-heparin asymmetric unit structures
were superimposed. An RMSD of 0.76 A suggested slight
changes may have occurred after heparin binding. Super-
imposing the HPV hexavalent and pentavalent capsomers on

Figure 4. The Molecule Building of L1 Protein and Putative L2
Densities in the Map
(A) The six copies of L1 (blue) were built into the HPV16 map density (gray) to
complete the asymmetric unit. The pentavalent and hexavalent capsomers are
indicated by a black pentagon and hexagon, respectively. The C-terminal arm
densities are continuous and clearly identified in the map.
(B) Difference density calculated from subtracting the L1-only capsid map
from the HPV16 experimental map (gray) showed difference densities (red) on
top view to show exposed densities on the surfaces of both pentavalent and
hexavalent capsomers, with wormlike shapes extending between capsid floor
and center of the capsomer.
(C) Cross-section view to show the L2 densities inside the capsomers.
the corresponding HPV-heparin capsomers resulted in RMSD
values of 0.51 and 1.12 A, respectively (Figure 6C). There was
no significant loop rearrangement among all HI, EF, DE, FG,
and BC loops (Figure 6C), and no significant change in the diam-
eter of the capsid.
By subtracting the calculated HPV-heparin L1-only map from
HPV-heparin experimental map, we found a difference density
that had significant conformational changes compared with the
difference densities from the HPV map (Figures 4B, 4C, 6D, 6E,
and S4). After binding with heparin, difference densities in the
HPV-heparin complex map included putative L2 density that map-
ped to both pentavalent and hexavalent capsomers. Compared to
virus alone, there was redisposition within the HPV-heparin cap-
somer and more putative L2 densities on the top surface of the
capsomer and inside the capsomers, especially for the pentava-
lent capsomers. These changes suggest that heparin binding
initiates conformational changes that may result in additional L2
residues becoming exposed at the capsid surface and the side
wall of capsomers, which is consistent with previous studies.
The HPV-heparin local resolution map was compared with the
HPV map to show that areas of poorer local resolution mapped
to the centers of the capsomers in the complex map (Figure 5C).
However, resolution at the rim of the BC and EF loops on penta-
valent capsomers improved slightly after binding with heparin,
possibly due to a stabilizing effect (Figure 5C, indicated by
arrow). Overall, both the pentavalent and hexavalent capsomers
showed regions of poorer resolution after heparin binding, sug-
gesting local flexible movements.
The Receptor Binding Site Is between Hexavalent and
Pentavalent Capsomers
The structures of multiple disaccharide units of heparin were
manually fitted one-by-one into the difference map calculated
by subtracting HPV from the HPV16-heparin reconstruction. The
fitting result for ten units was then refined in the HPV16-heparin
complex map. The resultant heparin model and the L1 protein
model were then used to describe the binding site (Figure 7A).
The HPV-heparin interaction appears to be conferred by sulfated
polysaccharide residues and six different side chains from two
contributing L1 proteins. Specifically, in the model two BC loops,
one EF loop, and one C-terminal arm from two neighboring L1 pro-
teins participated in the binding. One hexavalent L1 protein
contributed the 59.Lys of the BC loop and 176.Thr and 177.Asn
of the EF loop for interaction. All the other binding residues in the
model were from one pentavalent L1 that contributed a 57.Asn,
58.Asn, and 59.Lys from the BC loop, and C-terminal arm residues
of 428.Cys, 429.Gln, 431.His, 432.Thr, and 433.Pro (Figure 7B).
DISCUSSION
Variations in the size of the HPV capsid have been attributed pre-
viously to progressive stages of maturation of the capsid through
formation of disulfide linkages conferred by the C-terminal arm
using the pseudovirus model. Similarly, our 3D classification of
the quasi-HPV capsids used in our study (Experimental Proced-
ures) showed different-diameter capsids existing with similar
surface features (Figure S1). This size variation in capsids may
relate to the CRPV genome or maturation status. Variation also
suggests that heterogeneity is a natural attribute of HPV, which
may be linked to survival outside of the host in a hostile environ-
ment and may represent an evolving adaptability. For instance,
the range in diameter may be linked to stability, providing classes
of more (or less) stable particles. Once assembled into capsids,
the heterogeneous characteristic of HPV during and after the
maturation process may provide an advantage ensuring the sur-
vival of an existing sub-population, within the mixed population
of capsids.
Structure 25, 253263, February 7, 2017 257

Compared with the well-understood nature and structure
of the L1 major protein, the location and number of L2 minor
proteins per capsid have remained controversial, despite the
essential requirement for genome encapsidation and subcellular
trafficking. During the past 25 years, extensive biochemical and
structural studies have attempted to lift the veil shrouding L2
protein incorporation. Using the L1-only calculated map, we
identified non-L1 density in our experimental map and suggest
that difference density can be attributed to L2. L2 protein was
observed to intertwine with L1 protein at the base part of the
capsomer and extend along the side wall of the capsomer in
the canyon to the top surface until reaching the upper center
of the capsomer. This observation was consistent with a previ-
ous structural result that showed small L2 protein densities at
the lower center of each capsomer (Buck et al., 2008). With the
higher resolution that is now attainable, our map reveals more
about the location and features of the L2 protein. Because of
the quasi-equivalent 5-fold symmetry characteristics of the hex-
avalent capsomer, the features of putative L2 densities were
different between pentavalent and hexavalent capsomers. The
smaller potential density of L2 at the surface and inner parts of
pentavalent capsomer suggested a flexible interaction of L1
and L2 proteins in the pentavalent capsomer, which were aver-
aged out during the reconstruction process. Consistent with a
previous model, L1 residues 261269 map near the L2 density
that drapes over the outside of the capsomers, suggesting
possible interactions (Lowe et al., 2008). The variation of L2 dis-
tribution among capsomers suggested random L2 incorporation
during capsid formation, which may be related to the long virion
maturation time and the high heterogeneity of the population.
Among the 72 capsomers comprising the intact icosahe-
dral capsid, L2 molecules may be incorporated asymmetrically
and randomly distributed in both pentavalent and hexavalent
capsomers. Alternatively, L2 proteins may be located on each
of the 12 pentavalent capsomers with the other additional L2
copies randomly distributed in hexavalent positions. Finally,
258 Structure 25, 253263, February 7, 2017
Figure 5. Molecule Model Comparison and
Local Resolution of Pentavalent and Hexa-
valent Capsomers
(A) The L1 molecular structures of pentavalent
(red) and hexavalent (blue) capsomers are super-
imposed to show the structural differences,
especially between the C-terminal arms (blue ar-
row), with an RMSD value of 4.77 A between the
atom sets in two capsomers.
(B and C) The zoomed view of a pentavalent and
hexavalent capsomer of each local resolution map
of HPV16 (B) and HPV16-heparin (C). Resolution of
the map (color key in angstroms) shows that inner
and outermost densities have the lowest resolution
and, presumably, the most flexibility. (C) The res-
olution of the BC and EF loop densities (arrow)
improves after binding with heparin.
the third possibility for L2 incorporation
into the capsid is that all copies of L2
may cluster in a unique locus that includes
a pentavalent capsomer and some of the
surrounding five hexavalent capsomers.
An anisotropic distribution of L2 protein in the capsid implies
that a unique vertex may exist, which may function during host
cell binding and infection. However, due to the magnitude of
these difference densities they cannot be definitively appor-
tioned or assigned to L2 molecules.
The L2 N terminus has been shown previously to be accessible
at the surface of the capsomer (Conway et al., 2011; Lowe et al.,
2008), and such a location is suggested in our map (Figure 3B).
The incorporated L2 protein may start from the middle center
of the capsomer, extending to the surface along the side wall
to reach the capsid floor with most of the molecule including
the L2 C terminus intertwining with the L1 protein and interacting
with the genome at the base of the capsomer. At the current res-
olution we cannot solve the molecular structure of L2 peptide
within the density (Figure 4) or define the orientation. However,
the morphological features and conformational changes of the
putative L2 protein densities might be used to guide future
studies to contribute to understanding the mechanisms of entry
and infection.
Variations in the micro-environment of the T = 7d icosahedral
capsid (Guan et al., 2015a, 2015b; Lee et al., 2014; Xia et al.,
2016; Zhao et al., 2014) underlie functional differences between
pentavalent and hexavalent capsomers. Specifically, the U4
antibody binds preferentially at the icosahedral 5-fold (Guan
et al., 2015a), which is consistent with the previous studies where
U4 antibody prohibited the virus from binding cells (Day et al.,
2007; Deschuyteneer et al., 2010). Considering the functional dif-
ferences between pentavalent and hexavalent L1 environments,
preferential heparin binding, and the established importance of
L2 function during entry, we conclude that the 5-fold vertex
can be considered the functional site for binding host cells during
entry. This model suggests L2 incorporation must include the
vertex pentamers.
Previous work has shown that HS binding triggered conforma-
tional changes to the capsid, which resulted in reduced affinity to
the primary HS attachment receptor and more exposure of

L2 protein for furin cleavage (Day et al., 2008a, 2008b; Selinka
et al., 2003). The HPV-heparin complex captured in our study
showed no significant conformational changes of the L1 protein,
but suggested major L2 alteration. Post-attachment alterations
to the HPV capsid have been reported previously (Guan et al.,
2015a) and may correspond to the heparin-triggered conforma-
tional changes we report here that include small L1 protein
movements at the apical surface of the capsomers. The surface
loops on hexavalent capsomer had no significant changes
after binding heparin (Figure 6C), which coincides with the re-
tained ability of antibody HPV16.V5 to neutralize well after cell
attachment (Guan et al., 2015a; Lee et al., 2014). Compared
with HPV16.V5, the redisposition and more exposure of L2 den-
sities on the surface may affect other antibody binding, for
example, H33.J3, which displayed improved neutralizing
efficiency to cell-bound virions (Selinka et al., 2003). The lack
of global conformational changes of L1 protein after binding
heparin and the presence of possible L2 density near the
bound heparin molecule suggested that the L2 protein was likely
participating in the binding with heparin (Figures 6D and 6E). If
heparin binds to both L1 and L2 proteins, perhaps the L1 interac-
tions facilitate the orientation and the L2 binding with heparin
provides the contacts that triggered major conformational
Figure 6. Conformational Changes of HPV
Capsid Triggered by Heparin
(A) Radial density plots of HPV16 and HPV16-
heparin.
(B) The mass axis of L1 protein in pentavalent and
hexavalent capsomers for HPV (red) and HPV-HS
(blue) maps.
(C) The conformational changes of L1 surface
loops induced by heparin binding are indicated by
superimposing the pentavalent and hexavalent
capsomer models from HPV16 (red) and HPV16-
heparin (blue). The movement of FG, HI, BC, DE,
and EF loops were indicated. The RMSD value of
pentavalent and hexavalent capsomers before and
after heparin binding were measured as 1.12 and
0.51 A, respectively.
(D) L2 densities (blue) calculated by subtracting the
L1 only capsid model from HPV16-heparin map
was shown from top, whereas the heparin den-
sities were highlight in black.
(E) The cross-section view L2 densities (blue)
together with heparin (black) to show the densities
inside the capsomer. Pentavalent and hexavalent
capsomers were indicated by black pentagon and
hexagon.
changes. The movement of L2 after hepa-
rin binding also includes conformational
changes of L2 at the side wall of the cap-
somer near the canyon (Figures 6D, 6E,
and S4). These changes may indicate
that the binding sites for furin cleavage,
cyclophilin B, or other factors of the L2
protein are located at the side wall of the
capsomer, and the release of L2 and the
genome may be from the canyon during
the latter dis-assembly of the capsid;
however, further work remains to reveal the L2 structure and
subsequent changes.
Of the three heparin binding sites identified previously, the site
occupied by heparin in our density map overlapped with two res-
idues of site 3 (57.Asn, 59.Lys, 442.Lys, 443.Lys), whereas no
traces of bound heparin were found at the top of the capsomers
involving the FG and HI loops previously identified as sites 1 and
2 (Richards et al., 2013), and as the primary binding sites. How-
ever, considerable differences exist among these three experi-
ments including the form of HPV capsid (L1 only pentamers,
pseudovirus, or quasivirus) or the type of heparin (heparin oligo-
saccharide 810, heparin oligomers, or heparin 1030) used. The
seemingly disparate results suggest several possibilities. First,
due to the variations in the reagents, the heparin used in these
experiments was only capable of interacting at site 3 on our
quasivirus capsids due to steric limitations of binding. Certainly,
it seems likely from our structure that heparin binding to site 3
would interfere with binding at site 2. Second, a stepwise
sequence of events is conceivable. For example, heparin bound
HPV capsids at site 1 during the 1-hr incubation; the bound hep-
arin initiated the conformational changes revealed in the maps;
whereupon heparin dissociated from site 1 and then bound
site 3 as revealed in the HPV-heparin complex structure.
Structure 25, 253263, February 7, 2017 259

Figure 7. Epitope Analysis of Heparin Based on the Model and the
4.7-A Maps
The heparin binding site described by the model. (A) The fitted heparin
molecule (purple, yellow, and red) and L1 protein (blue) were used to locate the
contacts between heparin and L1. (B) The residues on the L1 protein are
indicated at their position with sequence number, residue name, and side
chain shown in red. The residues comprising the binding site identified here are
listed in the table along with their position on the L1 capsomer.
Until now, no structural tools to detect HPV conformational
changes during entry have been described. By using near-
atomic reconstruction maps, local conformational changes
were seen for the first time. Since heparin molecules were only
found at the pentavalent capsomer, triggering the conforma-
tional changes to expose more L2 densities in the canyon, these
changes suggested that HPV infection is initiated through a
pentavalent capsomer as a unique 5-fold vertex that may func-
tion during later stages of entry that include release of the DNA
genome tethered to L2. In support of this model is the possibility
that L2 might be incorporated into the HPV capsid to form a
unique vertex. Further studies will be needed to resolve these
questions.
EXPERIMENTAL PROCEDURES
Preparation of HPV16 Quasiviruses
HPV16 quasivirus containing L1 and L2 proteins and encapsidating a CRPV
genome having the SV40 origin of replication was prepared as described
previously (Brendle et al., 2010; Mejia et al., 2006; Pyeon et al., 2005). In
brief, HPV16 sheLL plasmid (kindly provided by John Schiller, NIH) was trans-
260 Structure 25, 253263, February 7, 2017
fected together with linear CRPV/SV40ori DNA into 293TT cells and prepared
as described previously (Buck et al., 2005a, 2005b; Pastrana et al., 2004).
HPV16 was allowed to mature overnight and then pelleted by centrifugation.
The centrifuged pellet was resuspended in 1 M NaCl and 0.2 M Tris (pH 7.4).
After CsCl gradient purification, the lower band was collected, concentrated,
and dialyzed against PBS, as described previously (Guan et al., 2015a). The
concentrated HPV16 particles were applied to Formavar-coated copper grids,
stained with 2% phosphotungstic acid, and analyzed for integrity and concen-
tration on a JEOL JEM-1400 electron microscope.
Cryo-EM
HPV16 was incubated with a 5-fold excess of heparin molecules (Sigma-
Aldrich, catalog no. H1027) according to an average mass of 16 kDa per mole-
cule and 360 binding sites predicted per capsid. HPV-heparin was incubated
for 1 hr at 37 C and concentrated to 0.8 mg/mL in PBS buffer. An aliquot of 3 mL
of virus-Fab complex was vitrified on QUANTIFOIL R2/1 holey carbon support
grids (Quantifoil) that were vitrified by plunge-freezing into liquid ethane using a
Cryoplunge 3 (Gatan). Low-dose micrographs were recorded using an FEI
Polara G2 microscope operating at 300 kV. Images were collected under the
software control of the EPU program using an FEI Falcon 2 direct electron
detector with a nominal magnification of 93,0003 yielding a calibrated pixel
size at the sample of 1.15 A. A similar procedure was followed to prepare
and collect data for untreated HPV particles. Defocus ranges of 1.045.19
and 0.784.85 mm (Table 1) were measured respectively for micrographs of
HPV16 and HPV16-heparin, respectively. RELION, AUTO3DEM, and EMAN2
program suites were used for image processing and 3D reconstructions
(Scheres, 2012; Tang et al., 2007; Yan et al., 2007).
Image Processing
Whole-frame alignment was carried out using MotionCorr to account for
stage drift (Li et al., 2013). The microscope contrast transfer function pa-
rameters were estimated for each micrograph using ctffind4 (Rohou and
Grigorieff, 2015). Semiautomatic particle selection was performed using
EMAN2 e2boxer.py to obtain the particle coordinates, followed by particle
extraction, linearization, normalization, apodization, and 3D reconstruction
of the images using AUTO3DEM 4.05 following the gold standard of initiating
the process by splitting the dataset into two independently refined halves
(Tang et al., 2007; Yan et al., 2007). The refinements for sharpening high-
resolution information were performed using EMBFACTOR (Fernandez
et al., 2008; Rosenthal and Henderson, 2003). Reported resolutions are esti-
mated according to the gold-standard FSC = 0.143 criterion (Scheres and
Chen, 2012).
For heterogeneity investigation, 3D classifications and refinements were
performed using RELION software (Scheres, 2012). A windowing operation
was applied to the particles in Fourier space to downsize the particle images
to yield a final box size of 260 pixels2 (corresponding to a pixel size of 2.3 A).
The final pixel size was chosen after optimization of the balance between
the Nyquist frequency limit and the memory requirements for computations.
Model Building
The atomic model of the HPV16 L1 capsomer with the C-terminal arm trun-
cated (PDB: 3OAE; residues 20403, 438474) was fitted as a rigid body
into the cryo-EM map using UCSF Chimera (Dasgupta et al., 2011; Pettersen
et al., 2004). Placement of the pseudoatomic model of an asymmetric unit of
HPV16 capsid (PDB: 3J6R; residues 9486) was used as a guideline during
building (Cardone et al., 2014). Model building proceeded de novo to place
the missing C-terminal arm residues 404437 and the N and C termini through
COOT (Emsley et al., 2010). Specifically, 17 residues were built into density at
the N-terminal end and 11 residues at the C-terminal end. Each L1 molecule in
the asymmetric unit was built independently using COOT (Emsley et al., 2010).
Icosahedral symmetry was applied to the refined asymmetric unit model to
generate the whole capsid through SITUS (Wriggers, 2010; Wriggers et al.,
1999), with the parameters of mass-weight of the atom, pixel size 1.147 A,
kernel width 4.8 A, Gaussian smoothing kernel, and scaling factor of 1.
The capsid atomic model was then further refined iteratively using the Phenix
Real Space Refinement program (Afonine et al., 2012). At each iteration, the
best model was visually inspected in COOT and adjusted manually accord-
ingly to the best fit into the density and guided by torsion restraints, planar
peptide restraints, and Ramachandran restrains. The quality of the final model
was evaluated by MolProbity (http://molprobity.biochem.duke.edu/) (Chen
et al., 2010) (Table S1). The same building and refinement procedures were
performed for the HPV-heparin complex map. Difference maps were calcu-
lated using SITUS (Wriggers, 2010; Wriggers et al., 1999).
Coordinates for the disaccharide unit of heparin isolated from (PDB: 3OAE)
were used as the basis for the model building (Dasgupta et al., 2011). The hep-
arin polysaccharide model was built manually using disaccharide units fitted
one-by-one into the difference map made by subtracting HPV16 from the
HPV16-heparin complex map. The result of initial fitting was refined using
the HPV16-heprin complex map. The final result included ten disaccharide
units of heparin built in the density with a correlation coefficient of 83% accord-
ing to Chimera (Pettersen et al., 2004). Contacts between the fitted heparin and
L1 protein structures were identified using Chimera with the criteria for van der
Waals overlap distances set at 0.4 and 0.0 A. Clashes between atoms were
defined by any overlap of 0.6 A or more.
ACCESSION NUMBERS
Cryo-EM maps for the HPV16 and HPV16-heparin complexes have been
deposited in the EM database (www.emdatabank.org/) with accession
numbers EMD: 6620 and 6619. Coordinates for the atomic model of the
asymmetric unit of HPV16 L1 proteins and the complex with the heparin
molecule have been deposited in the PDB under accession codes PDB:
5KEP and 5KEQ.
SUPPLEMENTAL INFORMATION
Supplemental Information includes four figures and one table and can be
found with this article online at http://dx.doi.org/10.1016/j.str.2016.12.001.
AUTHOR CONTRIBUTIONS
J.G., S.M.B., and S.A.B. performed the research; R.E.A. performed micro-
scopy and collected the cryo-EM data; A.M.M. and J.F.C. collected the
cryo-EM data; J.G. processed and analyzed the cryo-EM data; J.G., N.D.C.,
J.F.C., and S.H. wrote the paper.
ACKNOWLEDGMENTS
This work was supported in part by the J. Gittlen Memorial Golf Tournament
and the Pennsylvania Department of Health CURE funds. Research reported
in this publication was also supported by the Office of the Director, NIH, under
award numbers S10OD019995 and S10OD011986. The content is solely the
responsibility of the authors and does not necessarily represent the official
views of the NIH.
Received: June 8, 2016
Revised: October 7, 2016
Accepted: December 12, 2016
Published: January 5, 2017
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