Subcellular localization of ISAV M1 and NEP
In orthomyxoviruses, the segment 7 of influenza A and B viruses can generate a linear and a spliced transcript, which respectively encode M1 and M2 proteins [18, 19]. As the ISAV segment 7 also generates a linear and a spliced transcript with similar splicing strategy to the segment 7 of influenza viruses, the ISAV segment 7 was originally assumed to encode M1 and M2 proteins [20]. However, Kibenge et al. revealed that the two proteins encoded by the ISAV segment 7 were actually NS1 and NEP [21]. Instead, the ORF1 and ORF2 of ISAV segment 8 was confirmed to encode M1 and M2 proteins [12].
The ISAV M1 protein is 196 amino acids (aa) in length (Fig. 1a). To determine the subcellular localization of ISAV M1 protein, we constructed a plasmid pEGFP-M1, which expressed enhanced green fluorescent protein (EGFP)-tagged M1. In plasmid pEGFP-M1 or the empty vector pEGFP-N1 transfected SSN-1 cells (Fig. 1b), we found that EGFP-M1 showed green fluorescence in both cytosol and nucleus of the SSN-1 cells (Fig. 1b), similar to that observed in pEGFP-N1 transfected SSN-1 cells.
The ISAV NEP is encoded by a spliced ORF with 159-aa in length (Fig. 1a). To investigate the subcellular localization of NEP, the ISAV NEP gene was cloned into pDsRed2-N1 vector to generate plasmid pDsRed2-NEP, which expressed discosoma red fluorescent protein (DsRed)-tagged NEP. In transfected SSN-1 cells, the fluorescence protein DsRed itself showed red fluorescence throughout the SSN-1 cells, while the DsRed-NEP was localized only in the cytosol and accumulated adjacent to the nucleus of SSN-1 cells (Fig. 1c). Previous study have revealed that the ISAV NEP accumulated around the nucleus in plasmid pCDNA-Flag-NEP transfected Epithelioma papulosum cyprinid (EPC) cells [10]. Our results were consistent with the localization of NEP in EPC cells [10], indicating the accumulation of NEP around the nucleus was a common feature.
To illuminate the subcellular localization of M1 and NEP when they were co-expressed, the plasmids pEGFP-M1 and pDsRed2-NEP was co-transfected into SSN-1 cells (Fig. 1d). We found that M1 was co-localized with NEP in cytosol and accumulated adjacent to the nucleus (Fig. 1d). As control, in transfected SSN-1 cells, we found that EGFP itself did not co-localize with DsRed-NEP or DsRed (Fig. 1d). The results suggested that M1 probably interacted with NEP in SSN-1 cells.
Interaction between ISAV M1 and NEP
To further confirm the interaction between ISAV M1 and NEP, we generated plasmids expressing His-M1 or GST-NEP. These proteins were prokaryotically expressed and purified. Pull-down array showed that His-M1 could interact with GST-NEP, but not with GST. These data indicated that ISAV M1 could interact with NEP (Fig. 2).
Determination of the co-localization of Hsc70 with ISAV M1 or/and NEP
In addition to M1-NEP interaction that is essential for viral RNPs export, some other host factors such as Hsc70 has also been reported to facilitate viral RNPs export of influenza viruses [16, 17, 22]. Hsc70, a constitutive form of Hsp70 family, has been reported to be involved in the propagation of several viruses [23, 24]. Hsc70 contains a nuclear localization signal (NLS) and a NES (Fig. 3a) [25, 26], and was previously reported to bind with M1 protein of influenza A virus to facilitate RNP complex export from nucleus to cytosol [17]. In this study, the Hsc70 gene was amplified from SSN-1 cells and cloned into vector pEYFP-N1 to generate a plasmid pEYFP-Hsc70, which expressed enhanced yellow fluorescent protein (EYFP)-tagged Hsc70. In transfected SSN-1 cells, EYFP itself showed yellow fluorescence in both cytosol and nucleus of SSN-1 cells, while EYFP-Hsc70 showed fluorescence only in cytosol, the protein seemed to be excluded from the nucleus (Fig. 3b).
To determine the possible co-localization of Hsc70 with M1 or/and NEP, the plasmid pEYFP-Hsc70 was co-transfected with pEGFP-M1 or pDsRed2-NEP. The results showed that Hsc70 and M1 were still localized as they were expressed individually. However, Hsc70 was observed to co-localize with NEP in the cytosol and accumulated adjacent to the nucleus of SSN-1 cells (Fig. 4a). The results indicated that Hsc70 probably interacted with NEP, but whether it interacted with M1 needed to be determined further.
Interaction between Hsc70 and ISAV M1 or/and NEP
To investigate the interactions of Hsc70 with ISAV M1 or/and NEP, we generated two plasmids expressing GST-Hsc70 or His-Hsc70 respectively (Fig. 4b). GST-Hsc70 was used to study the interaction with His-M1, while His-Hsc70 was used to study the interaction with GST-NEP. Pull-down array showed that a weak band of GST-Hsc70 was pulled down by His-M1, indicating that Hsc70 could interact with M1 (Fig. 4b). It has been previously reported that Hsc70 could interact with M1 protein of influenza virus [17], indicating that Hsc70-M1 interaction might play a similar role in the life cycle of ISAV and influenza viruses. In addition, a band of GST-NEP, but not GST alone, was pulled down by His-Hsc70, indicating that Hsc70 could also interact with NEP (Fig. 4b). Therefore, it is hypothesized that the M1 and NEP of ISAV, and Hsc70 interact with each other to form a complex, which may play important roles in the export of vRNP of ISAV.
In order to exclude non-specific effects caused by cell origin, the subcellular localization of each protein as well as the co-localization of the proteins were investigated in human-origin Hela cells (Fig. 5a). Consistent with the observation in SSN-1 cells, the EGFP-M1 was localized throughout the cells, DsRed-NEP was localized in the cytosol and accumulated around the nucleus, while the Hsc70 was exclusively localized in the cytosol. Furthermore, Co-localization could also be observed between M1 and NEP as well as Hsc70 and NEP (Fig. 5b), which were consistent with the observations in SSN-1 cells.