Influenza virus ribonucleoprotein complex formation occurs in the nucleolus

TLDR: In this article, the subnuclear site of double-helical ribonucleoprotein complex (vRNP) formation in influenza virus was investigated and it was found that all vRNP components were colocalized in the nucleolus of virus-infected cells at early stage of infection.

Abstract: Influenza 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.

Figures

Figure 3 | Nucleolar disruption induced by an RNA polymerase I inhibitor impairs viral replication, transcription, and helical vRNP formation. a, b, f, A549 cells were pretreated with CX5461, 10 µg/mL actinomycin D (Act D), or 1% DMSO (Vehicle) for 2 h, followed by wild-type virus infection (MOI=5) for 5 h. a, Selectivity of the RNA polymerase inhibitors on Pol I and II activities. Total RNA was extracted and analysed by RT-qPCR. The expression levels were compared with that of vehicletreated cells using one-way ANOVA with Dunnett’s test; ***P<0.001. Data are presented as mean ± S.D. of three independent experiments. b, CX5461-induced nucleolar disruption and its effect on NP expression. Scale bars, 20 µm. Representative images from three independent experiments. c, Immunoprecipitation of vRNPs from the PB2-FLAG virus-infected A549 cells (MOI=5), followed by 10 µM CX5461 or

Figure 4b, and Supplementary movie 2). In contrast, NPNoLSmut were barely assembled 161 into double-helical structures and the resultant vRNPs showed pleomorphic morphology 162 with a height of ≤5 nm, where string-like structures, probably naked RNAs based on 163 their structure, were exposed (Figure 2c, 2d, 2e, and Supplementary movie 3). 164 Importantly, NoLS-NPNoLSmut was also assembled into double-helical vRNPs (~65% of 165 the vRNPs) (Figure 2c, 2d, 2e, and Supplementary movie 4). Immunoelectron 166 microscopy confirmed that both double-helical vRNPs and the pleomorphic aggregates 167 comprised NP and viral polymerase (Extended Data Figure 4a), indicating that the 168 pleomorphic aggregates are abortive vRNPs. Taken together, these data demonstrate 169 that the nucleolar NP localization is critical for functional double-helical vRNP 170 formation. 171

Full text

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Influenza virus ribonucleoprotein complex formation occurs
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in the nucleolus
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Sho Miyamoto
1#
, Masahiro Nakano
1,2,3
, Takeshi Morikawa
1
, Ai Hirabayashi
1,2,3
, Ryoma
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Tamura
1,2
, Yoko Fujita
1,2,3
, Nanami Hirose
1,2,3
, Yukiko Muramoto
1,2,3
, Takeshi Noda
1,2,3*
.
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Laboratory of Ultrastructural Virology, Institute for Frontier Life and Medical
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Sciences, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507,
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Japan
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2
Laboratory of Ultrastructural Virology, Graduate School of Biostudies, Kyoto
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University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
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3
CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama
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332-0012, Japan
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#
present address: Department of Pathology, National Institute of Infectious Diseases,
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Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan
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*correspondence to: t-noda@infront.kyoto-u.ac.jp
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preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted February 24, 2021. ; https://doi.org/10.1101/2021.02.24.432647doi: bioRxiv preprint
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted February 24, 2021. ; https://doi.org/10.1101/2021.02.24.432647doi: bioRxiv preprint
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted February 24, 2021. ; https://doi.org/10.1101/2021.02.24.432647doi: bioRxiv preprint
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted February 24, 2021. ; https://doi.org/10.1101/2021.02.24.432647doi: bioRxiv preprint
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted February 24, 2021. ; https://doi.org/10.1101/2021.02.24.432647doi: bioRxiv preprint
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted February 24, 2021. ; https://doi.org/10.1101/2021.02.24.432647doi: bioRxiv preprint
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted February 24, 2021. ; https://doi.org/10.1101/2021.02.24.432647doi: bioRxiv preprint
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted February 24, 2021. ; https://doi.org/10.1101/2021.02.24.432647doi: bioRxiv preprint
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted February 24, 2021. ; https://doi.org/10.1101/2021.02.24.432647doi: bioRxiv preprint
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted February 24, 2021. ; https://doi.org/10.1101/2021.02.24.432647doi: bioRxiv preprint

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preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted February 24, 2021. ; https://doi.org/10.1101/2021.02.24.432647doi: bioRxiv preprint

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Abstract
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Influenza A virus double-helical ribonucleoprotein complex (vRNP) performs
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transcription and replication of viral genomic RNA (vRNA). Unlike most RNA
20
viruses, vRNP formation accompanied by vRNA replication is carried out in the
21
nucleus of virus-infected cell. However, the precise subnuclear site remains
22
unknown. Here, we report the subnuclear site of vRNP formation in influenza
23
virus. We found that all vRNP components were colocalized in the nucleolus of
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virus-infected cells at early stage of infection. Mutational analysis showed that
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nucleolar localization of viral nucleoprotein, a major vRNP component, is critical
26
for functional double-helical vRNP formation. Furthermore, nucleolar disruption
27
of virus-infected cells inhibited vRNP component assembly into double-helical
28
vRNPs, resulting in decreased vRNA transcription and replication. Collectively,
29
our findings demonstrate that the vRNA replication-coupled vRNP formation
30
occurs in the nucleolus, demonstrating the importance of the nucleolus for
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influenza virus life cycle.
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preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted February 24, 2021. ; https://doi.org/10.1101/2021.02.24.432647doi: bioRxiv preprint

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Main
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Influenza A virus, belonging to the Orthomyxoviridae family, possesses
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eight-segmented, single-stranded, negative-sense RNA as its genome. Each viral
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genomic RNA (vRNA) segment exists as a ribonucleoprotein complex (vRNP)
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associated with multiple nucleoproteins (NPs) and a heterotrimeric RNA-dependent
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RNA polymerase complex composed of PB2, PB1, and PA subunits
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. The vRNPs,
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which are flexible double-stranded helices (width, ~10 nm; length, 30–120 nm)
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, are
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responsible for transcription and replication of the vRNAs. On transcription, vRNA is
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transcribed into 5′-capped and 3′-polyadenylated mRNA by the polymerase complex in
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a primer-dependent manner. During genome replication, the vRNA is copied into
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complementary RNA (cRNA) replicative intermediate by cis-acting viral polymerase
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complex, and the cRNA acts as a template for generating more vRNAs, with
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involvement of a trans-activating/trans-acting viral polymerase complex
3,4
. These
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replication processes are concomitant with ribonucleoprotein complex assembly; the 5′
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terminals of the nascent vRNA and cRNA are associated with a newly synthesized viral
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polymerase complex that is sequentially coated with multiple NPs and assembled into
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preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted February 24, 2021. ; https://doi.org/10.1101/2021.02.24.432647doi: bioRxiv preprint

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double-helical vRNPs and cRNPs, respectively
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.
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Unlike most RNA viruses, influenza A virus transcribes and replicates its
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genome in the nucleus of virus-infected cells
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. Accordingly, influenza A virus
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transcription, replication, and vRNP formation heavily rely on host nuclear machineries.
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Upon initiation of vRNA transcription, viral polymerase complex in the vRNP binds to
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carboxy-terminal domain of host RNA polymerase II (Pol II)
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. Then, the PB2 subunit
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binds to 5′-cap structure of host pre-mRNAs or small nuclear/nucleolar RNAs
8,9
, and
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the PA subunit cleaves and snatches the 5′-capped fragment for use as a primer
10-12
. The
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requirement of Pol II for initiation of viral mRNA synthesis indicates that the genome
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transcription takes place in the nucleoplasm, near host Pol II localization. Genome
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replication and double-helical vRNP formation reportedly involves several intranuclear
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host factors, such as minichromosome maintenance helicase complex, UAP56, Tat-SF1,
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and ANP32
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. Additionally, recent studies have demonstrated the importance of the
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intranuclear proteins fragile X mental retardation protein (FMRP), protein kinase C, and
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LYAR in the replication-coupled vRNP assembly
14-16
. However, since these host
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proteins are localized in different intranuclear domains, subnuclear site of vRNA
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preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted February 24, 2021. ; https://doi.org/10.1101/2021.02.24.432647doi: bioRxiv preprint