The Nucleolar Protein LYAR Facilitates Ribonucleoprotein Assembly of Influenza A Virus

Identification of LYAR as a putative vRNP interacting partner regulating IAV replication.

As detailed in the supplemental material using the reconstituted IAV vRNP (see Fig. S1 and S2 in the supplemental material), we identified 80 host proteins that copurified with vRNP by using affinity purification followed by mass spectrometry (Fig. S3 and Table S2); 61 of them are identified for the first time as potential vRNP-interacting partners despite their also interacting with other influenza viral proteins, and 21 are claimed to interact with all subunits of the RNP (Fig. S3 and Table S3). For further study, we focused on the nucleolar proteins due to their potential roles in vRNP function and poor knowledge of their interactions with IAV vRNP. Seventeen proteins identified were localized in the nucleolus (Fig. S4 and Table S4), and seven of them were ribosomal proteins (RPL family proteins) that were previously shown to bind viral RNP or RdRp (28, 29). NCL is the major nucleolar protein of growing eukaryotic cells with multiple functions whose roles in IAV replication are well established (26, 45, 46). Of the remaining proteins, only five of them, concerning influenza virus replication, were not determined (Table S5). Therefore, a short interfering RNA (siRNA) screen (LYAR, PPP1CA, HNRNPR, MCDRH, RPL19, and NCL) was performed to investigate their effects on IAV replication and polymerase activity, and NCL was used to serve as the positive control. Results showed that knockdown of LYAR, MCDRH, RPL19, or PPP1CA led to a significant decrease of virus titer at 24 h postinfection (hpi) compared to that of the NC control (Fig. 1A). Noticeably, a greater reduction in both virus titers (approximately 50-fold decrease) and polymerase activity (approximately 60% reduction) was observed in the si-LYAR group than in the NC controls (Fig. 1A and B). These results indicate an important role of LYAR in IAV replication. The siRNA silencing efficiency for tested genes was detected by quantitative reverse transcription-PCR (qRT-PCR), and the mRNA levels of each gene in silencing cells were reduced more than 50% compared to those in negative-control cells (Fig. 1C). Based on these results, we focused on the LYAR-vRNP interaction in our further studies.

LYAR interacts with IAV RNP subunits.

Interaction between LYAR and each individual component of the RNP was determined. Flag-LYAR and hemagglutinin (HA)-tagged PA, PB1, PB2, and NP, or HA-tagged green fluorescent protein (GFP) and HA (negative controls), were coexpressed in HEK293T cells, and a coimmunoprecipitation (Co-IP) assay was performed using an anti-HA tag monoclonal antibody. Results showed that LYAR was coprecipitated by PA, PB1, PB2, and NP but not the negative controls GFP and HA, suggesting that LYAR specifically interacts with all of the components of RNP (Fig. 2A). Since LYAR and all of the RNP components are RNA binding proteins, we hypothesized that interactions between LYAR and RNP subunits can be mediated by RNAs. To test our hypothesis, the same experiments were conducted using RNase A-treated cell lysates. The host protein PLSCR1, which is reported to interact with NP of A/WSN/33 (WSN, H1N1) in an RNA-independent manner (47), was used as a control. Results showed that PLSCR1 was coprecipitated with PR8 NP with or without RNase A treatment (Fig. 2A and B). In contrast, all of the RNP subunits failed to coprecipitate LYAR under RNase A treatment (Fig. 2B), indicating that LYAR interacts with RNP components in an RNA-dependent manner. The interaction between RNP components and endogenous LYAR was further studied by using influenza virus-infected A549 cells and coimmunoprecipitation with an anti-LYAR mouse antibody. The results revealed that PA, PB1, PB2, and NP were all coprecipitated by LYAR (Fig. 2C), demonstrating a real interaction between LYAR and RNP components during virus infection. Moreover, we found that RNase A treatment also disrupted the interaction between LYAR and RNP components in virus-infected cells (Fig. 2C), indicating that LYAR interaction with RNP components during virus infection is mediated by RNAs. To investigate the interaction between LYAR and the vRNP complex, we used a vRNP reconstitution system to construct vRNPs in which the NP was HA tagged. Previous studies claim that because NP and PA do not interact directly, their coprecipitation can only occur in the context of a vRNP (14, 48), which is also confirmed by our studies, which showed that NP did not coprecipitate PA when other vRNP subunits, including PB1, PB2, and vRNA, were absent (Fig. S6A and B). Our results showed that PA was specifically coprecipitated by HA-tagged NP, indicating that the vRNP complexes were immunoprecipitated, and LYAR was also detected in these immunoprecipitated complexes (Fig. 2D), indicating that LYAR associates with the reconstituted vRNPs. Additionally, when the lysine-rich region of LYAR (CTD, for C-terminal domain), which has RNA binding ability, was deleted, the interaction between LYAR (NTD, for N-terminal domain) and NP in the context of the vRNP was disrupted (Fig. 2D). Taken together, the results demonstrate that LYAR interacts with each RNP component and the C-terminal domain of LYAR plays a critical role in interaction with vRNP.

The colocalizations between LYAR and RNP components were further examined using immunofluorescence and confocal microscopy. In HeLa cells cotransfected with Flag-LYAR and either HA-PA, -PB1, -PB2, or -NP, LYAR localized to the nucleoli and colocalized with NPM1 (nucleolar maker) when expressed alone, and it colocalized to the nucleoli when expressed with PB1, PB2, or NP but diffused in the nucleoplasm when expressed with PA (Fig. 3A). In uninfected HeLa (Fig. 3B) and A549 cells (Fig. 3C), endogenous LYAR appeared as foci and localized in inside the region of NPM1 in the nucleoli. Interestingly, in cells infected with IAV PR8 for 6 h, some LYAR proteins were detected in nucleoplasm and cytoplasm and colocalized with NP (Fig. 3B and C), indicating that IAV infection triggers translocation of LYAR. Taken together, these data suggest that LYAR proteins have a role in IAV transcription and replication or vRNP nuclear export.

IAV infection increases the expression of LYAR.

To further explore the role of LYAR in the IAV life cycle, we next investigated the progress of expression of LYAR during the course of virus infection. A549 cells were infected with either the PR8 H1N1 or the HM H5N1 virus, and then the mRNA and protein levels of LYAR as well as the viral NP protein were analyzed. The data showed that the mRNA level of LYAR increased in both PR8 H1N1 (Fig. 4A) and HM H5N1 (Fig. 4D) virus-infected cells, which is correlated with virus replication during the infection course (Fig. 4B and E), suggesting that IAV infection upregulates the expression of LYAR at the transcriptional level. We noted that the protein levels of LYAR gradually increased until 24 h postinfection of both viruses (Fig. 4C and F) and were not consistent with the changes in mRNA levels. This may be due to protein degradation caused by cell apoptosis at late time points of virus infection. These results indicate that the expression of LYAR is increased during IAV infection, especially during the early period, implying a role of LYAR in IAV replication.

Knockdown of LYAR reduces IAV replication.

vRNPs are responsible for viral genome transcription and replication, and the LYAR interaction with each vRNP subunit as well as it expression level increasing with IAV replication suggest that LYAR plays a critical role in IAV replication. To determine its possible role in IAV replication, three specific siRNAs (si-1, si-2, and si-3) targeting LYAR were used to knock down LYAR in A549 cells. The silencing efficiency of LYAR-targeting siRNAs detected by Western blotting showed that LYAR protein levels in si-2- and si-3-treated A549 cells were significantly decreased compared to those of the negative control (Fig. 5A); therefore, these two siRNAs were used in our further experiments. Since LYAR can regulate cell growth, the siRNA-mediated knockdown of LYAR may influence cell viability, resulting in effects on virus replication. We determined the effect of LYAR silencing on cell viability in A549 cells and showed that the cell viability of si-LYAR-treated cells and mock-treated cells were comparable at 24, 36, and 48 h posttransfection (Fig. 5B), indicating that LYAR silencing does not have effects on A549 cell viability. Growth kinetics of both PR8 H1N1 and HM H5N1 viruses showed that virus titers of both viruses were significantly reduced in LYAR-silenced cells, in contrast to that in control cells (Fig. 5C and D), indicating that knockdown of LYAR reduces IAV replication. In addition, the LYAR-KO A549 cells were produced and confirmed by Western blotting (Fig. 5E) to determine the effect of LYAR on IAV replication. Results showed that virus titers of both PR8 H1N1 and HM H5N1 viruses were dramatically decreased in the LYAR-KO A549 cells compared with those in the WT A549 cells (Fig. 5F and G), indicating that LYAR does play a critical role in IAV replication.

To examine whether LYAR specifically regulates IAV replication, we explored the effect of LYAR on two RNA viruses with a cytoplasmic replication cycle (vesicular stomatitis virus [VSV] and Japanese encephalitis virus [JEV]). Results showed that siRNA treatment significantly suppressed the replication of the recombinant VSV-GFP virus in A549 cells, which was determined by detecting GFP expression by Western blotting and was visualized by fluorescence microscopy (Fig. S7A). The effect of LYAR knockdown on JEV replication was determined by measuring the mRNA level of core protein C. The results showed that the mRNA level of JEV core protein C was significantly reduced in LYAR-silenced cells compared to that in control cells (Fig. S7B), indicating that LYAR knockdown inhibits JEV replication. These results indicate that LYAR regulates the replication of both nuclear and cytoplasmic replicating RNA viruses.

LYAR facilitates viral RNA synthesis.

To determine the mechanisms of LYAR-influenced IAV replication, we investigated the effects of LYAR on viral RNA and protein syntheses. LYAR-silenced A549 cells were infected with the PR8 virus, and the vRNA, cRNA, and mRNA levels of NP and the protein levels of PB2, NP, and M1 were determined. The levels of all three species of viral RNA were significantly lowered in LYAR-silenced cells compared to those of the control cells at 12, 24, and 36 hpi (Fig. 6A). As expected, expression levels of PB2, NP, and M1 proteins were reduced in LYAR-silenced cells, in contrast to those in the control cells at all tested time points (Fig. 6C). In contrast, the levels of all three species of viral RNA, as well as PB2, NP, and M1 proteins, were significantly increased in cells overexpressing LYAR (Fig. 6B and D). These data indicate that LYAR promotes viral RNA and protein synthesis.

IAV genome primary transcription is independent of de novo viral protein synthesis, while viral genome replication requires newly synthesized viral proteins (34). Therefore, we investigated the effects of LYAR on viral RNA genome transcription and replication separately. CHX, an inhibitor of protein synthesis in eukaryotic cells, was used to block the synthesis of viral proteins, thereby inhibiting the synthesis of cRNA and the replication of vRNA but not the level of primary transcription from incoming vRNPs (49). In LYAR-silenced cells without CHX, vRNA, cRNA, and mRNA levels were significantly reduced at 6 and 8 hpi compared to those in mock-silenced cells (Fig. 7A), whereas in the presence of CHX, similar levels of vRNA and mRNA were detected in both virus-infected LYAR-silenced cells and mock-silenced cells (Fig. 7B), suggesting that LYAR does not participate in primary transcription. To verify the inhibition of CHX treatment on virus multiplication, NP mRNA levels in CHX-treated and untreated infected cells were determined, and the results showed that NP mRNA levels were reduced approximately 60-fold after CHX treatment (Fig. 7C). Taken together, these findings demonstrate that LYAR facilitates RNA synthesis after primary transcription.

LYAR enhances viral polymerase activity.

Since LYAR facilitates viral RNA synthesis in IAV-infected cells, we further determined whether LYAR regulates viral RNA synthesis by influencing polymerase activity. For this purpose, a well-established minireplicon assay was applied to examine the effect of LYAR on the polymerase activity of PR8 H1N1 virus. HEK293T cells were cotransfected with si-LYAR (si-2 and si-3) or HA-LYAR (increasing amount of LYAR), together with plasmids encoding PB1, PB2, PA, and NP, as well as a PolI-driven RNA expression plasmid encoding the NS vRNA segment. The data showed that knockdown of LYAR resulted in an approximately 80% reduction of the polymerase activity (Fig. 8A), while overexpressing LYAR led to increased polymerase activity in a dose-dependent manner (Fig. 8B). To determine whether the LYAR-promoted polymerase activity is due to an increase of the expression levels of the RNP subunits, the expression levels of the RNP components were determined. Western blot data showed that the expression levels of of all the RNP subunits, including PA, PB1, PB2, and NP, were unchanged (Fig. 8C), although the GFP expression driven by PolI was increased in LYAR-overexpressed cells and decreased in LYAR-silenced cells. These results indicate that the polymerase activity enhanced by LYAR is not caused by increasing expression of RNP subunits. To assess the viability of HEK293T cells treated with si-LYAR, a cell-counting kit 8 (CCK-8) assay was used to measure cell viability. Results showed that there was no significant difference between LYAR knockdown cells and control cells at any tested time point (Fig. 8D). These data indicate that LYAR promotes viral polymerase activity, thereby facilitating viral RNA synthesis.

LYAR facilitates viral RNP assembly.

Influenza virus genome replication depends on the assembly of progeny vRNPs by newly translated NP and polymerase complex proteins. We therefore determined whether LYAR participates in the viral RNP assembly process, specifically, NP oligomerization (NP self-association), 3P (PB1, PB2, and PA) formation, and viral RNP (viral RNAs, NP, and 3P) formation. The effect of LYAR on NP self-association was tested by coimmunoprecipitation of HA-NP with Flag-NP. The data showed that HA-NP associated with Flag-NP equally with or without LYAR overexpression (Fig. 9A), indicating that LYAR does not affect NP oligomerization. The effect of LYAR on 3P formation was also determined by coimmunoprecipitation, and Flag-PB1 was used as bait to immunoprecipitate PA and PB2. We showed that PA and PB2 were precipitated by PB1 equally with or without HA-LYAR (Fig. 9B), demonstrating that LYAR does not affect 3P formation, as NP directly interacts with PB1 and PB2 but not PA, and the NP-PA interaction occurs only in the context of a vRNP (14, 48). Therefore, a vRNP reconstitution system in which the NP was HA tagged was used to form the functional viral RNPs, and the Co-IP experiment was performed by using an anti-HA antibody. The amount of PA coprecipitated by HA-NP represents the efficiency of vRNP assembly. The data showed the amount of PA precipitated by HA-NP was significantly increased with an increasing amount of Flag-LYAR (Fig. 9C). Moreover, HA-NP coprecipitated much less PA in LYAR-silenced cells than the negative-control cells (Fig. 9D). The expression of GFP driven by vRNP was enhanced in LYAR-overexpressed cells and reduced in LYAR-silenced cells compared to that in the control cells, while the expression of all RNP components was almost not changed (Fig. 9C and D), suggesting that LYAR directly affects vRNP assembly. Taken together, these results provide evidence that LYAR facilitates vRNP assembly.

LYAR knockdown leads to nuclear retention of vRNPs.

Influenza vRNPs are assembled in the nucleus and subsequently exported to the cytoplasm as a complex, where new viral particles are assembled. Because LYAR silencing has been shown to significantly inhibit IAV replication, whether LYAR knockdown has effects on the nuclear export of vRNPs was determined in A549 cells infected with the PR8 H1N1 virus. Results showed that approximately 30% of mock-silenced infected cells (NC) displayed NP nuclear localization at 7 hpi (Fig. 10A and B), indicating that the nuclear export of vRNPs had occurred in most of the infected cells. In contrast, nearly 80% of LYAR-silenced infected cells (si-LYAR) had an NP nuclear localization (Fig. 10A and B), indicating vRNP nuclear export was largely delayed due to LYAR silencing. Furthermore, the results of Western blotting on nuclear and cytoplasmic fractions of these infected cells were consistent with the indirect immunofluorescence assay (IFA) data, i.e., the NP was predominantly in the cytoplasmic fraction of the mock-silenced cells, in contrast to that in the nuclear fraction of the LYAR-silenced cells (Fig. 10C). Meanwhile, a significant reduction of NP protein level was detected in the whole-cell lysis of LYAR-silenced cells (Fig. 10C), indicating the viral protein synthesis was also inhibited when LYAR was knocked down. In addition, the distribution of vRNAs was detected by using an in situ hybridization assay. The results showed that vRNAs of the viral M segment were markedly retained in the nucleus in LYAR-silenced infected cells at 7 hpi (Fig. 10D and E), and the total amounts of M vRNAs in the LYAR-silenced cells were markedly reduced compared to those in the control cells (Fig. 10D), indicating LYAR knockdown inhibits the nuclear export of vRNPs as well as vRNA synthesis. Collectively, these results demonstrate that LYAR knockdown results in a marked retention of vRNPs in the nucleus.

Cells and viruses.

Human embryonic kidney 293T cells (HEK293T) and Madin-Darby canine kidney (MDCK) cells were purchased from the ATCC (American Type Culture Collection, Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS) (PAN-Biotech, Germany). The Henrietta Lacks strain of cancer cells (HeLa) and adenocarcinomic human alveolar basal epithelial cells (A549) were purchased from the ATCC and maintained in RPMI 1640 and F12 media (HyClone, Beijing, China), respectively, supplemented with 10% FBS. All cells were cultured at 37°C in a 5% CO₂ humidified atmosphere. The IAVs used in this study were A/Puerto Rico/8/1934 (PR8, H1N1) and A/duck/Hubei/Hangmei01/2006 (HM, H5N1). All viruses were amplified using 10-day-old embryonic chicken eggs and then titrated by determining log₁₀ PFU/ml values on MDCK cells. All cell experiments with H5N1 virus were performed in an animal biosafety level 3 (BSL-3) laboratory. This study was carried out in accordance with the recommendations for BSL-3 of Huazhong Agricultural University (HZAU). The protocol was approved by the BSL-3 laboratory of HZAU. The recombinant vesicular stomatitis virus expressing green fluorescence protein (VSV-GFP) was a gift from the Harbin Veterinary Research Institute (Harbin, China). Japanese encephalitis virus (JEV) was kindly provided by Shengbo Cao (Huazhong Agricultural University, Wuhan, China).

Plasmids and small interfering RNAs.

For construction of p3XFlag-LYAR (Flag-LYAR) and pCAGGS-HA-LYAR (HA-LYAR), the full-length cDNA of LYAR amplified by PCR was cloned into vectors p3XFlag (Flag) and pCAGGS-HA (HA), digested by BglII/XbaI and ECORI/XhoI, respectively. The Flag-tagged, HA-tagged, and nontagged pCDNA3.1 plasmids encoding PB1, PB2, PA, and NP were derived from the H1N1 (A/Puerto Rico/8/1934) virus. siRNAs targeting human gene LYAR, NCL, HNRNPR, MCDRH, PPP1CA, or RPL19, with a nontarget siRNA (si-NC) as a negative control, were synthesized by GenePharma (Shanghai, China) and used in this study. PCR primers and siRNA sequences are shown in Table S1 in the supplemental material.

Antibodies and reagents.

Antibodies used for Western blotting, immunoprecipitation, and indirect immunofluorescence were anti-Flag M2 mouse monoclonal antibody (F3165; Sigma, USA); anti-LYAR mouse polyclonal antibody (H00055646-B01P; Abnova, China); anti-NPM1 rabbit polyclonal antibody (AP2834a; ABGENT, USA); anti-HA, -GFP, and -glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibodies (PMK013C, PKM009S, and PMK043F; PMK Bio, China); anti-Histone 3.1 polyclonal rabbit antibody (p30266; Abmart, USA); rabbit polyclonal antibodies against influenza A viral proteins PB1, PB2, PA, NP, and M1 (GTX125923, GTX125926, GTX118991, GTX125989, and GTX125928; GeneTex, USA); and Alexa Fluor 488-conjugated AffiniPure goat anti-rabbit and Alexa Fluor 594-conjugated affinipure goat anti-mouse secondary antibodies (SA00006-2 and SA00006-3; Proteintech, USA). The small-molecule compounds used in this study were CHX (cycloheximide; 100 μg/ml; 66819; Sigma, USA) and DAPI (4′,6′-diamidino-2-phenylindole dihydrochloride; 1:1,000) (C1002; Beyotime, China).

Transfections.

Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Briefly, plasmids, siRNAs, and Lipofectamine were diluted to equal volumes with Opti-MEM and incubated for 5 min at room temperature. The diluted Lipofectamine and the diluted DNA (or RNA) were mixed and incubated for 20 min at room temperature. The mixture was added to cells and incubated for 6 h, and cells were then cultured in fresh medium supplemented with 10% FBS.

Generation of LYAR-KO A549 cells.

LYAR-KO A549 cells were generated by using the CRISPR/Cas9 system as described previously (69, 70). The single guide RNA (sgRNA) sequence targeting the human LYAR gene (5′-TGAGCATCACAGATCCGAAG-3′) was cloned into lentiCRISPR v2 vector and applied for producing the recombined lentivirus. A549 cells were infected with the LYAR lentiCRISPR v2 lentivirus or the empty vector lentiCRISPR v2 lentivirus (negative control). After 48 to 60 h postinfection, puromycin (2.5 mg/ml) was added to select the positive clones. Finally, the monoclonal cells acquired by using the limiting dilution method were expanded and the knockout of LYAR was confirmed by Western blotting.

Co-IP assay.

HEK293T cells were transfected with the indicated plasmids, and then cells were washed with cold phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay buffer (V900854; Sigma, USA) containing Complete protease inhibitor cocktail (B14001; Biotool, USA) at 24 to 48 h posttransfection. The lysates were pretreated with 20 μl of protein A/G agarose (sc-2003; Santa Cruz, USA) for 1 h at 4°C, and then protein A/G agarose was removed by centrifugation. Two to three μg of the indicated antibody was added to the pretreated lysates, followed by overnight incubation at 4°C. Protein A/G agarose was added to the lysates and incubated at 4°C for another 2 h with rotation. The agarose beads were collected by centrifugation and washed four times with lysis buffer. The beads were resuspended in 1× SDS loading buffer and proteins were resolved by SDS-PAGE, followed by transferring to nitrocellulose and Western blotting.

IFA and confocal microscopy.

Indirect immunofluorescence and confocal microscopy were performed as described previously (71). Briefly, HeLa or A549 cells were fixed with 4% paraformaldehyde (PFA) for 10 min, treated with 0.2% (vol/vol) Triton X-100 for 10 min, and then incubated with 1% (wt/vol) bovine serum albumin (BSA) for 1 h at room temperature. Samples were then incubated with the indicated primary antibody for 2 h, followed by incubation with the appropriate Alexa Fluor-conjugated secondary antibody, and stained with DAPI to visualize DNA. Images were acquired using a confocal microscope (LSM510 or LSM880; Zeiss, Germany).

Minireplicon assay.

Polymerase activity was measured by a minireplicon assay. In brief, HEK293T cells were cotransfected with pPolI-luc or pPolI-eGFP, a Renilla luciferase expression plasmid, and four RNP expression plasmids, pCDNA3.1-PB1, pCDNA3.1-PB2, pCDNA3.1-PA, and pCDNA3.1-NP. Luciferase activity was measured using a Dual-Luciferase assay kit (Promega) according to the manufacturer's protocol at 24 h posttransfection. Renilla luciferase activity was used as an internal control and to normalize transfection efficiency.

Virus titration.

A549 cells in 12-well plates were transfected with the indicated plasmids (1 μg) or the indicated siRNA (40 pmol). At 24 h posttransfection, cells were infected with the indicated influenza virus at a multiplicity of infection (MOI) of 0.01. Viral supernatants were harvested at the indicated time points postinfection, and plaque assays were performed on MDCK cells to titrate virus titer as described previously (72).

RNA isolation and qRT-PCR.

For quantitative reverse transcription-PCR (qRT-PCR), cells were lysed with TRIzol reagent (Invitrogen, USA), and total RNA was extracted according to the manufacturer's instructions. One to two micrograms of RNA was used to generate cDNA using reverse transcriptase (AMV XL; TaKaRa, Tokyo). Real-time PCR (Vii7A; ABI, USA) was performed using FastStart Universal SYBR green Master (Roche). The PCR conditions were 2 min at 50°C, 10 min at 95°C, and then 40 cycles of 15 s at 95°C and 1 min at 60°C. The levels of viral NP vRNA, cRNA, and mRNA were determined by using a strand-specific real-time RT-PCR as described previously (73). To ensure the specific amplification of vRNA, cRNA, and mRNA, primers complementary to each type of RNA added a tag of 18 to 20 nucleotides unrelated to the influenza virus to the 5′ end were used in reverse transcription, and the tagged cDNA was amplified by real-time PCR using the tag portion as the forward primer and a segment-specific reverse primer. 18S rRNA was used as a control for the normalization of cellular mRNA and intracellular viral RNA. The sequences of the primers used for qRT-PCR are shown in Table S1.

Nuclear and cytoplasmic fractionation.

Subcellular fractions were extracted as described previously (74). A total of 10⁶ A549 cells treated accordingly were harvested and lysed with 100 μl of cytoplasmic extraction buffer [10 mM HEPES, 10 mM KCl, 2 mM Mg(Ac)₂, 3 mM CaCl₂, 340 mM sucrose, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.9] on ice for 20 min, followed by the addition of NP-40 (Amresco, Solon, OH, USA) to a final concentration of 0.25% (vol/vol). Samples were then vortexed for 15 s and centrifuged for 10 min at 3,500 × g at 4°C. Supernatants (the cytoplasmic fraction) were collected and stored at −80°C. Pellets were dissolved in 80 μl nuclear extraction buffer [50 mM HEPES, 500 mM NaCl, 1.5 mM MgCl₂, 0.1% (vol/vol) Trition-X100, 1 mM DTT, 1 mM PMSF, pH 7.9], incubated on ice for 10 min, and then centrifuged at 14,000 × g for 10 min at 4°C. Supernatants (the nuclear fraction) were collected and stored at −80°C.

FISH.

Fluorescence in situ hybridization (FISH) probes labeled with the Quasar 570 fluorophore for detecting PR8 H1N1 M segment vRNA were designed by using an online probe designer (Stellaris Probe Designer version 4.2), and the sequences are presented in Table S1. The probes were purchased from Biosearch Technologies (Novato, CA, USA). For in situ hybridization analysis, A549 cells in a 24-well plate were fixed for 10 min with 4% PFA and then washed with PBS three times. Cells were then permeabilized with 0.2% Triton X-100 for 10 min and washed briefly. Cells next were incubated with 200 μl wash buffer A (SMF-WA1-60; Biosearch Technologies, USA) for 5 min. Wash buffer A was removed, and 200 μl hybridization buffer (SMF-HB1-10; Biosearch Technologies, USA) containing 2 μl FISH probes (final concentration, 12.5 nM) was added and incubated for 16 h at 37°C in the dark. The hybridization buffer was removed, and then cells were incubated with wash buffer A for 30 min at 37°C. DAPI was added to counterstain the nuclei, and then cells were incubated in wash buffer B (SMF-WB1-20; Biosearch Technologies, USA) for 5 min and washed briefly with PBS. Images were obtained with a confocal microscope (LSM 880; Zeiss, Germany).

Cell viability assay.

To detect the effect of LYAR silencing on cell proliferation, the cell viability of HEK293T cells and A549 cells transfected with si-LYAR or si-NC were measured by CCK-8 activity according to the manufacturer's instructions (Dojindo Molecular Technologies). In brief, cells in 96-well plates were transfected with si-LYAR or si-NC, and cell viability was measured at 24, 36, and 48 h posttransfection. CCK-8 reagent was added to each well of a plate, and the absorbance at 450 nm was measured by a microplate reader after 1 h of incubation.

Statistical analysis.

The data are presented as means ± standard deviations (SD) from three independent experiments. Statistical significance was determined using two-tailed Student's t test. A P value of less than 0.05 was considered statistically significant, and a P value of less than 0.01 was considered highly significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001).