Discussion
While several studies have identified NLSs in pVII from human mastadenovirus C, the presence, specific contribution, as well as their role in nuclear import of various other adenovirus members remained elusive. Here we have combined bioinformatics, microscopy imaging, biochemical and structural approaches to identify and characterize four putative NLSs in Pre-pVII from the ancient siadenovirus FrAdV1. Further, by co-expression of wild type and NLS substitution derivatives thereof in the presence and absence of several nuclear import pathways inhibitors (Fig. 2, 6, 7, 8) we have characterized the contribution of each NLS to the subcellular localization of FrAdV1 Pre-pVII, thus unveiling the contribution of different nuclear import pathways. This is the first study to characterize and dissect the functional contribution of individual sequences to the subcellular localization of an AdV pVII protein and may provide a basis for future evolutionary comparisons regarding different AdV pVII proteins 8,10,12,50.
When expressed in the absence of other viral proteins, FrAdV1 Pre-pVII strongly accumulated in cell nucleoli (Fig. 2), in stark contrast to what was reported for Pre-pVII from both PsSiAdV which is known to accumulate in the nucleoplasm 47 and from HAdV2, which has been shown to localize in the nucleoplasm and in the nucleoli8. The reason and functional significance for such discrepancies remain elusive, but it is noteworthy that in the case of FrAdV-1, nucleolar accumulation is dependent on a highly basic sequence (NLSc:111GRK IKK AR AP120), which is the main determinant of nucleolar targeting, and a bipartite sequence located upstream (NLSb:74RKK IPK TPVGVVGPLWQGTRKR 95; see Fig. 7), which also contributes to the process, although to a lesser extent. On the other hand, only one NoLS (93MRR YAK MKRRRRR VARR HRRR 112) has been identified in HAdV2 Pre-pVII 8, while none have been identified in PsSiAdV Pre-pVII 47. Therefore, it is likely that the number and strength of NoLSs on AdV Pre-pVII proteins influences their degree of nucleolar targeting. Our findings strengthen the hypothesis that Pre-pVII from different AdVs are endowed with specific subcellular localization abilities, as exemplified by the ability of bovine adenovirus 3 Pre-pVII to localize to mitochondria during viral infection 51.
We propose a model for Frog Pre-pVII nuclear transport where the strong nucleolar targeting is due to the concerted action of the four NLSs identified here, each playing a specific role in protein subcellular localization (Fig. 9). The N-terminal NLSa appears to be a non-classical NLS, binding with high affinity to IMPβ1 directly (Fig. 3 and 4), similarly to HIV-1 tat and Rev NLSs 52. NLSb is poorly active outside of its physiological context, both in terms of IMP binding (Fig. 3) and nuclear targeting activity (Fig. 4 and Supplementary Fig. S2). However, it is sufficient to confer IMPα/β1 dependent nuclear targeting to full-length FrAdV1 Pre-pVII once all other NLSs have been inactivated (Fig. 7 and Fig. 8). This suggest that NLSb might be an atypical NLS which binds IMPs thanks to its specific three-dimensional conformation, rather than in a liner fashion, such as reported for the NLS described in the intestinal fatty acid binding protein 53. NLSc can partially target a heterologous protein to the nucleolus in an IMPα/β1 independent fashion, does not bind either IMPα2 nor IMPβ1 with high affinity (Fig. 3 and Supplementary Table S4), but substitution of its basic residues in the context of full-length Pre-pVII completely ablates nucleolar targeting (Fig. 7), suggesting that it is important for nucleolar localization, but by itself, not sufficient to localise proteins to the nucleolus. It therefore has similarities to regions within HAdV2 that act as an NoLS8. Finally, the C-terminal NLSd is a bona fidaemonopartite cNLS mediating IMPα/β1 dependent nuclear transport, by directly interacting with IMPα major binding site (Figs. 3-5).
Therefore, our data are consistent with previous studies that reported the ability of Pre-pVII proteins to bind multiple IMPs and be important through multiple pathways 8,10,12,50. Whilst HAdV5 Pre-pVII nuclear transport is dependent on IMPα/β1 12, wild-type FrAdV1 Pre-pVII can accumulate in the nucleolus even in the presence of the highly efficient IMPα/β1 inhibitor Bimax2. Since simultaneous mutation of NLSc (conferring nucleolar accumulation) and NLSa (binding to IMPβ1 with high affinity) renders FrAdV1 Pre-pVII nuclear transport sensitive to Bimax2, FrAdV1 can be actively imported in the nucleus by IMPα/β1 (via NLSd) and IMPβ1 (via NLSa). The evidence that FrAdV1 Pre-pVII nuclear and nucleolar localization was not impacted by the transdominant negative RanQ69L, strongly supports the idea that it can enter the nucleus by passive diffusion and be therein retained after interaction with dsDNA and other nuclear components. Indeed, HAdV2 Pre-pVII has been shown to interact with cellular DNA and co-localize with human chromosomes 8, and mutation of the highly basic NLSc, which results in loss of nucleolar localization, is sufficient to render Frog Pre-pVII sensitive to RanQ69L, but not to Bimax2.
In conclusion, our findings revealed important differences between the import of FrAdV1 Pre-pVII in comparison to what has been reported for HAdV and PsSiAdV. In particular the protein accumulated in the nucleolus more strongly as compared to its orthologues, and its nuclear targeting is insensitive to inhibition of IMPα/β1. The findings of this study provide valuable insights into how different adenoviruses interact with the host cell’s nuclear transport machinery. Understanding these interactions may have broader implications for viral replication and infection strategies across different adenovirus genera, and potential applications in therapeutic approaches.