Migration of influenza virus nucleoproteins to the nucleolus is critical for the formation of ribonucleoprotein complexes

The influenza A virus duplex ribonucleoprotein complex (RNP) is responsible for the transcription and replication of viral genomic RNA (vRNA) and plays a crucial role in the viral life cycle
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RNP formation occurs within the nucleus of virus-infected cells, and the nuclear domains involved in this process remain unclear
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A study published in ASM Journal by researchers from Japan shows that the nucleolus is an important site for the formation of functional RNPs
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The researchers used several microscopy techniques to reveal that the main component of the viral ribonucleoprotein complex, the viral nucleoprotein (NP), temporarily resides in the nucleolus, the site of ribosomal RNA/protein assembly, where it forms Depends on nucleolar localization signals
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Mutation of the nucleolar localization signal (NoLS) on NP blocks the formation of duplex RNPs, resulting in loss of viral RNA synthesis capacity, whereas ectopic fusion of NoLS enables NP mutants to form functional duplex RNPs
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Furthermore, nucleolar disruption in virus-infected cells inhibits the assembly of NPs into duplex RNPs, resulting in a marked reduction in viral RNA synthesis and replication
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thisThe findings demonstrate that the nucleolus is an important location for RNP formation and viral replication,Migration of NPs to the nucleolus is a critical step in the formation of functional RNPs, showing the importance of the nucleolus in the influenza virus life cycle
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Influenza A virus isSingle-stranded negative-sense RNA virus
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Viral genomic RNA (vRNA) fragments exist in the form of ribonucleoprotein complexes (RNPs) containing multiple nucleoproteins, an RNA-dependent RNA polymerase complex composed of PB2, PB1, and PA subunits
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The ribonucleoprotein complex RNP is responsible for transcription and replicationvRNAs
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in transcriptionDuring the process, vRNA is transcribed into mRNA with a 5'-cap and a 3'-polyA tail; whileDuring replication, vRNA is replicated as a cRNA replication intermediate,while cRNA acts as a template to generate more vRNAs
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These replication processes are accompanied by RNP assembly,The 5′ ends of nascent vRNA and cRNA are linked to a newly synthesized viral polymerase complex, which is sequentially coated with multiple NPs and assembled into duplex vRNPs and cRNPs, respectively.

Unlike most RNA viruses, influenza A virus transcribes and replicates its genome in the nucleus of infected cells,Therefore, the transcription, replication and RNP formation of influenza A virus are heavily dependent on the nuclear machinery of the host
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Since NPs are localized to the nucleolus, the researchers hypothesized that the assembly of NPs into double-helical RNPs is dependent on the nucleolus
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thenResearchersSeveral microscopic and biochemical approaches were employed to investigate the importance of the nucleolus in the formation of functional RNPs—including the use of NP mutants lacking nucleolar localization signals, as well as compounds that cause nucleolar rupture
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The importance of nucleolar NP localization for functional RNP formation
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Only NPs among RNPs have one nuclear localization signal, one nucleolar localization signal (NOLS)
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To investigate the importance of nucleolar NP localization for RNP formation, we constructed two NoLS mutant NPs: one NP NoLSmut with an alanine substitution in NoLS, localized only in the nucleoplasm, and the other a reverse mutant NoLS - NP NoLSmut, which incorporates full NoLS
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The amino terminus of NP NoLSmut, facilitating its nucleolar localization
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The experimental results demonstrate that the nucleolar localization of NPs is critical for both transcription and replication of viral RNAs
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Effects of nucleolar disruption on the formation of functional RNPs
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Considering the necessity of nucleolar NP localization for correct RNP formation, disruption of nucleolar structure would severely affect RNP formation
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To test this hypothesis, the researchers used the selective RNA polymerase I (Pol I) inhibitor CX5461
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CX5461 causes nucleolar disruption through inhibition of Pol I activity
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The experimental results show that the nucleolus is necessary for the correct assembly of NPs into functional double-helical RNPs
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Further experimental analysis demonstrated that the formation of functional double-helical RNPs of influenza A virus depends on the nucleolar migration of NPs
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The findings underscore the importance of the nucleolus in the influenza virus life cycle
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It is necessary to further study the intranucleolar host factors that lead to RNP formation to understand the detailed mechanism of RNP formation, which will facilitate the development of novel antiviral drugs against influenza virus
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