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.