MOLECULAR CHARACTERIZATION OF THE NUCLEOCAPSID PROTEIN OF ARTERIVIRUSES

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MOLECULAR CHARACTERIZATION OF THE NUCLEOCAPSID PROTEIN OF ARTERIVIRUSES

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Title: MOLECULAR CHARACTERIZATION OF THE NUCLEOCAPSID PROTEIN OF ARTERIVIRUSES
Author: MOHAMMADI, HAKIMEH
Abstract: Nuclear localization of the nucleocapsid (N) protein of arteriviruses and the possible function of N in the nucleus for host cell function modulation were studied. Subcellular localization of N of lactate dehydrogenase-elevating virus (LDV) was determined by tagging N with enhanced green fluorescence protein (EGFP) on the N- and C-termini. Both N-EGFP and EGFP-N fusion proteins were found to localize to the nucleus and nucleolus of gene-transfected cells. A ‘pat4’ motif was identified in N as a potential nuclear localization signal (NLS), and its functional significance was determined by expressing a series of deletion constructs. The results showed that the ‘pat4’ NLS in LDV N was essential for nuclear translocation. LDV N interacted with importin-α and -β proteins suggesting that its nuclear localization is mediated through the importin-dependent nuclear transport pathway. This study was expanded to the equine arteritis virus (EAV) N protein. The EAV N gene was fused with EGFP at the N- or C-terminus, and its cellular distribution was investigated in gene-transfected cells. Both N-EGFP and EGFP-N fusion proteins of EAV N accumulated in the nucleus and nucleolus in addition to the cytoplasm. A series of deletion mutants were made by progressively deleting amino acids from the C- and N-termini of EAV N to determine the functional significance of the NLS-motif at amino acids positions 4-16. Studies using the mutant genes showed that the EAV N nuclear localization required the entire amino acid sequence from 4-20 position. EAV N interacted with both importin-α and -β proteins but also with some other fusion proteins suggesting that factors other than the importin-regulated nuclear transport pathway may be involved in this process. The simian hemorrhagic fever virus (SHFV) N gene was cloned as a fusion protein with EGFP. The functionality of the potential ‘pat7’ type NLS present at amino acid positions 22-28 of SHFV N was determined by constructing a series of deletion mutants and individually expressing them in cells. The results showed that the ‘pat7’ NLS was essential for nuclear translocation of SHFV N. The interaction of SHFV N with importin-α suggests that N recruits this chaperone protein for its nuclear localization. These finding confirm that the nuclear localization of N is a common property of arteriviruses. Next, modulation of cell cycle by arteriviruses was investigated. Replication of PRRSV and EAV in cells lead to accumulation of cells at the G2/M phase. Furthermore, cells expressing N protein from all four arteriviruses indicated that the number of cells in the G2/M phase was higher compared to controls. The modulatory effect of N protein on G2/M arrest was similar for all four N proteins, although PRRSV N and EAV N showed stronger effects on the cell number increase at the G2/M phase than LDV N and SHFV N. The results obtained from this research improve our understanding of N protein nuclear localization and its biological role during infection by members of the Arteriviridae family.
URI: http://hdl.handle.net/10214/3335
Date: 2012-02-23


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