Phylogeny and primary structure analysis of fiber shafts of all human adenovirus types for rational design of adenoviral gene therapy vectors.

Short
Communication
Phylogeny and primary structure analysis of fiber
shafts of all human adenovirus types for rational
design of adenoviral gene-therapy vectors
Sebastian Darr, Ijad Madisch, So¨ren Hofmayer, Fabienne Rehren
and Albert Heim
Correspondence
Albert Heim
Heim.Albert@mh-hannover.de
Institut fu¨r Virologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany
Received 19 June 2009
Accepted 4 August 2009
The fiber shaft of human adenoviruses (HAdVs) is essential for bringing the penton base into
proximity to the secondary cellular receptor. Fiber shaft sequences of all 53 HAdV types were
studied. Phylogeny of the fiber shaft revealed clustering corresponding to the HAdV species
concept. An intraspecies recombination hot spot was found at the shaft/knob boundary, a highly
conserved sequence stretch. For example, HAdV-D20 clustered with HAdV-D23 in the fiber
shaft, but with HAdV-D47 in the fiber knob. Although all shafts exhibited the typical
pseudorepeats, amino acid sequence identity was found to be as high as 92% (interspecies) and
54% (intraspecies). In contrast to a previous study, a flexibility motif (KXGGLXFD/N) was found in
eight HAdV-D types, whereas the putative heparan sulfate-binding site (KKTK) was only found in
species HAdV-C. Our results suggest that pseudotyping of gene-therapy vectors at the shaft/
knob boundary is feasible, but that flexibility data of shafts should be considered.
Members of the family Adenoviridae are double-stranded DNA
viruses with a non-enveloped, icosahedral capsid (Swenson et
al., 2003). The 53 human adenovirus (HAdV) types are
grouped into six recognized species (HAdV-A to -F) and a
proposed seventh species (HAdV-G), which were defined
historically on the basis of haemagglutination properties,
oncogenic properties in rodents and DNA homology. The
viruses are mainly responsible for causing respiratory-tract
illness (HAdV-B3, -B7, -B21, -E4, -C1, -C2 and -C5),
gastroenteric infections (HAdV-A12, -A18, -A31, -F40 and
-F41) and ocular infections (HAdV-D8, -D19 and -D37).
Adenovirus capsids consist in total of 240 copies of
trimeric hexon protein, and a penton complex at each of
the 12 vertices. The penton is made of a pentameric base
and a trimeric fiber. The fiber is organized in three well-
defined regions that play an important role in adenovirus
infectivity (Shenk, 2001). These are the amino-terminal
‘tail’ that binds to the penton base, a central ‘shaft’ of
variable length and a carboxy-terminal ‘knob’ domain. The
HAdV-C2 fiber shaft was the first to be analysed in detail
by Green et al. (1983), who suggested pseudorepeats of 15
residues consisting of two short b-strands and two b-
bends. The fiber knob interacts with primary cellular
receptors, e.g. coxsackievirus–adenovirus receptor (CAR),
CD46, CD86 and sialic acid (Marttila et al., 2005; Roelvink
et al., 1998; Seiradake et al., 2009; Short et al., 2004; Sirena
et al., 2004). Primary binding to cells is followed by cell
entry after interaction of the RGD tripeptide loop
extending from the penton base with cellular avb3 or avb5
integrins (Bai et al., 1994; Mathias et al., 1994; Schoehn
et al., 1996; Wickham et al., 1993). The flexibility of the
fiber shaft seems to be essential to bring the penton base
into close proximity to the secondary receptor after the
primary receptor interaction. Two flexibility loops have
been described in the fiber shaft of most HAdV types, but
they have not been found in species HAdV-D (Chroboczek
et al., 1995). Furthermore, a putative heparan sulfate-
binding site [KKTK or, in general, BBXB and BBBXXB,
where ‘B’ stands for a basic amino acid such as Lys, Arg or
His (Templeton, 1992)] was found in the fiber shafts of
species HAdV-C serotypes, but not in other HAdV species.
The virus enters host cells by a process termed receptor-
mediated endocytosis (Maxfield & McGraw, 2004) via
clathrin-coated pits using dynamin-mediated endocytosis
(Li et al., 1998; Morgan et al., 1969; Nemerow & Stewart,
1999; Wang et al., 1998). Cell entry is followed by an
interaction with cytosolic molecular motors, which drive
the capsid along microtubules (Mabit et al., 2002;
Suomalainen et al., 2001) through the microtubule-
organizing centre to the nucleus (Kelkar et al., 2004).
Even though the adenovirus fiber shaft is essential for
infectivity and understanding of virus tropism (Nakamura
et al., 2003; Roelvink et al., 1998; Seki et al., 2002;
The GenBank/EMBL/DDBJ accession numbers for newly generated
sequences are FM210540–FM210562 and FN397565–FN397571.
Supplementary figures and tables are available with the online version of
this paper.
Journal of General Virology (2009), 90, 2849–2854 DOI 10.1099/vir.0.014514-0
014514 G 2009 SGM Printed in Great Britain 2849

Shayakhmetov & Lieber, 2000; Smith et al., 2003; Vigne
et al., 2003), a complete dataset of fiber shaft sequences has
not been available to date. In this study, fiber shaft
sequences were generated and a complete dataset of all 53
HAdV types and eight simian adenovirus (SAdV) types,
which belong to the HAdV species, was used to study the
primary structure of the fiber shaft and to predict flexibility
loops and heparan sulfate-binding sites. Furthermore,
clustering of the fiber shaft region was compared with
that of the fiber knob in order to search for recombination
events in the evolution of HAdV types.
HAdV prototype strains were obtained from the ATCC,
except for HAdV-B14, -D10, -D13 and -D30 prototype
strains, which were obtained from our collection at the
German National Reference Laboratory for Adenoviruses,
Hannover Medical School, Germany. DNA was extracted
with a Qiagen blood kit. Fiber shaft amplicons for species
HAdV-D were generated by PCR using primers displayed
in Supplementary Table S1 (available in JGV Online). PCR
products were separated in 2% agarose gel and extracted
from the gel with a Qiagen gel extraction kit. Cycle
sequencing was performed with PCR primers and rhoda-
mine-labelled dideoxynucleotide chain terminators (ABI)
and an ABI Prism 310 or ABI 3130 automatic sequencer.
The generated fiber shaft sequences were deposited in
GenBank under accession numbers FM210540–FM210562
(see Supplementary Table S2, available in JGV Online).
Multiple alignments were generated by sequential pairwise
alignment using the CLUSTAL algorithm implemented in the
BioEdit software package (version 6.0.5; Hall, 1999) and
adjusted manually to conform to the optimized alignment
of deduced amino acid sequences. Phylogenetic trees of the
fiber shaft region based on nucleotide positions 31162–
32239 (referring to the HAdV-C2 fiber shaft sequence;
GenBank accession no. J01917) were constructed with the
help of MEGA software (version 4; Tamura et al., 2007) with
several phylogenetic-reconstruction algorithms (neighbour
joining, minimum evolution and maximum parsimony)
and were congruent in overall structure (Fig. 1 depicts
neighbour-joining trees).
Sequences of adenovirus fiber shaft clustered according to
the species concept, as observed previously for the hexon e
determinant, the penton base d determinant and the c
determinant of the fiber knob (Madisch et al., 2005, 2007).
Clustering was supported by high bootstrap values.
Interspecies variability in the shaft region was higher (55–
93% amino acid sequence divergence) than in the fiber
knob region (50–59%) (Madisch et al., 2007); in contrast,
intraspecies divergence was higher in the knob region (18–
53% amino acid sequence divergence) than in the fiber shaft
(18–44%). Surprisingly, all HAdV species except for HAdV-
B showed a lower intraspecies ratio of synonymous/non-
synonymous (S/N) mutations in the fiber shaft (e.g. HAdV-
D, 2.2) than in the fiber knob (e.g. HAdV-D, 3.03). This
suggests that more positive selection occurs in the fiber shaft
region, although the fiber knob contains a well-established
immunogenic determinant (c).
Both the tail/shaft boundary and the shaft/knob boundary
were highly conserved throughout all adenovirus subtypes,
with a completely conserved GVL sequence and a conserved
TLWT motif, respectively. However, species HAdV-F has
two fibers of different lengths expressed at a relative ratio of
1 : 1 (Albinsson & Kidd, 1999; Favier et al., 2002; Kidd et al.,
1990, 1993; Yeh et al., 1994) (Supplementary Fig. S1,
available in JGVOnline). Surprisingly, the short fibers have a
TIWS sequence as the fiber shaft/knob boundary and an
amino acid sequence identity of only 41% to the long shafts,
suggesting that one of the fibers was acquired from another
HAdV species (Kidd et al., 1993) (Fig. 1a). As only the long
fiber is able to interact with CAR (Roelvink et al., 1998), the
short one may bind to an additional, as-yet-unknown
receptor mainly expressed in the gastrointestinal tract
(Favier et al., 2002), explaining the tropism of HAdV-F.
Comparison of phylogenetic trees of fiber shaft and knob
suggested several intraspecies-recombination events in the
phylogeny of 10 (of 33) HAdV-D prototypes, because of
different clustering confirmed by high bootstrap values
(Fig. 1). For example, HAdV-D20 clustered with HAdV-
D23 in the fiber shaft region, whereas it clustered with
HAdV-D47 in the fiber knob region. Bootscans were
performed with the software SimPlot (version 3.5.1; Lole
et al., 1999) with a window of 200 bp (20 bp step) based on
a Kimura two-parameter substitution model (Kimura,
1980) with a transition/transversion ratio of 2.0 (Fig. 2).
Bootscan results determined a recombination hot spot at
the shaft/knob boundary [nucleotide position 440, referring
to HAdV-D20 (GenBank accession no. AJ811444) in a
complete fiber alignment], a highly conserved region
that is prone to promoting homologous recombination
(Supplementary Fig. S2, available in JGV Online). These
recombination events in species HAdV-D, with several
prototypes sharing identical shaft sequences, suggested the
feasibility of a fiber knob-replacement strategy for
pseudotyping of gene-therapy vectors with a species
HAdV-D-derived fiber gene. As the fiber knob contains
the major receptor-binding site, fiber knob pseudotyping
holds promise to modify tropism. Species HAdV-D-
derived gene-therapy vectors were developed recently for
the treatment of malignant melanoma and soft-tissue
sarcoma (Hoffmann et al., 2007, 2008).
Furthermore, we included seven intermediate HAdV-D
strains (HAdV-D15H9, -D17H29, -D19H30, -D30H44,
-D37H13, -D37H17 and -D46H13) in our phylogenetic
analysis. Previously, these strains had been typed with
contradictory results by neutralization testing (hexon e
determinant) and haemagglutination-inhibition testing
(c determinant of fiber knob), indicating at least one
recombination event between the hexon gene and the fiber
knob-encoding region. However, a detailed genetic analysis
of these intermediate strains had not yet been performed.
Only one of seven HAdV-D intermediate strains (HAdV-
D19H30) was recombinant at the fiber shaft/knob
boundary hot spot (Supplementary Fig. S3, available in
JGV Online). Surprisingly, the fiber shaft of HAdV-
S. Darr and others
2850 Journal of General Virology 90

D19H30 was related closely to that of HAdV-D49, whereas
it clustered immediately adjacent to HAdV-D30 in the fiber
knob region, supported by high bootstrap values
(Supplementary Fig. S4, available in JGV Online). This
result indicated another recombination event in the
genome of HAdV-D19H30 between the hexon gene and
the fiber gene, suggesting a multiple-recombinant HAdV
isolate similar to the recently published novel HAdV type
D53 (Walsh et al., 2009). Recently, frequent recombination
events were also described in the phylogeny of species
HAdV-C field isolates (Lukashev et al., 2008).
A complete overview of the length of all human adenovirus
fiber shafts was generated (Supplementary Fig. S1). All
fiber shaft sequences were congruent to the cross-b model
of pseudorepeats, which contains two b-strands and two
turns (Green et al., 1983). The highest number of
pseudorepeats was found in species HAdV-A (23) and
the lowest in species HAdV-B (six). Several HAdV types
revealed differences in the length of their fiber shafts in
comparison to other HAdV types of their species, e.g.
HAdV-B16 (30 aa, about two pseudorepeats longer),
HAdV-A31 (31 aa, about two pseudorepeats shorter) and
Fig. 1. Phylogenetic trees (neighbour joining) of the nucleic acid sequences of (a) the fiber shaft domain (1077 bp, referring to
HAdV-C1; GenBank accession no. AC000017) and (b) the fiber knob domain (522 bp, referring to HAdV-C1). Bootstrap
values (%) were generated with 1000 pseudoreplicates. In addition to the newly generated fiber shaft sequences, reference
sequences from GenBank were used; see Supplementary Table S2 (available in JGV Online) for details.
Adenovirus fiber shaft phylogeny
http://vir.sgmjournals.org 2851

HAdV-C6 (51 aa, about three pseudorepeats shorter).
These differences in length may also influence flexibility
of the fiber shaft.
All HAdV-D shafts consisted of eight pseudorepeats with a
length between 140 and 148 aa. HAdVs of species D can be
divided into three groups according to the length of their
fiber shaft. The first group, with fiber shaft lengths between
140 and 142 aa, consisted of types HAdV-D8, -D9, -D15,
-D28, -D43, -D44 and -D48; the second, with a length of
145 aa, consisted of types HAdV-D17, -D19, -D37, -D36
and -D45, and the third group, with a length of 147 or
148 aa, consisted of all remaining HAdV-D types.
The entire fiber shaft dataset finally allows determination of
the absence of a heparan sulfate-binding site (KKTK motif)
in all HAdVs other than species HAdV-C. This included a
search for any general motif, BBXB or BBBXXB (where ‘B’
stands for a basic amino acid such as Lys, Arg or His).
The heparan sulfate-binding site was formerly described as
a sufficient receptor for initial binding of several HAdVs
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Fig. 2. Bootscan analysis performed with SimPlot revealed recombination events at the shaft/knob boundary in the phylogeny
of HAdV-D types HAdV-D47 (a), -D26 (b), -D20 (c) and -D30 (d).
Fig. 3. Porcupine molecular-dynamic repres-
entation of the fiber shaft/knob boundary
including the second flexibility loop of HAdV-
C2 (a), which was also predicted for HAdV-
D23 (b). For comparison, HAdV-D17 (c) is
depicted, which also had differences in the
adjacent amino-terminal loop (red circle).
S. Darr and others
2852 Journal of General Virology 90

(Dechecchi et al., 2001). In contrast, recent studies
demonstrated that the KKTK motif of the fiber shaft plays
only a minimal role, if any, in the binding of heparan
sulfate glycosaminoglycans, but is important for post-
internalization steps of virus infection, especially trafficking
to the nucleus (Kritz et al., 2007).
Adenovirus fiber shafts of all HAdVs except species HAdV-
D contain a different number of residues in the third
pseudorepeat that are thought to allow bending of the fiber
shaft (Chroboczek et al., 1995; Wu et al., 2003). Our
complete sequence dataset of fiber shaft sequences now
demonstrates the absence of this flexibility loop in any of
the 33 species D adenoviruses.
A second flexibility loop (the KLGXGLXFD/N sequence,
where X stands for any amino acid) is located in the
penultimate pseudorepeat of the fiber shaft. After analysing
the fiber shaft sequences of HAdV-D8, -D9 and -D15, it
was assumed that all HAdV-D adenoviruses lack this
second flexibility loop, in contrast to all other HAdV
species (Chroboczek et al., 1995). However, our complete
dataset of adenovirus fiber shaft sequences demonstrated a
KDGGLXFD/N motif in eight HAdV-D prototypes
(HAdV-D20, -D23, -D24, -D32, -D33, -D46, -D47 and
-D51), which is only 1 aa shorter than the formerly
described motif.
The flexibility of the proposed KDGGLXFD/N motif in
HAdV-D23 was analysed in silico in comparison to HAdV-
D17 (without a flexibility loop) and HAdV-C2 (with the
flexibility-loop sequence KLGXGLXFD/N). For this pur-
pose, homology models of the partial fiber protein were
initially produced and visualized by using the Swiss-
PdbViewer protein-modelling environment (version 4.01;
Guex & Peitsch, 1997) and VMD (version 1.8.6; Humphrey
et al., 1996). Based on the crystallography fiber data of
HAdV-C2 (Pdb accession no. 1QIU), predictions for
HAdV-D23 and -D17 were performed by using a structural
alignment to guide the threading of model sequences onto
the known molecular structures using the Swiss-PdbViewer
software. A CONCOORD-based simulation and principal-
component analysis on the resulting ensemble was
performed with the Dynamite web server (Barrett et al.,
2004). A higher flexibility was displayed by the length of
the dark-blue spikes of both HAdV-D23 and HAdV-C2
compared with HAdV-D17 in porcupine molecular-
dynamic diagrams of the fiber shaft/knob boundary
(Fig. 3). This suggested that the proposed flexibility motif
KDGGLXFD/N of species HAdV-D is functional. This may
also imply higher infectivity, as previous studies suggested
that the flexibility of adenovirus fiber shafts was crucial for
adenovirus–receptor interaction (Wu et al., 2003).
In conclusion, positive selection of fiber shaft mutations as
strong as seen in the fiber knob region was indicated by the
high intra- and interspecies divergence and low S/N values.
Moreover, a recombination hot spot was found at the
shaft/knob boundary of species HAdV-D and may be used
in future for efficient pseudotyping in the design of HAdV-
D gene-therapy vectors. Further work is needed to
determine whether differences in HAdV-D fiber structure
influence organ tropism.
Acknowledgements
We would like to thank Mark van Raaij (Instituto de Biologia
Molecular de Barcelona, Barcelona, Spain) for his help in modelling
the HAdV-C2 fiber X-ray diffraction file and Kelly Te for critical
reading of the manuscript.
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