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The Extended C-Terminal Region of Influenza C Virus Nucleoprotein Is Important for Nuclear Import and Ribonucleoprotein Activity.

ABSTRACT: The influenza C virus (ICV) is a human-pathogenic agent, and the infections are frequently identified in children. Compared to influenza A and B viruses, the nucleoprotein of ICV (NPC) has an extended C-terminal region of which the functional significance is ill defined. We observed that the nuclear localization signals (NLSs) found on the nucleoproteins of influenza A and B virus subtypes are absent at corresponding positions on ICV. Instead, we found that a long bipartite nuclear localization signal resides at the extended C-terminal region, spanning from R513 to K549. Our experimental data determined that the KKMK motif within this region plays important roles in both nuclear import and polymerase activity. Similar to the influenza A viruses, NPC also binds to multiple human importin ? isoforms. Taken together, our results enhance the understanding of the virus-host interaction of the influenza C virus.IMPORTANCE As a member of the Orthomyxoviridae family, the polymerase complex of the influenza C virus structurally resembles its influenza A and influenza B virus counterparts, but the nucleoprotein differs by possessing an extra C-terminal region. We have characterized this region in view of nuclear import and interaction with the importin ? protein family. Our results demonstrate the functional significance of a previously uncharacterized region on Orthomyxoviridae nucleoprotein (NP). Based on this work, we propose that importin ? binding to influenza C virus NP is regulated by a long bipartite nuclear localization signal. Since the sequence of influenza D virus NP shares high homology to that of the influenza C virus, this work will also shed light on how influenza D virus NP functions.

SUBMITTER: Tang YS

PROVIDER: S-EPMC6475786 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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The Extended C-Terminal Region of Influenza C Virus Nucleoprotein Is Important for Nuclear Import and Ribonucleoprotein Activity.

Tang Yun-Sang YS Lo Chun-Yeung CY Mok Chris Ka-Pun CK Chan Paul Kay-Sheung PK Shaw Pang-Chui PC

Journal of virology 20190417 9

The influenza C virus (ICV) is a human-pathogenic agent, and the infections are frequently identified in children. Compared to influenza A and B viruses, the nucleoprotein of ICV (NPC) has an extended C-terminal region of which the functional significance is ill defined. We observed that the nuclear localization signals (NLSs) found on the nucleoproteins of influenza A and B virus subtypes are absent at corresponding positions on ICV. Instead, we found that a long bipartite nuclear localization ...[more]

PMID: 30814281

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Project description:The viral ribonucleoprotein (vRNP) of the influenza A virus (IAV) is responsible for the viral RNA transcription and replication in the nucleus, and its functions rely on host factors. Previous studies have indicated that eukaryotic translation elongation factor 1 delta (eEF1D) may associate with RNP subunits, but its roles in IAV replication are unclear. Herein, we showed that eEF1D was an inhibitor of IAV replication because knockout of eEF1D resulted in a significant increase in virus yield. eEF1D interacted with RNP subunits polymerase acidic protein (PA), polymerase basic 1 (PB1), polymerase basic 2 (PB2), and also with nucleoprotein (NP) in an RNA-dependent manner. Further studies revealed that eEF1D impeded the nuclear import of NP and PA-PB1 heterodimer of IAV, thereby suppressing the vRNP assembly, viral polymerase activity, and viral RNA synthesis. Together, our studies demonstrate eEF1D negatively regulating the IAV replication by inhibition of the nuclear import of RNP subunits, which not only uncovers a novel role of eEF1D in IAV replication but also provides new insights into the mechanisms of nuclear import of vRNP proteins.IMPORTANCE Influenza A virus is the major cause of influenza, a respiratory disease in humans and animals. Different from most other RNA viruses, the transcription and replication of IAV occur in the cell nucleus. Therefore, the vRNPs must be imported into the nucleus for viral transcription and replication, which requires participation of host proteins. However, the mechanisms of the IAV-host interactions involved in nuclear import remain poorly understood. Here, we identified eEF1D as a novel inhibitor for the influenza virus life cycle. Importantly, eEF1D impaired the interaction between NP and importin α5 and the interaction between PB1 and RanBP5, which impeded the nuclear import of vRNP. Our studies not only reveal the molecular mechanisms of the nuclear import of IAV vRNP but also provide potential anti-influenza targets for antiviral development.

Project description:UnlabelledThe nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex. During the replication of influenza virus, the vRNP complex undergoes nuclear-cytoplasmic shuttling, during which NP serves as one of the determinants. To date, many phosphorylation sites on NP have been identified, but the biological functions of many of these phosphorylation sites remain unknown. In the present study, the functions of the phosphorylation sites S9, Y10, and Y296 were characterized. These residues are highly conserved, and their phosphorylation was essential for virus growth in cell culture and in a mouse model by regulating the activity of the viral polymerase and the nuclear-cytoplasmic shuttling of NP. The phosphorylation and dephosphorylation of S9 and Y10 controlled nuclear import of NP by affecting the binding affinity between NP and different isoforms of importin-α. In addition, the phosphorylation of Y296 caused nuclear retention of NP by reducing the interaction between NP and CRM1. Furthermore, tyrosine phosphorylation of NP during the early stage of virus infection was ablated when Y296 was mutated to F. However, at later stages of infection, it was weakened by the Y10F mutation. Taken together, the present data indicate that the phosphorylation and dephosphorylation of NP control the shuttling of NP between the nucleus and the cytoplasm during virus replication.ImportanceIt is well known that phosphorylation regulates the functions of viral proteins and the life cycle of influenza A virus. As NP is the most abundant protein in the vRNP complex of influenza A virus, several phosphorylation sites on this protein have been identified. However, the functions of these phosphorylation sites were unknown. The present study demonstrates that the phosphorylation status of these sites on NP can mediate its nuclear-cytoplasmic shuttling, which drives the trafficking of vRNP complexes in infected cells. The present data suggest that the phosphorylated residues of NP are multistep controllers of the virus life cycle and new targets for the design of anti-influenza drugs.

Dataset Information

The Extended C-Terminal Region of Influenza C Virus Nucleoprotein Is Important for Nuclear Import and Ribonucleoprotein Activity.

Publications

The Extended C-Terminal Region of Influenza C Virus Nucleoprotein Is Important for Nuclear Import and Ribonucleoprotein Activity.

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