Serine 105 and 120 are important phosphorylation sites for porcine reproductive and respiratory syndrome virus N protein function

Abstract

The nucleocapsid (N) protein is the most abundant protein of porcine reproductive and respiratory syndrome virus (PRRSV). It has been shown to be multiphosphorylated. However, the phosphorylation sites are still unknown. In this study, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to analyze the phosphorylation sites of N protein expressed in Sf9 cells. The results showed that N protein contains two phosphorylation sites. Since N protein can regulate IL-10, which may facilitate PRRSV replication, we constructed four plasmids (pCA-XH-GD, pCA-A105, pCA-A120 and pCA-A105-120) and transfected them into Pig alveolar macrophages (PAMs,3D4/2). The qPCR results showed that mutations at residues 105 and 120 were associated with down-regulation of the IL-10 mRNA level, potentially decreasing the viral growth ability. Then, we mutated the phosphorylation sites (S105A and S120A) and rescued three mutated viruses, namely, A105, A120 and A105-120. Compared with wild-type virus titers, the titers of the mutated viruses at 48 hpi were significantly decreased. The Nsp(non-structural protein) 9 qPCR results were consistent with the multistep growth kinetics results. The infected PAMs (primary PAMs) results were similar with Marc-145.The findings indicated that the mutations impaired the viral replication ability. The confocal microscopy results suggested that mutations to residues 105 and 120 did not affect N protein distribution. Whether the mutations affected other functions of N protein and what the underlying mechanisms are need further investigation. In conclusion, our results show that residues 105 and 120 are phosphorylation sites and are important for N protein function and for viral replication ability.

A novel amino acid site of N protein could affect the PRRSV-2 replication by regulating the viral RNA transcription

“…Regulating host cytokines, such as IL-10 or IRF3, is one of the major functions of the N protein [ 20 , 21 ]. N protein has ten serine sites, three of them (serine 36 , serine 105 and serine 120 ) have confirmed to be involved in regulating IL-10 mRNA or IL-10 expression [ 18 , 19 , 22 ]. These results indicated that the serine of N protein could affect the N-induced expression of IL-10.…”

“…Previous studies have shown that mutating the serine 105 and serine 120 of the N protein can reduce viral replication and virulence [ 18 ]. However, mutations to serine 36 and serine 122 do not change the viral replication ability [ 18 , 19 , 22 , 23 ]. Therefore, we questioned whether the serine 78 or serine 99 of the N protein could change PRRSV replication efficiency.…”

“…The nucleocapsid (N) protein is the most abundant protein in the PRRSV and plays a vital role in the viral life cycle, including regulating cytokine levels in the host, connecting with other viral proteins and combining with viral RNA [ 17 ]. As a protein with 123 or 128 amino acids, a mutation to an amino acid site may alter the functions of the N protein or affect viral replication ability and virulence [ 18 , 19 ].…”

Hyper-phosphorylation of nsp2-related proteins of porcine reproductive and respiratory syndrome virus

“…Phosphorylation by the host GSK3 kinase affects conformational transitions of the MHV N protein and regulates its interaction with the DDX1 helicase to fine-tune the discontinuous transcription of sgRNAs (Wu et al, 2014). Phosphorylation of the N protein of PRRSV has also been reported (Wootton et al, 2002;Yoo et al, 2003;Chen et al, 2018Chen et al, , 2019. Phospho-ablatant mutations on two PRRSV N protein phosphorylation sites attenuated viral replication capability both in vitro and in vivo (Chen et al, 2018(Chen et al, , 2019.…”

“…Phosphorylation of the N protein of PRRSV has also been reported (Wootton et al, 2002;Yoo et al, 2003;Chen et al, 2018Chen et al, , 2019. Phospho-ablatant mutations on two PRRSV N protein phosphorylation sites attenuated viral replication capability both in vitro and in vivo (Chen et al, 2018(Chen et al, , 2019. However, specific biological roles of PRRSV N protein phosphorylation remain largely unclear.…”