Importin α/β-dependent nuclear transport of human parvovirus B19 nonstructural protein 1 is essential for viral replication

Alvisi, Gualtiero; Manaresi, Elisabetta; Cross, E.M.; Hoad, Mikayla; Akbari, Nasim; Pavan, Silvia E.; Ariawan, A. Daryl; Bua, Gloria; Petersen, Gayle F; Forwood, Jade K.; Gallinella, Giorgio

doi:10.1016/j.antiviral.2023.105588

Cited by 11 publications

(7 citation statements)

References 92 publications

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“…These can function independently as a monopartite cNLS (Figure 2D,E ), but preferentially work as a bipartite cNLS, with the upstream and downstream stretches of basic aas binding to the IMPα minor and major binding sites, respectively (Figure 4 ), in a similar manner to that reported for the nucleoplasmin bipartite cNLS (Conti & Kuriyan, 2000 ). Most monopartite cNLSs are fully functional, perfectly fit to the consensus (K‐K/R‐X‐K/R, Figure 1 , Figure S1 ), and can be expected to bind to the IMPα major binding site in an extended conformation, similar to JCPyV LTA (Figure 4 ), SV40 LTA, and several other monopartite cNLSs (Alvisi et al, 2023 ; Conti et al, 1998 ; Smith et al, 2018 ). However, HPyV7 LTA contains an extremely weak cNLS (143‐PP K Q KK PN‐152), which does not fit the cNLS consensus but is still capable of partially relocating GFP to the nucleus (Figure 2Dl,E ) and weakly binding IMPα3ΔIBB in vitro (Figure S5B ).…”

Section: Discussionmentioning

confidence: 82%

A functional and structural comparative analysis of large tumor antigens reveals evolution of different importin α‐dependent nuclear localization signals

Cross,

Akbari,

Ghassabian

et al. 2024

Protein Science

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Nucleocytoplasmic transport regulates the passage of proteins between the nucleus and cytoplasm. In the best characterized pathway, importin (IMP) α bridges cargoes bearing basic, classical nuclear localization signals (cNLSs) to IMPβ1, which mediates transport through the nuclear pore complex. IMPα recognizes three types of cNLSs via two binding sites: the major binding site accommodates monopartite cNLSs, the minor binding site recognizes atypical cNLSs, whilst bipartite cNLSs simultaneously interact with both major and minor sites. Despite the growing knowledge regarding IMPα‐cNLS interactions, our understanding of the evolution of cNLSs is limited. We combined bioinformatic, biochemical, functional, and structural approaches to study this phenomenon, using polyomaviruses (PyVs) Large Tumor Antigens (LTAs) as a model. We characterized functional cNLSs from all human (H)PyV LTAs, located between the LXCXE motif and origin binding domain. Surprisingly, the prototypical SV40 monopartite NLS is not well conserved; HPyV LTA NLSs are extremely heterogenous in terms of structural organization, IMPα isoform binding, and nuclear targeting abilities, thus influencing the nuclear accumulation properties of full‐length proteins. While several LTAs possess bipartite cNLSs, Merkel Cell (MC) PyV contains a hybrid bipartite cNLS whose upstream stretch of basic amino acids can function as an atypical cNLS, specifically binding to the IMPα minor site upon deletion of the downstream amino acids after viral integration in the host genome. Therefore, duplication of a monopartite cNLS and subsequent accumulation of point mutations, optimizing interaction with distinct IMPα binding sites, led to the evolution of bipartite and atypical NLSs binding at the minor site.This article is protected by copyright. All rights reserved.

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Section: Discussionmentioning

confidence: 82%

A functional and structural comparative analysis of large tumor antigens reveals evolution of different importin α‐dependent nuclear localization signals

Cross,

Akbari,

Ghassabian

et al. 2024

Protein Science

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“…Plasmid pcDNA3.1-NT-GFP-TOPO, mediating the expression of GFP cycle3, and pcDNA3.1-NT-GFP-TOPO-SV40-NLS, mediating the expression of a fusion protein between GFP cycle3 and Simian vacuolating virus (SV) 40 large tumour antigen NLS (PKKKRKV-132), were described previously [26]. Plasmids pEGFP-N1-H1E and pEPI-GFP-human cytomegalovirus (HCMV)-UL44, encoding control GFP fusion proteins localizing to the nucleus via different IMPα/β-independent and IMPα/β-dependent pathways, respectively were described previously [27–29].…”

Section: Methodsmentioning

confidence: 99%

Structural and functional characterization of siadenovirus core protein VII nuclear localization demonstrates the existence of multiple nuclear transport pathways

Athukorala,

Donnelly,

Pavan

et al. 2024

Journal of General Virology

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Adenovirus protein VII (pVII) plays a crucial role in the nuclear localization of genomic DNA following viral infection and contains nuclear localization signal (NLS) sequences for the importin (IMP)-mediated nuclear import pathway. However, functional analysis of pVII in adenoviruses to date has failed to fully determine the underlying mechanisms responsible for nuclear import of pVII. Therefore, in the present study, we extended our analysis by examining the nuclear trafficking of adenovirus pVII from a non-human species, psittacine siadenovirus F (PsSiAdV). We identified a putative classical (c)NLS at pVII residues 120–128 (120PGGFKRRRL128). Fluorescence polarization and electrophoretic mobility shift assays demonstrated direct, high-affinity interaction with both IMPα2 and IMPα3 but not IMPβ. Structural analysis of the pVII-NLS/IMPα2 complex confirmed a classical interaction, with the major binding site of IMPα occupied by K124 of pVII-NLS. Quantitative confocal laser scanning microscopy showed that PsSiAdV pVII-NLS can confer IMPα/β-dependent nuclear localization to GFP. PsSiAdV pVII also localized in the nucleus when expressed in the absence of other viral proteins. Importantly, in contrast to what has been reported for HAdV pVII, PsSiAdV pVII does not localize to the nucleolus. In addition, our study demonstrated that inhibition of the IMPα/β nuclear import pathway did not prevent PsSiAdV pVII nuclear targeting, indicating the existence of alternative pathways for nuclear localization, similar to what has been previously shown for human adenovirus pVII. Further examination of other potential NLS signals, characterization of alternative nuclear import pathways, and investigation of pVII nuclear targeting across different adenovirus species is recommended to fully elucidate the role of varying nuclear import pathways in the nuclear localization of pVII.

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“…N‐terminally FITC‐tagged peptide UL44_410‐433 corresponding to the HCMV C‐terminal 24 residues (410‐KEESDSEDSVTFEFVPNTKKQKCG‐433) and its phosphorylated variant UL44_410‐433_pT (410‐KEESDSEDSVTFEFVPNTKKQKCG‐433) were synthesized by the Dementia Research Centre, Macquarie University and used for fluorescence polarization assays as described in Ref. [6]. For the phosphorylated variant, phosphorylated threonine amino acid (Fmoc‐Thr(PO(OBzl)OH)‐OH) was employed for the 427aa site.…”

Section: Methodsmentioning

confidence: 99%

“…The best‐characterized NLSs are highly basic sequences recognized by IMPβ1 through the adapter molecule IMPα [2], and are known as classical (c)NLSs. Human IMPαs exist as seven isoforms (hIMPα1/3/4/5/6/7/8), with the mouse homolog of hIMPα1, “mIMPα2”, frequently used for structural studies [3–7]. cNLSs can either be monopartite or bipartite.…”

Section: Figmentioning

confidence: 99%

Structural determinants of phosphorylation‐dependent nuclear transport of HCMV DNA polymerase processivity factor UL44

Cross,

Marin,

Ariawan

et al. 2024

FEBS Letters

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Human cytomegalovirus (HCMV) DNA polymerase processivity factor UL44 is transported into the nucleus by importin (IMP) α/β through a classical nuclear localization signal (NLS), and this region is susceptible to cdc2‐mediated phosphorylation at position T427. Whilst phosphorylation within and close to the UL44 NLS regulates nuclear transport, the details remain elusive, due to the paucity of structural information regarding the role of negatively charged cargo phosphate groups. We addressed this issue, studying the effect of UL44 T427 phosphorylation on interaction with several IMPα isoforms by biochemical and structural approaches. Phosphorylation decreased UL44/IMPα affinity 10‐fold, and a comparative structural analysis of UL44 NLS phosphorylated and non‐phosphorylated peptides complexed with mouse IMPα2 revealed the structural rearrangements responsible for phosphorylation‐dependent inhibition of UL44 nuclear import.

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Importin α/β-dependent nuclear transport of human parvovirus B19 nonstructural protein 1 is essential for viral replication

Cited by 11 publications

References 92 publications

A functional and structural comparative analysis of large tumor antigens reveals evolution of different importin α‐dependent nuclear localization signals

A functional and structural comparative analysis of large tumor antigens reveals evolution of different importin α‐dependent nuclear localization signals

Structural and functional characterization of siadenovirus core protein VII nuclear localization demonstrates the existence of multiple nuclear transport pathways

Structural determinants of phosphorylation‐dependent nuclear transport of HCMV DNA polymerase processivity factor UL44

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