Introduction
Adenoviruses (AdVs) are an intensively studied group of viruses due to their remarkable diversity, pathogenic relevance and prominent role as leading candidates as viral vectors in gene therapy. AdVs are classified into six genera: Mastadenovirus , Aviadenovirus ,Atadenovirus , Siadenovirus , Ichtadenovirus , andTestadenovirus , and their infections have been reported in almost all vertebrates. Siadenovirus , in particular, is known for its distinctive genomic characteristics and has been identified from reptiles and avian species 1-5. GenusSiadenovirus is proposed to have an amphibian origin. Frog siadenovirus 1 (FrAdV1), identified in the northern leopard frog (Lithobates pipiens ), was the first siadenovirus to be sequenced 1 and serves as the common ancestor for all siadenoviruses. However, despite extensive investigations, no further evidence of FrAdV1 infections in frogs or any other animal has been uncovered 6. The evolution of adenoviruses with an amphibian origin to infect distantly related host species including, critically endangered bird species, emphasises potential host-switching7. Their initial detection in a cell line of reptilian origin, and the absence of further evidence concerning frog siadenoviruses presents them as a compelling area of study.
During AdV infection, viral fiber and penton proteins bind and interact with the coxsackie adenoviral receptor and integrin V respectively, triggering virus uptake into clathrin-coated early endosomes (EE)7-10. In the EE, a drop in the pH induces conformational changes in the capsid and facilitates the release of the proteins VI and penton, disrupting the endosomal membrane, thus allowing the release and translocation of nucleocapsid to the nucleus in a microtubule and dynein dependent fashion 11. The capsid consists of three cationic proteins termed protein V, VII, and mu, with protein VII (pVII) being the most abundant. Shortly after viral infection, pVII is expressed as a precursor (Pre-pVII) and transported into the cell nucleus where it tightly interacts with the viral genome, preparing it for assembly 11. Before genome packaging, Pre-pVII is cleaved by the adenoviral protease, which removes 24 amino acid residues from the N-terminus, resulting in mature pVII, which will be packaged into the new viral particle. Both proteins are highly basic due to the presence of arginine and lysine residues and can be considered as functional analogues of histones, condensing the viral genome to be positioned inside the viral particle, with mature pVII being involved in nuclear delivery of viral DNA upon infection of new cells 10. Accordingly, both pVII and Pre-pVII are endowed with nuclear targeting abilities during viral infection8,10,12.
Molecules smaller than 70 kDa are believed to passively diffuse through the nuclear pore complex (NPC); on the other hand, larger ones and those which need to quickly accumulate in the cell nucleus require energy dependent transport, mediated by cellular transporters belonging to the importin (IMP) superfamily, which recognize nuclear localization signals (NLS) on cargoes 13,14. The best characterized NLS are termed “classical” (c)NLS and are short sequences enriched in basic amino acids such as arginine and lysine. cNLSs can either be monopartite, consisting of a single stretch of basic amino acids, or consist of two basic sequences separated by a short linker in a bipartite arrangement 15-17. cNLSs directly interact with IMPα, which functions as an adapter bridging the cargo to IMPβ1, which in turn mediates translocation of the complex across the NPC. Non-classical (nc)NLSs, on the other hand, are able to bind directly to IMPβ1 or one of its several homologues such as IMPβ2, IMP7 and many others, without the need for IMPα 18-20. In recent years, several studies have investigated the structural, functional, and cellular properties of HAdV pVII, and began the characterization of its nuclear targeting abilities. HAdV pVII proteins are able to interact with cellular components such as IMP7, IMPβ2, IMPα/β1, hsp70 and histone H1, so that pVII and Pre-pVII are believed to be able to use multiple nuclear import pathways. Furthermore, HAdV Pre-VII and pVII appear to have different nuclear import preferences, with cleavage believed to alter its IMPs interaction properties. Indeed, while pVII preferentially interacts with IMPβ2, Pre-pVII has a higher affinity for IMPα/β. Unfortunately, despite several NLSs have been described within pVII, their contribution to nuclear transport is still unclear, and the molecular determinants of nuclear import are not well characterized8,10,12. Additionally, nothing is known regarding nuclear import of adenoviruses of animal origin, including siadenoviruses, with the exception of a recent report from psittacine siadenovirus F (PsSiAdV). To address this issue, we thoroughly characterized the nuclear import process of Pre-pVII from FrAdV1, which can be considered the most ancient member of the Siadenovirusgenus. We identified four putative NLSs and characterized their structural, functional and biochemical properties utilising a wide range of approaches and dissected the contribution of each to Pre-VII subcellular localization. Upon expression in mammalian cells in the absence of any other viral protein, a GFP Pre-pVII fusion protein strongly accumulated in the nucleolus. The subcellular localization of several full-length Pre-pVII derivatives carrying amino acid substitutions within key NLSs basic residues revealed that a total impairment on nuclear localization was detected only upon inactivation of all NLSs, while co-expression with selective nuclear transport inhibitors highlighted an important role for IMPα/β and IMPβ1 but not IMPβ2 for nuclear targeting. By combining quantitative confocal laser scanning microscopy (CLSM) with biochemical and crystallographic assays we could further show that specific NLSs selectively bind different IMPs, and differently contribute to Pre-pVII subcellular localization. Indeed, NLSd (PPRKRRR VA-149) binds with high affinity to IMPα, in analogous fashion to several cNLSs, while NLSa (GYWRRKR SKK A-53) preferentially binds to IMPβ1. On the other hand, NLSc (GRK IKK AR AP-120) is crucial in mediating nucleolar targeting and can, therefore, be considered a nucleolar localization sequence (NoLS), while NLSb plays a minor but significant role by contributing to nucleolar and nuclear targeting.