Cryoelectron Microscopy of Protein IX-Modified Adenoviruses Suggests a New Position for the C Terminus of Protein IX

Abstract

Recombinant human adenovirus is a useful gene delivery vector for clinical gene therapy. Minor capsid protein IX of adenovirus has been of recent interest since multiple studies have shown that modifications can be made to its C terminus to alter viral tropism or add molecular tags and/or reporter proteins. We examined the structure of an engineered adenovirus displaying the enhanced green fluorescent protein (EGFP) fused to the C terminus of protein IX. Cryoelectron microscopy and reconstruction localized the C-terminal EGFP fusion between the H2 hexon and the H4 hexon, positioned between adjacent facets, directly above the density previously assigned as protein IIIa. The original assignment of IIIa was based largely on indirect evidence, and the data presented herein support the reassignment of the IIIa density as protein IX.The adenoviral capsid is a complex assembly of seven polypeptides, organized into an ϳ900-Å-diameter icosahedral shell. Twelve trimers of hexon, the major capsid component, are arranged onto each of 20 interlocking triangular facets, with penton capsomeres and their protruding fibers occupying each of the 12 vertex positions (24). The icosahedral lattice of hexons is stabilized by a number of so-called "minor" capsid proteins, including IIIa, VI, VIII, and IX. With the exception of protein IX, these minor components provide vital functions and are absolutely necessary for the adenovirus (Ad) life cycle (35). The presence of protein IX is not required for the encapsidation of Ad genomes of subgenomic size (Ͻ36 kb), but virions with capsids lacking IX (⌬IX) are more thermolabile than wild-type virions (5, 6). Previous work (13) suggests that deletion of protein IX prevents the encapsidation of genomes of wild-type or larger sizes (36 to 38 kb). A more recent study indicates that the absence of IX does not prevent encapsidation but rather that the resulting ⌬IX virions are defective in infectivity assays (27).Central to each facet are the groups-of-nine hexons (GONs) that are observed after various methods of capsid disruption (6, 10). The crystal structure of the hexon trimer (25) reveals a pseudohexagonal base with three towers extending upwards, creating a triangular top approximately 113 Å tall. The maximum radius of the hexon trimer is 50 Å. The triangular top is rotated roughly 10 degrees counterclockwise with respect to the hexagonal base. The hexon trimers present within each facet are closely packed along their hexagonal bases, creating a continuous protein shell that is approximately 33 to 44 Å thick. The three towers of each hexon trimer extend roughly 69 Å above the capsid floor, thereby creating cavities between the towers of different hexon trimers. Due to the different relative angles between the bases and towers of individual hexon trimers, each GON contains four large and three small cavities between the nine different hexon trimers.Structurally, protein IX is thought to function as capsid cement, and trimers of IX are thought to reside at the bottom of the four large cavitie...

Current Advances and Future Challenges in Adenoviral Vector Biology and Targeting

“…The new model (Fig. 1B) is in agreement with our Ad-IX-EGFP reconstruction [Marsh et al, 2006] and has four trimers of the Nterminal portion of protein IX residing between hexons of the GON with the C-terminal portions present as four-helix bundles at the position originally assigned as IIIa [Saban et al, 2006]. This exposed position of the C-terminus of protein IX is consistent with its exposed display of peptides and proteins from the Ad virion , Li et al, 2005, Vellinga et al, 2004.…”

“…Two independent cryoEM reconstructions of Ad virions devoid of protein IX (AdΔIX) have revealed a correlation between this assigned IIIa density and protein IX; AdΔIX virions were always lacking the IIIa density [Fabry et al, 2005, Scheres et al, 2005. More recent cryoEM studies in our laboratory on Ad5 vectors displaying either biotin acceptor proteins (BAPs) or EGFP on the C-terminus of protein IX shows the added BAP/ EGFP density appearing above the density assigned as protein IIIa, strongly suggesting that this density may be attributable to the C-terminal portion of protein IX [Marsh et al, 2006]. This observation, taken together with the observed loss of putative IIIa density upon IX deletion argue strongly that assigned IIIa density is actually protein IX.…”

Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

“…pIX is a minor capsid protein that stabilizes the hexon on the facets of the capsid (14,23). Recent studies showed that the N-terminal domains of three pIX monomers form a triskelion structure that cements three hexon proteins together, whereas the C-terminal domains are located near the edge between two facets and form a tetramer (22,45,60,68). Three of the four C-terminal domains associate together in a parallel structure, whereas the fourth domain, which stretches across from one facet to the adjacent facet, associates with the trimer in an antiparallel manner.…”

Assessment of Genetic Shielding for Adenovirus Vectors

“…The ABD ELISA data also indicates that there may be spatial factors playing a role with albumin, in that longer linkers were required to see good conjugation to albumin, thus suggesting the natural globular configuration of albumin may prevent it from fitting into the pIX cavity. Currently the classical configuration of the Ad capsid and position of pIX is being questioned [54] and therefore spatial elements of the capsid may play a bigger role in determining suitable large pIX-protein fusions to develop as pIX-modified vectors. Despite the failure with albumin a smaller self-protein alpha-1-antitrypsin was successfully fused to the pIX protein and virus was generated with this fusion capsid incorporated.…”