Heterogeneous Nuclear Ribonucleoprotein A1 and Lamin A/C Modulate Nucleocytoplasmic Shuttling of Avian Reovirus p17

Abstract

Avian reovirus (ARV) p17 protein continuously shuttles between the nucleus and the cytoplasm via transcription-dependent and chromosome region maintenance 1 (CRM1)-independent mechanisms. Nevertheless, whether cellular proteins modulate nucleocytoplasmic shuttling of p17 remains unknown. This is the first report that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 serves as a carrier protein to modulate nucleocytoplasmic shuttling of p17. Both in vitro and in vivo studies indicated that direct interaction of p17 with hnRNP A1 maps within the amino terminus (amino acids [aa] 19 to 40) of p17 and the Gly-rich region of the C terminus of hnRNP A1. Furthermore, our results reveal that the formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Utilizing sequence and mutagenesis analyses, we have identified nuclear export signal (NES) 19 LSLRELAI 26 of p17. Mutations of these residues causes a nuclear retention of p17. In this work, we uncovered that the N-terminal 21 amino acids (aa 19 to 40) of p17 that comprise the NES can modulate both p17 and hnRNP A1 interaction and nucleocytoplasmic shuttling of p17. In this work, the interaction site of p17 with lamin A/C was mapped within the amino terminus (aa 41 to 60) of p17 and p17 colocalized with lamin A/C at the nuclear envelope. Knockdown of hnRNP A1 or lamin A/C led to inhibition of nucleocytoplasmic shuttling of p17 and reduced virus yield. Collectively, the results of this study provide mechanistic insights into hnRNP A1 and lamin A/Cmodulated nucleocytoplasmic shuttling of the ARV p17 protein.IMPORTANCE Avian reoviruses (ARVs) cause considerable economic losses in the poultry industry. The ARV p17 protein continuously shuttles between the nucleus and the cytoplasm to regulate several cellular signaling pathways and interacts with several cellular proteins to cause translation shutoff, cell cycle arrest, and autophagosome formation, all of which enhance virus replication. To date the mechanisms underlying nucleocytoplasmic shuttling of p17 remain largely unknown. Here we report that hnRNP A1 and lamin A/C serve as carrier and mediator proteins to modulate nucleocytoplasmic shuttling of p17. The formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Furthermore, we have identified an NES-containing nucleocytoplasmic shuttling domain (aa 19 to 40) of p17 that is critical for binding to hnRNP A1 and for nucleocytoplasmic shuttling of p17. This study provides novel insights into how hnRNP A1 and lamin A/C modulate nucleocytoplasmic shuttling of the ARV p17 protein. RESULTSCellular proteins hnRNP A1 and lamin A/C coimmunoprecipatate with p17. As shown previously, we identified several cellular proteins, such as nucleoporin Tpr, vimentin, and lamin A/C, that interact with the ARV p17 protein (27,28,30). To explore whether and how cellular proteins are involved in mediating nucleocytoplasmic shut-Chiu et al. FIG 1 p17 interacts with hnRNP A1. (A) Identification of poten...

The multifarious roles of heterogeneous ribonucleoprotein A1 in viral infections

“…ARV p17 protein interacts with hnRNPA1 and is indirectly shuttled across the nuclear pore complex, alongside the hnRNPA1‐transportin‐1 cargo . C‐terminal, glycine‐rich region (195‐268 aa) of hnRNPA1 is deemed crucial for its interaction with ARV p17 (19‐40 aa) . This is supported by site‐directed mutagenesis, wherein mutant hnRNPA1 195‐268 with intact M9 NLS (195‐268 aa) retained its binding ability to p17, whereas mutant hnRNPA1 1‐184 (1‐184 aa) lost its capacity to interact with p17, thereby leading to the nuclear accumulation of ARV p17 protein .…”

“…Chiu et al reported that in avian reovirus (ARV) infection, nonstructural protein p17 is shuttled to and fro from the nucleus in an hnRNPA1‐dependent manner . hnRNPA1 is constantly shuttled to and fro the nucleus by an active import/export mechanism which relies on its interaction with the F‐peptide (301‐318 aa) of Transportin‐1, a member of the importin family .…”

“…hnRNPA1 is constantly shuttled to and fro the nucleus by an active import/export mechanism which relies on its interaction with the F‐peptide (301‐318 aa) of Transportin‐1, a member of the importin family . ARV p17 protein interacts with hnRNPA1 and is indirectly shuttled across the nuclear pore complex, alongside the hnRNPA1‐transportin‐1 cargo . C‐terminal, glycine‐rich region (195‐268 aa) of hnRNPA1 is deemed crucial for its interaction with ARV p17 (19‐40 aa) .…”

Identification and phylogenetic characterization of bovine ephemeral fever virus (BEFV) of Middle Eastern lineage associated with 2018‐2019 outbreaks in India

“…The interactions of N-P-L proteins form the ribonucleoprotein (RNP) complex that holds the viral RNA genome. Additionally, matrix protein performs multifunctional roles, as it acts as a nucleocytoplasmic shuttling protein (Chiu et al, 2019). It plays a regulatory role in the expression of viral and host proteins (Delmas et al, 2008) causes cytopathic effect (CPE) and induces apoptosis (Chen et al, 2010;Lin et al, 2009) even when expressed independent to other viral proteins (Gaddy & Lyles, 2005) and also leads to autophagy, thereby suppressing the anti-viral immune pathway ultimately benefiting in the virus production (Cheng et al, 2019).…”

Cyclin-dependent kinases and CDK inhibitors in virus-associated cancers

“…Furthermore, avian reovirus p17 protein has been demonstrated to function like v-CDKI, [28] which shuttles between the Fig. 5 The regulation of cells CDK and CIKs expression by HBV and HCV cytoplasm and the nucleus [128] to perform specific duties in mediating cancer cell cycle and growth [28]. Avian reovirus protein p17 is a nucleoporin Tpr Suppressor and can activate p53, p21 and PTEN and inactivates the PI3K/AKT/mTOR and ERK signaling pathways [79,129].…”