scholarly journals Migration of Influenza Virus Nucleoprotein into the Nucleolus Is Essential for Ribonucleoprotein Complex Formation

mBio ◽  
2022 ◽  
Author(s):  
Sho Miyamoto ◽  
Masahiro Nakano ◽  
Takeshi Morikawa ◽  
Ai Hirabayashi ◽  
Ryoma Tamura ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Life Cycle ◽  
Viral Genome ◽  
Influenza A ◽  
Genome Replication ◽  
Ribonucleoprotein Complex ◽  
Virus Life Cycle ◽  
Viral Genome Replication ◽  
Infected Cells ◽  
Nuclear Domains

Influenza A virus ribonucleoprotein complex (RNP) is responsible for viral genome replication, thus playing essential roles in the virus life cycle. RNP formation occurs in the nuclei of infected cells; however, little is known about the nuclear domains involved in this process.

scholarly journals DDX3 Interacts with Influenza A Virus NS1 and NP Proteins and Exerts Antiviral Function through Regulation of Stress Granule Formation

2016 ◽  
Vol 90 (7) ◽  
pp. 3661-3675 ◽  
Author(s):  
Sathya N. Thulasi Raman ◽  
Guanqun Liu ◽  
Hyun Mi Pyo ◽  
Ya Cheng Cui ◽  
Fang Xu ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Life Cycle ◽  
Virus Infection ◽  
Influenza A ◽  
Influenza Virus Infection ◽  
Ns1 Protein ◽  
Antiviral Protein ◽  
Virus Life Cycle ◽  
Interaction Partners ◽  
Infected Cells

ABSTRACTDDX3 belongs to the DEAD box RNA helicase family and is a multifunctional protein affecting the life cycle of a variety of viruses. However, its role in influenza virus infection is unknown. In this study, we explored the potential role of DDX3 in influenza virus life cycle and discovered that DDX3 is an antiviral protein. Since many host proteins affect virus life cycle by interacting with certain components of the viral machinery, we first verified whether DDX3 has any viral interaction partners. Immunoprecipitation studies revealed NS1 and NP as direct interaction partners of DDX3. Stress granules (SGs) are known to be antiviral and do form in influenza virus-infected cells expressing defective NS1 protein. Additionally, a recent study showed that DDX3 is an important SG-nucleating factor. We thus explored whether DDX3 plays a role in influenza virus infection through regulation of SGs. Our results showed that SGs were formed in infected cells upon infection with a mutant influenza virus lacking functional NS1 (del NS1) protein, and DDX3 colocalized with NP in SGs. We further determined that the DDX3 helicase domain did not interact with NS1 and NP; however, it was essential for DDX3 localization in virus-induced SGs. Knockdown of DDX3 resulted in impaired SG formation and led to increased virus titers. Taken together, our results identified DDX3 as an antiviral protein with a role in virus-induced SG formation.IMPORTANCEDDX3 is a multifunctional RNA helicase and has been reported to be involved in regulating various virus life cycles. However, its function during influenza A virus infection remains unknown. In this study, we demonstrated that DDX3 is capable of interacting with influenza virus NS1 and NP proteins; DDX3 and NP colocalize in the del NS1 virus-induced SGs. Furthermore, knockdown of DDX3 impaired SG formation and led to a decreased virus titer. Thus, we provided evidence that DDX3 is an antiviral protein during influenza virus infection and its antiviral activity is through regulation of SG formation. Our findings provide knowledge about the function of DDX3 in the influenza virus life cycle and information for future work on manipulating the SG pathway and its components to fight influenza virus infection.

scholarly journals Influenza virus ribonucleoprotein complex formation occurs in the nucleolus

2021 ◽  
Author(s):  
Sho Miyamoto ◽  
Masahiro Nakano ◽  
Takeshi Morikawa ◽  
Ai Hirabayashi ◽  
Ryoma Tamura ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Mutational Analysis ◽  
Early Stage ◽  
Ribonucleoprotein Complex ◽  
Virus Life Cycle ◽  
Infected Cells ◽  
Component Assembly ◽  
Viral Genomic Rna ◽  
Transcription And Replication

AbstractInfluenza A virus double-helical ribonucleoprotein complex (vRNP) performs transcription and replication of viral genomic RNA (vRNA). Unlike most RNA viruses, vRNP formation accompanied by vRNA replication is carried out in the nucleus of virus-infected cell. However, the precise subnuclear site remains unknown. Here, we report the subnuclear site of vRNP formation in influenza virus. We found that all vRNP components were colocalized in the nucleolus of virus-infected cells at early stage of infection. Mutational analysis showed that nucleolar localization of viral nucleoprotein, a major vRNP component, is critical for functional double-helical vRNP formation. Furthermore, nucleolar disruption of virus-infected cells inhibited vRNP component assembly into double-helical vRNPs, resulting in decreased vRNA transcription and replication. Collectively, our findings demonstrate that the vRNA replication-coupled vRNP formation occurs in the nucleolus, demonstrating the importance of the nucleolus for influenza virus life cycle.

scholarly journals Mutational Analysis of Conserved Amino Acids in the Influenza A Virus Nucleoprotein

2009 ◽  
Vol 83 (9) ◽  
pp. 4153-4162 ◽  
Author(s):  
Zejun Li ◽  
Tokiko Watanabe ◽  
Masato Hatta ◽  
Shinji Watanabe ◽  
Asuka Nanbo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Life Cycle ◽  
Viral Replication ◽  
Viral Genome ◽  
Influenza A ◽  
Mutational Analysis ◽  
Viral Rna ◽  
Genome Replication ◽  
Viral Genome Replication ◽  
Conserved Amino Acids

ABSTRACT The nucleoprotein (NP), which has multiple functions during the virus life cycle, possesses regions that are highly conserved among influenza A, B, and C viruses. To better understand the roles of highly conserved NP amino acids in viral replication, we conducted a comprehensive mutational analysis. Using reverse genetics, we attempted to generate 74 viruses possessing mutations at conserved amino acids of NP. Of these, 48 mutant viruses were successfully rescued; 26 mutants were not viable, suggesting a critical role of the respective NP amino acids in viral replication. To identify the step(s) in the viral life cycle that is impaired by these NP mutations, we examined viral-genome replication/transcription, NP localization, and incorporation of viral-RNA segments into progeny virions. We identified 15 amino acid substitutions in NP that inhibited viral-genome replication and/or transcription, resulting in significant growth defects of viruses possessing these substitutions. We also found several NP mutations that affected the efficient incorporation of multiple viral-RNA (vRNA) segments into progeny virions even though a single vRNA segment was incorporated efficiently. The respective conserved amino acids in NP may thus be critical for the assembly and/or incorporation of sets of eight vRNA segments.

scholarly journals Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes

2021 ◽  
Vol 12 ◽  
Author(s):  
Lisett Liblekas ◽  
Alla Piirsoo ◽  
Annika Laanemets ◽  
Eva-Maria Tombak ◽  
Airiin Laaneväli ◽  
...  
Keyword(s):  
Life Cycle ◽  
Viral Genome ◽  
Maintenance Phase ◽  
Viral Life Cycle ◽  
Human Papillomaviruses ◽  
Genome Replication ◽  
Viral Genomes ◽  
Viral Genome Replication ◽  
Infected Cells ◽  
Cell Genome

The life-cycle of human papillomaviruses (HPVs) includes three distinct phases of the viral genome replication. First, the viral genome is amplified in the infected cells, and this amplification is often accompanied by the oligomerization of the viral genomes. Second stage includes the replication of viral genomes in concert with the host cell genome. The viral genome is further amplified during the third stage of the viral-life cycle, which takes place only in the differentiated keratinocytes. We have previously shown that the HPV18 genomes utilize at least two distinct replication mechanisms during the initial amplification. One of these mechanisms is a well-described bidirectional replication via theta type of replication intermediates. The nature of another replication mechanism utilized by HPV18 involves most likely recombination-dependent replication. In this paper, we show that the usage of different replication mechanisms is a property shared also by other HPV types, namely HPV11 and HPV5. We further show that the emergence of the recombination dependent replication coincides with the oligomerization of the viral genomes and is dependent on the replicative DNA polymerases. We also show that the oligomeric genomes of HPV18 replicate almost exclusively using recombination dependent mechanism, whereas monomeric HPV31 genomes replicate bi-directionally during the maintenance phase of the viral life-cycle.

scholarly journals Dimerisation of the influenza virus RNA polymerase during viral genome replication

2019 ◽  
Vol 1 (1A) ◽  
Author(s):  
Alexander Walker ◽  
Haitian Fan ◽  
Loic Carrique ◽  
Jeremy Keown ◽  
David Bauer ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Viral Genome ◽  
Genome Replication ◽  
Virus Rna ◽  
Viral Genome Replication

scholarly journals Regulation of Influenza A Virus Nucleoprotein Oligomerization by Phosphorylation

2014 ◽  
Vol 89 (2) ◽  
pp. 1452-1455 ◽  
Author(s):  
Lauren Turrell ◽  
Edward C. Hutchinson ◽  
Frank T. Vreede ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Serine Residue ◽  
Reversible Phosphorylation ◽  
Viral Growth ◽  
Ribonucleoprotein Complex ◽  
Infected Cells ◽  
Neighboring Molecule

In the influenza virus ribonucleoprotein complex, the oligomerization of the nucleoprotein is mediated by an interaction between the tail-loop of one molecule and the groove of the neighboring molecule. In this study, we show that phosphorylation of a serine residue (S165) within the groove of influenza A virus nucleoprotein inhibits oligomerization and, consequently, ribonucleoprotein activity and viral growth. We propose that nucleoprotein oligomerization in infected cells is regulated by reversible phosphorylation.

scholarly journals Phosphorylation Controls the Nuclear-Cytoplasmic Shuttling of Influenza A Virus Nucleoprotein

2015 ◽  
Vol 89 (11) ◽  
pp. 5822-5834 ◽  
Author(s):  
Weinan Zheng ◽  
Jing Li ◽  
Shanshan Wang ◽  
Shuaishuai Cao ◽  
Jingwen Jiang ◽  
...  
Keyword(s):  
Life Cycle ◽  
Influenza A Virus ◽  
Influenza A ◽  
Early Stage ◽  
Phosphorylation Sites ◽  
Nuclear Retention ◽  
Virus Growth ◽  
Virus Life Cycle ◽  
Importin Α ◽  
Infected Cells

ABSTRACTThe 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.

scholarly journals SARS-CoV-2: from its discovery to genome structure, transcription, and replication

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ayslan Castro Brant ◽  
Wei Tian ◽  
Vladimir Majerciak ◽  
Wei Yang ◽  
Zhi-Ming Zheng
Keyword(s):  
Viral Genome ◽  
Genome Structure ◽  
Membrane Vesicles ◽  
Regulatory Sequence ◽  
Atypical Pneumonia ◽  
Genome Replication ◽  
Long Distance ◽  
Viral Genome Replication ◽  
Infected Cells ◽  
Rna Interaction

AbstractSARS-CoV-2 is an extremely contagious respiratory virus causing adult atypical pneumonia COVID-19 with severe acute respiratory syndrome (SARS). SARS-CoV-2 has a single-stranded, positive-sense RNA (+RNA) genome of ~ 29.9 kb and exhibits significant genetic shift from different isolates. After entering the susceptible cells expressing both ACE2 and TMPRSS2, the SARS-CoV-2 genome directly functions as an mRNA to translate two polyproteins from the ORF1a and ORF1b region, which are cleaved by two viral proteases into sixteen non-structural proteins (nsp1-16) to initiate viral genome replication and transcription. The SARS-CoV-2 genome also encodes four structural (S, E, M and N) and up to six accessory (3a, 6, 7a, 7b, 8, and 9b) proteins, but their translation requires newly synthesized individual subgenomic RNAs (sgRNA) in the infected cells. Synthesis of the full-length viral genomic RNA (gRNA) and sgRNAs are conducted inside double-membrane vesicles (DMVs) by the viral replication and transcription complex (RTC), which comprises nsp7, nsp8, nsp9, nsp12, nsp13 and a short RNA primer. To produce sgRNAs, RTC starts RNA synthesis from the highly structured gRNA 3' end and switches template at various transcription regulatory sequence (TRSB) sites along the gRNA body probably mediated by a long-distance RNA–RNA interaction. The TRS motif in the gRNA 5' leader (TRSL) is responsible for the RNA–RNA interaction with the TRSB upstream of each ORF and skipping of the viral genome in between them to produce individual sgRNAs. Abundance of individual sgRNAs and viral gRNA synthesized in the infected cells depend on the location and read-through efficiency of each TRSB. Although more studies are needed, the unprecedented COVID-19 pandemic has taught the world a painful lesson that is to invest and proactively prepare future emergence of other types of coronaviruses and any other possible biological horrors.

scholarly journals Structures of influenza A virus RNA polymerase offer insight into viral genome replication

Nature ◽  
2019 ◽  
Vol 573 (7773) ◽  
pp. 287-290 ◽  
Author(s):  
Haitian Fan ◽  
Alexander P. Walker ◽  
Loïc Carrique ◽  
Jeremy R. Keown ◽  
Itziar Serna Martin ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Influenza A Virus ◽  
Viral Genome ◽  
Influenza A ◽  
Genome Replication ◽  
Virus Rna ◽  
Viral Genome Replication ◽  
Insight Into

scholarly journals Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

2020 ◽  
Vol 94 (20) ◽  
Author(s):  
Alla Piirsoo ◽  
Martin Kala ◽  
Eve Sankovski ◽  
Mart Ustav ◽  
Marko Piirsoo
Keyword(s):  
Life Cycle ◽  
Viral Genome ◽  
Copy Number ◽  
Genome Replication ◽  
Dependent Manner ◽  
Infection Cycle ◽  
Viral Genomes ◽  
E1 Protein ◽  
Viral Genome Replication

ABSTRACT The life cycle of human papillomaviruses (HPVs) comprises three distinct phases of DNA replication: initial amplification, maintenance of the genome copy number at a constant level, and vegetative amplification. The viral helicase E1 is one of the factors required for the initiation of HPV genome replication. However, the functions of the E1 protein during other phases of the viral life cycle are largely uncharacterized. Here, we studied the role of the HPV18 E1 helicase in three phases of viral genome replication by downregulating E1 expression using RNA interference or inducing degradation of the E1 protein via inhibition of casein kinase 2α expression or catalytic activity. We generated a novel modified HPV18 genome expressing Nanoluc and tagged E1 and E2 proteins and created several stable HPV18-positive cell lines. We showed that, in contrast to initial amplification of the HPV18 genome, other phases of viral genome replication involve also an E1-independent mechanism. We characterize two distinct populations of HPV18 replicons existing during the maintenance and vegetative amplification phases. We show that a subset of these replicons, including viral genome monomers, replicate in an E1-dependent manner, while some oligomeric forms of the HPV18 genome replicate independently of E1 function. IMPORTANCE Human papillomavirus (HPV) infections pose serious medical problem. To date, there are no HPV-specific antivirals available due to poor understanding of the molecular mechanisms of virus infection cycle. The infection cycle of HPV involves initial amplification of the viral genomes and maintenance of the viral genomes with a constant copy number, followed by another round of viral genome amplification and new viral particle formation. The viral protein E1 is critical for the initial amplification of the viral genome. However, E1 involvement in other phases of the viral life cycle has remained controversial. In the present study, we show that at least two different replication modes of the HPV18 genome are undertaken simultaneously during the maintenance and vegetative amplification phases, i.e., replication of the majority of the HPV18 genome proceeds under the control of the host cell replication machinery without E1 function, whereas a minority of the genome replicates in an E1-dependent manner.

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