Influenza virus ribonucleoprotein complex formation occurs in the nucleolus

TL;DR: 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.

Summary

Main

• Accordingly, influenza A virus transcription, replication, and vRNP formation heavily rely on host nuclear machineries.
• Upon initiation of vRNA transcription, viral polymerase complex in the vRNP binds to carboxy-terminal domain of host RNA polymerase II (Pol II) 7 .
• Since these host proteins are localized in different intranuclear domains, subnuclear site of vRNA replication and vRNP formation remains unidentified.

Result Nucleolar localization of vRNP components

• Given that the nucleolus is a site of vRNP formation, de novo synthesized vRNP components (NP, PB2, PB1, PA, and vRNA) should simultaneously exist in the nucleolus of virus-infected cell.
• To examine their localization in virus-infected cells, Madin-Darby Canine Kidney (MDCK) cells were infected with influenza A virus and fixed over time.
• Immunostaining with an anti-NP antibody showed that NP was co-localized with nucleolin/C23, a nucleolar marker, 5-7 h post-infections (hpi) .
• In contrast, although PB2 subunits were localized in the GC regions, they were mainly localized in periphery, but not central region, of the nucleolus .
• These results suggest that the vRNP components are assembled into vRNP in the GC region of the nucleolar periphery.

Importance of nucleolar NP localization for functional vRNP formation

• To investigate the importance of NP nucleolar localization for the vRNP formation, the authors constructed mutant vRNPs using two NoLS-mutant NPs: NP NoLSmut with alanine substitutions in the NoLS localizes only in the nucleoplasm and a reverse mutant NoLS-NP NoLSmut , with an intact NoLS fused to the amino-terminus of NP NoLSmut that causes its nucleolar localization 18 .
• Then, the cells were subjected to immunoprecipitation using anti-FLAG antibody, and the precipitates were assessed by western blotting and RT-PCR .
• Furthermore, full-length HA vRNA was barely detected in the precipitate, and the immunoprecipitated vRNPs did not produce HA mRNA , indicating that the NP NoLSmut was not properly assembled into functional vRNPs, although heterotrimeric viral polymerase subunit was assembled.
• Intriguingly, NoLS-NP NoLSmut , PB1, and PA were adequately coprecipitated with PB2, from which full-length HA vRNA was detected .
• Taken together, these results indicate that nucleolar localization of NP is indispensable for functional vRNP formation.

Impact of nucleolar disruption on functional vRNP formation

• Considering the necessity of nucleolar NP localization for proper vRNP formation, nucleolar structure disruption would heavily impact the vRNP component assembly.
• Actinomycin D, which inhibits both Pol I and Pol II activities, was used as control.
• CX5461 treatment modestly decreased the amount of immunoprecipitated NP as well as PB1 and PA subunits in these cells, although viral protein expression levels were comparable, or marginally lower, compared to those in control cells , suggesting that nucleolar disruption impacted the vRNP component assembly.
• Thus, these results demonstrate that the nucleolus is required for proper assembly of the vRNP components into functional double-helical vRNPs.

Discussion

• The authors showed that the nucleolus is the site for formation of functional vRNPs with double-helical structure.
• Inhibition of nucleolar NP localization and nucleolar structure disruption affected vRNP component assembly, resulting in defective vRNP formation.
• Thus, interaction between viral polymerase and NP is likely involved in its nucleolar import and subsequent vRNP formation.
• Khatchikian et al. reported that host 28S rRNA-derived 54 nucleotides are inserted into the HA vRNA during viral replication via genetic recombination 35 .
• Moreover, the authors detected not only vRNA but also anti-genomic RNA in the nucleolus of virus-infected cells, supporting that the nucleolus is the site of vRNA replication and vRNP formation.

Plasmid construction

• PCAGGS/NP NoLSmut and pCAGGS/NoLS-NP NoLSmut were constructed using inverse PCR 38 with sequences similar to those previously reported (pCAGGS/NP-NLS2mut and pCAGGS/NLS2-NP-NLS2mut, respectively) 18 .
• To generate pCAGGS/PB2-FLAG, the PB2 ORF and FLAG were linked with a linker (AAA).
• PPol I/PB2-FLAG was constructed by inserting the PB2-FLAG ORF with stop codon into a truncated pPol I/PB2 plasmid with 3′ non-coding region and additional 143 nucleotides of 5′ terminal coding and non-coding regions 39 .

Immunofluorescence

• Cells were plated in 8-well chamber slides (Matsunami, Japan) coated with rat collagen I (Corning, NY USA).
• Infected or transfected cells were fixed in 4% paraformaldehyde (PFA) in phosphate buffer (PB) (Nacalai Tesque, Japan) for 10 min and then permeabilized with 0.5% Triton-X in PBS for 5 min.
• The cells were blocked with Blocking One (Nacalai Tesque) for 30 min followed by incubation with primary antibodies overnight at 4 °C and secondary antibodies for 1 h at room temperature.
• For nuclei and rRNA staining, cells were treated with Hoechst 33342 (Thermo Fisher Scientific) and Nucleolus Bright Red (Dojindo, Japan), respectively, for 10 min.
• Section images were recorded using DeltaVision Elite (GE healthcare) with a 60× oil objective, deconvolved and projected using 'Quick Projection' tool by softWoRx (GE Healthcare).

Protease treatment

• As the optimal condition for protease treatment depends on the protease type, lot, and cell strain 20 , the authors recommend verifying the protease concentration and incubation time.
• After permeabilization, the cells were washed twice in cold-PBS on ice and placed in cold 5 µg/mL TPCK-Trypsin in PBS.
• Thereafter, the cells were washed in PBS and blocked as described above.

Fluorescence in situ hybridisation (FISH)

• Briefly, probes were transcribed in vitro using digoxigenin (DIG)-11-UTP and RiboMAX Large Scale RNA Production System-T7 (Promega, WI USA).
• The template of positive-and negative-sense PB2 genome segment (~300 bp) was PCR amplified using pPol I/PB2.
• The infected cells were fixed with 4% PFA in PB for 10 min and permeabilized with 0.5% Triton X-100 for 5 min at room temperature.
• Cells were then blocked with prehybridization buffer (50% formamide [Fujifilm], 2× SSC, 5× Denhardt's solution [Fujifilm], 20 μg/mL salmon sperm DNA [BioDynamics Laboratory, Japan]) for 1 h at room temperature and then incubated with 200 ng/mL of DIG-labelled RNA probe diluted in prehybridization buffer overnight at 60 °C on a shaker.

Western blotting

• Briefly, cells or samples described below were dissolved with 2× Tris-Glycine SDS Sample Buffer (Thermo Fisher Scientific), boiled for 5 min in absence of a reducing agent, and subjected to SDS-PAGE.
• Proteins were electroblotted onto Immobilon-P transfer membranes .
• The membranes were blocked with Blocking One for 30 min at room temperature and then incubated with primary antibodies overnight at 4 °C.
• After incubation with HRP-conjugated secondary antibodies for 1 h at room temperature, the blots were developed using Chemi-Lumi One Super (Nacalai Tesque).

Cell viability

• Cell viability was assessed with CellTiter-Glo Luminescent Cell Viability Assay according to the manufacturer's instructions.
• Briefly, CellTiter-Glo reagent (equal in volume to the culture medium) was added to A549 cells.
• Plates were shaken on a plate shaker for 2 min to induce cell lysis, incubated at room temperature for 10 min, and subjected to luminescence measurement.

vRNP purification

• To prepare virion-derived vRNPs, MDCK cells were infected with the virus and incubated at 37 °C for two days.
• Virions in the supernatants were purified by ultracentrifugation through a 30% (w/w) sucrose cushion.
• Each fraction was electrophoresed with SDS-polyacrylamide gel and immunoblotted with an anti-NP antibody .
• NP-enriched fractions 7 and 8 were used for vRNP observations.

In vitro transcription of vRNPs

• RNA was purified using RNeasy Mini kit, mixed with equal volume of 2× RNA Loading Dye (New England Biolabs), heated at 90 °C for 2 min, and immediately chilled on ice.
• For preparation of vRNA markers, all eight vRNA segments of the influenza virus were transcribed using 0.25 μCi/μL [α-32 P] UTP and RiboMAX Large Scale RNA Production System-T7 as described above.
• The transcribed RNAs were purified and mixed before electrophoresis.

High-speed atomic force microscopy (HS-AFM)

• The samples were prepared in a microcentrifuge tube, dropped onto freshly cleaved mica without any surface modification, and incubated for 1-5 min at room temperature (~24°C).
• Images were taken at a 2 images/sec rate using cantilevers (BL-AC10DS, Olympus, Japan) with a 0.1 N/m spring constant and a resonance frequency in water of 0.6 MHz.
• To increase the resolution, the electron-beam deposited tips were fabricated using phenol or ferrocene powder 45 .
• A low-pass filter and a flattening filter were applied to individual images to remove spike noise and flatten the xy-plane, respectively.
• Rod-like and helical structures with a uniform height of 9.0 ± 1.5 nm were defined as helical vRNPs.

Immuno-electron microscopy

• Purified vRNPs were adsorbed onto carbon-coated nickel grids and fixed with 2% PFA for 5 min.
• After washing, the grids were incubated with 6-nm gold conjugated anti-mouse or anti-rabbit antibodies for 1 h at room temperature.
• The images were acquired with an HT7700 (Hitachi High-Tech Corporation, Japan).
• For thin-section preparations, infected and mock-infected MDCK cells were fixed with 1.5% PFA and 0.025% glutaraldehyde in 0.1 M PB for 1 h.
• Ultrathin sections (80 nm) were cut with Leica EM UC7 (Leica, Germany) and collected on a nickel grid.

RT-PCR

• Total RNAs were extracted using an RNeasy Mini Kit with on-column DNase digestion .
• Ten nanograms of the extracted RNA samples were reverse-transcribed using a Uni-12 primer (5′-AGCRAAAGCAGG-3′) and Superscript III reverse transcriptase (Thermo Fisher Scientific).
• Ten-fold diluted cDNAs were PCR amplified using KOD FX (Toyobo, Japan) and 0.25 μM HA segment-specific primers according to manufacturer's protocol.
• The PCR products were electrophoresed on 1.0% agarose gels containing 0.01% (w/v) ethidium bromide in 0.5× TBE.

RT-qPCR

• Two-hundred nanograms of total RNAs were reverse-transcribed using Random primer 6 (New England Biolabs) and Superscript III reverse transcriptase.
• The relative expression levels of target genes were normalized to that of GAPDH.
• A primer set for pre-rRNA, described previously 47 , was used.

Strand-specific RT-qPCR

• Briefly, total RNA was extracted from cells using an RNeasy Mini Kit.
• CDNAs complementary to the three types of HA genome were synthesized with tagged primers at the 5′ end.

Subcellular fractionation

• Total protein concentrations were measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and adjusted to approximately 0.5 µg/mL.
• The samples (~0.5 µg) were subjected to western blotting.

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

1
Influenza virus ribonucleoprotein complex formation occurs
1
in the nucleolus
2
3
Sho Miyamoto
1#
, Masahiro Nakano
1,2,3
, Takeshi Morikawa
1
, Ai Hirabayashi
1,2,3
, Ryoma
4
Tamura
1,2
, Yoko Fujita
1,2,3
, Nanami Hirose
1,2,3
, Yukiko Muramoto
1,2,3
, Takeshi Noda
1,2,3*
.
5
6
1
Laboratory of Ultrastructural Virology, Institute for Frontier Life and Medical
7
Sciences, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507,
8
Japan
9
2
Laboratory of Ultrastructural Virology, Graduate School of Biostudies, Kyoto
10
University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
11
3
CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama
12
332-0012, Japan
13
#
present address: Department of Pathology, National Institute of Infectious Diseases,
14
Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan
15
*correspondence to: t-noda@infront.kyoto-u.ac.jp
16
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

2
17
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

3
Abstract
18
Influenza A virus double-helical ribonucleoprotein complex (vRNP) performs
19
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
24
virus-infected cells at early stage of infection. Mutational analysis showed that
25
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
31
influenza virus life cycle.
32
33
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

4
Main
34
Influenza A virus, belonging to the Orthomyxoviridae family, possesses
35
eight-segmented, single-stranded, negative-sense RNA as its genome. Each viral
36
genomic RNA (vRNA) segment exists as a ribonucleoprotein complex (vRNP)
37
associated with multiple nucleoproteins (NPs) and a heterotrimeric RNA-dependent
38
RNA polymerase complex composed of PB2, PB1, and PA subunits
1
. The vRNPs,
39
which are flexible double-stranded helices (width, ~10 nm; length, 30–120 nm)
2
, are
40
responsible for transcription and replication of the vRNAs. On transcription, vRNA is
41
transcribed into 5′-capped and 3′-polyadenylated mRNA by the polymerase complex in
42
a primer-dependent manner. During genome replication, the vRNA is copied into
43
complementary RNA (cRNA) replicative intermediate by cis-acting viral polymerase
44
complex, and the cRNA acts as a template for generating more vRNAs, with
45
involvement of a trans-activating/trans-acting viral polymerase complex
3,4
. These
46
replication processes are concomitant with ribonucleoprotein complex assembly; the 5′
47
terminals of the nascent vRNA and cRNA are associated with a newly synthesized viral
48
polymerase complex that is sequentially coated with multiple NPs and assembled into
49
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

5
double-helical vRNPs and cRNPs, respectively
5
.
50
Unlike most RNA viruses, influenza A virus transcribes and replicates its
51
genome in the nucleus of virus-infected cells
6
. Accordingly, influenza A virus
52
transcription, replication, and vRNP formation heavily rely on host nuclear machineries.
53
Upon initiation of vRNA transcription, viral polymerase complex in the vRNP binds to
54
carboxy-terminal domain of host RNA polymerase II (Pol II)
7
. Then, the PB2 subunit
55
binds to 5′-cap structure of host pre-mRNAs or small nuclear/nucleolar RNAs
8,9
, and
56
the PA subunit cleaves and snatches the 5′-capped fragment for use as a primer
10-12
. The
57
requirement of Pol II for initiation of viral mRNA synthesis indicates that the genome
58
transcription takes place in the nucleoplasm, near host Pol II localization. Genome
59
replication and double-helical vRNP formation reportedly involves several intranuclear
60
host factors, such as minichromosome maintenance helicase complex, UAP56, Tat-SF1,
61
and ANP32
13
. Additionally, recent studies have demonstrated the importance of the
62
intranuclear proteins fragile X mental retardation protein (FMRP), protein kinase C, and
63
LYAR in the replication-coupled vRNP assembly
14-16
. However, since these host
64
proteins are localized in different intranuclear domains, subnuclear site of vRNA
65
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