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Nguyen, Le Tam Nhan

University of British Columbia

Influenza viruses must deliver their genome into the nucleus of infected cells for replication. This process is mediated by the viral nucleoprotein (NP), containing two nuclear localization signals (NLSs): NLS1 at the N-terminus and a newly identified NLS2 spanning residues 212-216 (²¹²GRKTR²¹⁶). Most studies have concentrated on the role of NP's NLSs using in vitro-assembled ribonucleoproteins (RNPs) or purified viral ribonucleoproteins (vRNPs), which may differ structurally from vRNPs within mammalian cells during an infection. To determine the role of NLS1 and NLS2 in the nuclear import of vRNPs in a naturally-occurring infection system, tissue culture cells were infected with mutant NLS1 (NLS1MT) and NLS2 (NLS2MT) viruses. I hypothesize that the mutations of NLSs of NP inhibit the nuclear import of the influenza genome, thereby impairing viral replication and infection. I found that cells infected with these mutant viruses were defective in the nuclear import of incoming vRNPs and produced reduced amounts of newly synthesized NP, which is needed for progeny vRNPs assembly. NLS2MT-infected cells were also defective in nucleolar localization of NP and produced less progeny virus than NLS1MT-infected cells. To determine whether NLS2 is present in other viral and/or cellular proteins, I used bioinformatics analyses and found that only 0.4% of human nuclear proteins contained the putative NLS2. From these, I chose to study the nucleolus protein 14 (NOP14), which possesses a putative NLS2 at its N-terminus and whose nuclear import mechanism has not yet been characterized. Mutations of NLS2 did not affect the NOP14 nuclear import, but its nucleolar localization, indicating that NLS2 is not the only NLS of NOP14 and that NLS2 is a functional nucleolar localization signal of NOP14. According to NLS prediction tools, NOP14 has three additional putative NLSs. Using functional assays, I demonstrated that one of these is a novel NLS located at the C-terminus of NOP14. Collectively, my work dissects the contributions of NLS1 and NLS21 to the nuclear import of influenza A vRNPs and demonstrates the importance of NLS2 in nucleolar NP localization in a naturally-infection system. These findings provide better insights into the viral-host interaction during influenza A virus infection.

Text

2024-04-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/84467

Zoology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/