Recombination Hotspot Institute of Virology S. Darr, I. Madisch, A. Heim Phylogeny and primary structure analysis of all Human Adenovirus (HAdV) Fiber shafts for rational design of adenoviral gene therapy vectors Phylogenetic trees of the fiber shaft and fiber knob confirmed the HAdV species concept For example, HAdV-D20 clusters to HAdV-D23 in the fiber shaft whereas it clusters to HAdV-D47 in the fiber knob (Fig. 4). Another example is HAdV-D26 that clusters to HAdV-D25 in the shaft region, whereas it clusters to HAdV-D27 in the fiber knob. A bootscan analysis demonstrated a recombination hotspot in a conserved region at the shaft/knob intersection at amino acid position 446 referring to HAdV-D30 in a complete fiber alignment. Inroduction HAdV have an icosahedral capsid which consists of 240 copies of the trimeric hexon protein, and a penton complex at each of the twelve vertices. The penton is made of a trimeric fiber which interacts with the cellular attachment receptor (e.g. CAR) and a pentameric base which binds to the internalization receptor (v3 or v5 integrins ) . The fiber is organized in three well-defined regions, the amino-terminal “tail” which binds to the penton base, a central shaft of variable length and a carboxy-terminal “knob” domain (Fig. 1). Heparan Binding Site Penton Base Fiber Shaft Penton Base: Secondary receptor Fiber Knob: Primary receptor Fig. 3 Neighbor joining trees containing all 52 human adenovirus fiber shafts (left) and fiber knobs (right). CAR Cell membrane Fig. 1 Primary and secondary receptor interaction of human adenovirus (Wu et al. (2003)) Sequences of adenovirus fiber shaft clustered strictly according to the species concept and were supported by high bootstrap values. The only exception was HAdV-E4 that clustered together with adenoviruses of species A in the fiber shaft region while it clustered with species C in the knob region of the fiber suggesting a recombination event in the phylogeny of HAdV-E4. Fig. 5 Multiple alignment of deduced amino acid sequences The flexibility of the fiber shaft seems to be essential to bring the penton base in close proximity to the secondary receptor after the primary receptor interaction. The complete data set of all human adenovirus fiber shafts determines the absence of this heparan binding site in all human adenoviruses except those of species C. A heparan binding site consisting of a KKTK (Lys-Lys-Thr-Lys) motif was formerly described as sufficient receptor for initial binding of several HAdV. Recombination Hotspot Results Bootscan of HAdV-D47 Bootscan of HAdV-D20 Bootscan of HAdV-D26 Bootscan of HAdV-D30 Length of adenovirus fiber shafts correlates with the species affiliation Conclusion Adenoviruses are highly diverse in the length of their fiber shafts. Recombination events in human adenoviruses of species D revealed a recombination hotspot suggesting that an intraspecies pseudotyping by fiber knob exchange should be feasible. Adenoviruses of species B, that are not primarily interacting with the CAR-receptor had the shortest, most rigid fiber shaft. Therefore, the usage of species B fiber shafts holds promise with regards to non-CAR-binding adenoviral vectors. Fig. 4 Bootscan analysis of several species D adenoviruses confirmed a recombination hotspot Moreover, comparison of phylogenetic trees of shaft and knob regions of species HAdV-D suggested a multitude of intraspecies recombination events in the phylogeny of HAdV types because of different clustering. Fig. 2 Length of all 52 human adenovirus fiber shafts MHH / Institute of Virology , OE 5230 Carl-Neuberg-Strasse 1, 30625 Hannover darr.sebastian@mh-hannover.de