Cellular microRNA let-7c inhibits M1 protein expression of the H1N1 influenza A virus in infected human lung epithelial cells

The influenza virus (IV) triggers a series of signalling events inside host cells and induces complex cellular responses. Studies have suggested that host factors play an essential role in IV replication. MicroRNAs (miRNAs) represent a class of small non-coding RNAs that target mRNAs, triggering either translation repression or RNA degradation. Emerging research suggests that host-derived cellular miRNAs are involved in mediating the host–IV interaction. Using miRNA microarrays, we identified several miRNAs aberrantly expressed in IV-infected human lung epithelial cells (A549). Specifically, miR-let-7c was highly up-regulated in IV-infected A549 cells. PITA and miRanda database screening indicated that the let-7c seed sequence is a perfect complementary sequence match to the 3′ untranslated region (UTR) of viral gene M1 (+) cRNA, but not to PB2 and PA. As detected by a luciferase reporter system, let-7c directly targeted the 3′-UTR of M1 (+) cRNA, but not PB2 and PA. To experimentally identify the function of cellular let-7c, precursor let-7c was transfected into A549 cells. Let-7c down-regulated IV M1 expression at both the (+) cRNA and protein levels. Furthermore, transfection with a let-7c inhibitor enhanced the expression of M1. Therefore, let-7c may reduce IV replication by degrading M1 (+) cRNA. This is the first report indicating that cellular miRNA regulates IV replication through the degradation of viral gene (+) cRNA by matching the 3′-UTR of the viral cRNA. These findings suggest that let-7c plays a role in protecting host cells from the virus in addition to its known cellular functions.


Introduction
Influenza A viruses (IAVs) can infect a broad spectrum of hosts (birds, humans and other mammals) and evolve into highly pathogenic strains [1,2]. Influenza A virus is an RNA virus that encodes up to 11 proteins. The small coding capacity demands that the virus exploits the host's cellular factors for many vital aspects of its life cycle. Influenza A viruses are among the most common causes of human respiratory infections and are associated with high morbidity and mortality. Previous studies indicated that many mammalian host proteins mediate the IAV-induced host response. Moreover, these protein-protein interactions between virus and host are critical for viral propagation. Furthermore, efficient influenza virus propagation critically depends upon the activation of host caspase-3, as the presence of a caspase-3 inhibitor in cells strongly impairs viral replication. In the present study, caspase-3 was partially knocked down by small interfering RNAs [3]. Turpin et al. showed that p53 levels and activity were up-regulated during influenza infection, whereas inhibition of p53 led to increased viral titres [4]. Recently, cyclophilin A was found to interact with the IAV-encoded M1 protein and impair early viral replication [5]. Based on a genome-wide RNA interference screening, Konig et al. identified 295 cellular cofactors required for early stage replication of the influenza virus [6].
MicroRNAs are single-stranded RNA molecules that are 21-23 nucleotides in length [7]. Several hundred miRNAs have been identified in plants, animals and viral RNA genomes [8,9]. In animals, miRNAs regulate many cellular processes by binding to 3′-UTRs of their target mRNAs, causing translational repression of the target mRNA [8]. Recent studies showed that viral miRNAs play an important role in regulating viral infection in host cells by targeting the cellular or viral genes. MicroRNAs encoded by Simian virus 40 (SV40) were reported to target their own virally encoded T antigen, leading to a decrease in cytotoxic T lymphocyte-mediated lysis of infected cells [10]. miR-latency-associated transcript (LAT), an miRNA produced from the viral LAT of herpes simplex virus type 1, targets the cellular mRNAs encoding transforming growth factor b and SMAD3 that results in inhibition of cell proliferation and apoptosis [11]. These two examples clearly indicate that viral miRNAs are capable of controlling expression of viral or cellular genes.
Conversely, cellular miRNAs can regulate viral infections. Hostderived miR-24 and miR-93 have been found to target the viral large protein (L protein) and phosphoprotein (P protein) genes, respectively. A deficiency in miR-24 and miR-93 was responsible for increased vesicular stomatitis virus (VSV) propagation in Dicer1 knockout cells [12]. Furthermore, Lecellier et al. reported that host miR-32 effectively inhibits the accumulation of the retrovirus primate foamy virus type 1 (PFV-1) in human cells by targeting a sequence in the genome of the PFV-1 [13]. Huang et al. reported that a cluster of cellular miRNAs, including miR-28, miR-125b, miR-150, miR-223 and miR-382, targets the 3′ ends of various human immunodeficiency virus 1 (HIV-1) mRNAs. These host miRNAs are enriched in resting CD4 + T cells as compared to activated CD4 + T cells, indicating that these cellular miRNAs are important to maintain HIV-1 latency [14]. Inhibition of miR-122, a cellular miRNA highly and specifically expressed in the human liver, resulted in a marked loss in autonomous replication of hepatitis C viral RNAs, suggesting that miR-122 likely enhances propagation of the virus [15,16].
Most recently, Li et al. reported that cellular miRNA expression could be altered during influenza virus infection, irrespective of the lethality of the virus [17]. In the present study, a miRNA array was used to screen and identify possible cellular miRNAs involved in regulating influenza virus infection in human lung epithelial cells; let-7c was identified to specifically interact with IAV matrix protein (M1) (+) cRNA. Functional analysis indicated that let-7c inhibited the replication of the virus in human cells. As M1 is the most abundant protein in the IAV viral particle [18], and M1 mutation studies indicate that M1 is involved in virus-host protein interactions [19], the findings of the present study suggest that cellular let-7c may act as a potential therapeutic target to inhibit influenza virus infection through its direct interaction with the viral M1 mRNA.

Viral infections
Host cells were washed with phosphate-buffered saline and infected with influenza virus at a multiplicity of infection of 5 for 1 hr at 37°C. After infection, the inocula were removed, and cells were incubated with F12K containing 0.2% bovine serum albumin (BSA) (Gibco, Invitrogen Incorporated, Carlsbad, CA, USA) for the time indicated.

Cell viability assay
The A549 cells were seeded in 48-well plates. After overnight incubation in a 5% CO 2 incubator at 37°C, cells were transfected with the miRNA expression vectors or an empty vector and infected with influenza A/ JingFang/86-1(H1N1) virus or influenza virus A/FM/1/47 (H1N1) 24 hrs later. Each transfection was performed at least in triplicate. Cell viabilities were assessed by Cell Counting Kit-8 (CCK-8) assay (Dojindo, Shanghai, China) 24 hrs post-infection.

Viral titres
To measure viral titres, transfected A549 cells were infected with IAV for 1 hr at 37°C. After a 1-hr adsorption, the inocula were removed, and cells were incubated with DMEM containing 0.2% BSA for the indicated times. The virus titres from culture supernatants of A549 cells were determined by measuring 50% Tissue Culture Infective Dose (TCID 50 ) in MDCK cells. The titres were evaluated by the method described by Reed and Muench [22].

Vector construction and transfection
Genomic fragments of Homo sapiens let-7a, let-7c, let-7d, mir-7-1, mir-26b, mir-146b and mir-192 precursors were amplified by PCR from A549 cells. The PCR product was cloned into pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA) digested by HindIII and XbaI. The 3′-UTR segment of influenza virus M1 was amplified by PCR from H1N1 viral cDNA and subcloned into the pMIR-Report luciferase reporter vector (Ambion) digested by HindIII and SpeI. The 3′-UTR segment of influenza virus PA, PB1 and PB2 was synthesized directly. The annealed oligonucleotides were used to ligate into the HindIII and SpeI sites of the pMIR-Report luciferase. Vector transfection was performed with FuGene ® HD transfection reagent (Roche, Indianapolis, IN, USA) according to the manufacturer's protocol.

siRNAs and transfection
The let-7c inhibitor was synthesized by GenePharma. siRNA transfection was performed using Lipofectamine 2000 (Invitrogen). In brief, cells were first plated in a six-well plate to 60% confluence, and 0.4 lM siR-NA was then dissolved in 2% Lipofectamine 2000/Opti-MEM medium (Invitrogen) and added into each well. The cells were incubated with the mixture for 6 hrs before replacing the medium. Total RNAs and proteins were prepared 48 hrs after transfection and were used for qRT-PCR and western blotting analysis.

Western blot analysis
Cell fractions were prepared using M-PER Mammalian Protein Extraction Reagent based on the manufacturer's instruction (Pierce, Rockford, IL, USA). Western blot analysis was performed as described previously [17].

Luciferase reporter assay
The A549 cells were seeded at 20,000 cells per well in 24-well plates and cotransfected with 100 ng of firefly luciferase reporter vector containing the influenza M1 (+) cRNA 3′-UTR and 8 ng of the b-gal Control Plasmid (Ambion) in a final volume of 0.5 ml using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, both luciferase and b-gal activity were measured and the relative level of luciferase was normalized against the b-gal readings (Promega, Madison, WI, USA).

Results miRNA expression profile analysis of IAV-infected human cells
To determine the ability of IAV (JingFang/86-1(H1N1) virus) to modulate the expression of cellular miRNAs involved in its replication, the expression profile of cellular miRNA in infected and uninfected A549 human lung epithelial cells was compared by microarray analysis (Table 1). Twenty-eight miRNAs were found to be up-regulated or down-regulated upon infection.

Effect of miRNA overexpression on the viability of IAV-infected A549 cells
Based on the profile screening results, miRNA-expressing vectors were constructed for each of the 28 identified miRNAs. A549 cells were then transfected with each of these miRNA expression vectors or the control empty vector followed by influenza A/JingFang/86-1 (H1N1) virus infection 24 hrs later. Cell viability was assessed 24 hrs post-infection. Five miRNAs (miR-10a, miR-146a, let-7a and let-7c) significantly increased cell viability as compared to the cells treated with the control empty vector, whereas two miRNAs (miR-7d and miR-146b) decreased cell viability (Fig. 1).

miRNA overexpression inhibited IAV titres in infected A549 cells
Viral titres were examined in the infected A549 cells after transfection with the miRNA expression vectors that significantly affected cell viability. The supernatants of influenza A/JingFang/86-1(H1N1) virusinfected cells were collected and transferred onto MDCK cells; viral titres were determined by evaluating the TCID 50 of the MDCK cells. Among all the miRNA expression vectors that significantly affected cell viability, only the let-7c expression vector significantly decreased viral titres by 50% in comparison to the control vector (Fig. 2).

Let-7c overexpression inhibited M1 vRNA synthesis
Providing further support that host let-7c played a role in regulating IAV infection, let-7c was significantly up-regulated in infected A549 over uninfected A549 cells as measured by the expression of mature let-7c transcript using real-time PCR (Fig. 3). To explore whether or not overexpression of let-7c had any effect on vRNA synthesis, vRNA levels were evaluated in H1N1 virusinfected A549 cells transfected with miRNA vectors (Fig. 4A and   ª 2012 The Authors B). The quantity of M1 vRNA was analysed at 4, 6 and 24 hrs after infection. As shown in Fig. 4C and D, the level of M1 vRNA was reduced in the cells overexpressing let-7c compared with empty vector-transfected cells. Specifically, an approximate 87% reduction in M1 vRNA at 4 hrs was observed in influenza A/JingFang/86-1(H1N1) virus-infected A549 cells.

Let-7c regulated the expression of M1 vRNA and protein
To further explore whether let-7c could directly regulate M1 protein expression, the effect of let-7c expression vector and let-7c inhibitors on the expression of M1 protein in influenza A/JingFang/86-1(H1N1) virus or influenza A/FM/1/47 (H1N1) virus-infected A549 cells was investigated. Western blot assay indicated that M1 protein was dramatically reduced after pcDNA3.1(+)/let-7c transfection, whereas the control pcDNA3.1(+) vector did not significantly affect M1 protein expression (Fig. 7A). As shown in Fig. 7B, transfection with a let-7c inhibitor enhanced M1 expression as compared to transfection with vehicle alone [23].

Discussion
In the present study, we indicate that the expression of let-7c is upregulated in IAV-infected human lung epithelial cells and that overexpression of let-7c results in the inhibition of IAV replication in host cells. This study provides a better understanding of the interaction between IAV and host cells.
Matrix protein (M1) is the most abundant conservative protein in the viral particle. It is a multifunctional protein that plays a role in influenza virus replication and consists of 252 amino acids that are relatively conserved among influenza subtypes [18]. A number of published reports have shown that several host cell factors may be required for regulation of influenza virus replication through interaction with M1 protein during different stages of infection. Viral ribonucleoprotein (RNP)/M1 and NP in the cytoplasm were reported to associate with host cytoskeletal components that may help the viral morphogenesis [24,25]. Watanabe et al. indicated that heat shock cognate protein 70 (Hsc70) was involved in the influenza virus life cycle by interacting with M1 [26]. Intracellular caspase-8 was also found to bind M1 to trigger the caspase-8-mediated apoptosis pathway in influenza virus-infected cells [27,28]. Furthermore, the cellular receptor of activated C kinase 1 is able to interact with and phosphorylate M1. In the present study, we observed that host cells can also regulate influenza virus replication at the transcriptional level, as human cellular miR-let-7c were shown to degrade M1 (+) cRNA by binding to its 3′-UTR. Therefore, miR-let-7c mediates anti-IAV activity in the lung epithelial cells, and the 3′-UTR terminal sequences of the M1 (+) cRNA can be used as an effective target against viral replication. Furthermore, we indicate that miR-let-7c is another host factor involved in the replication of IAV. The present data suggest that upregulation of let-7c in lung epithelial cells upon IAV infection may be considered as a self-protective mechanism mediated by the host cells during the infection.
The exact function of let-7 in human development and normal physiology is not yet obvious. Two members of the let-7 family, let-7a and let-7c, have been shown to regulate cell proliferation and to inhibit expression of the oncogenes ras [29] and c-myc [30]. As the influenza virus life cycle involves a large number of cellular proteins and biological pathways, we postulate that let-7c may indirectly affect Let-7c was detected and presented relative to GAPDH mRNA. Data are expressed as mean ± S.E., n = 3, *P < 0.05, #P < 0.01.
ª 2012 The Authors the influenza virus propagation by directly targeting host protein transcripts as well. Therefore, bioinformatic analysis was performed to identify the host protein targets of let-7c. Let-7c was shown to bind to 3′-UTR residues of the EIF2AK2, MCM5, PA2G4 and caspase-8 host mRNAs (Supplementary Fig. 1). Although a trend towards reduction was observed, pcDNA3.1(+)/let-7c did not produce a significant reduction of EIF2AK2, MCM5, PA2G4 or caspase-8 as measured by the luciferase reporter system ( Supplementary Figs 2-3). Thus, it is still worthwhile to investigate whether or not let-7c also mediates the virus-host interaction by acting on host proteins/pathways. Several recent reports have investigated whether or not cellular microRNA is involved in influenza virus infection. Li et al. reported that cellular miRNA expression is altered during influenza virus infection, irrespective of the lethality of the virus [17]. Song et al. screened host miRNAs and found that miR-323, miR-491 and miR-654 inhibit the replication of the H1N1 IAV in MDCK cells by binding to the 3′ coding region rather than the 3′-UTR of the PB1 gene. Generally, a target mRNA is degraded if the miRNA and the mRNA are a perfect complementary sequence match, whereas translational repression is the most common effect if the miRNA and the mRNA are not a perfect complementary sequence match. However, this previous study showed that the imperfect binding between the miRNAs and PB1 mRNA could down-regulate PB1 expression through mRNA degradation [31]. They explained that the PB1 gene is not of sufficient length to contain miRNA-binding sites in the 3′-UTR sequence and postulated that other mechanisms may play a role in directing the action of miRNAs. In our study, cellular miR-let-7c is a perfect complementary sequence match to the 3′-UTR of the M1 gene and could therefore  Relative luciferase activity (%) * Fig. 6 Let-7c target analysis using luciferase reporter assays in A549 cells Luciferase reporter assays of A549 cells 48 hrs after cotransfection of the influenza virus M1, PA, PB1 and PB2 3′-UTR reporter constructs and let-7c expression vector. Data were normalized to bgalactosidase activity. Data are expressed as mean ± S.E., n = 3, *P < 0.05.
reduce M1 expression at both the (+) cRNA and protein level. The differences in the results between the Song et al. study and our study may derive from the different role and replication process that PB1 and M1 genes have in regulating the IAV life cycle in host cells. Moreover, the A549 cells used in the present study differed from the MDCK cells; the two cell types were derived from different animal species as well as from different tissues. Therefore, it is still worthwhile to investigate the regulation mechanism(s) of cellular miRNA on the IAV genes.
In conclusion, our results indicate that cellular let-7c suppresses IAV replication in host cells by targeting the 3′-UTR of M1 (+) cRNA. The findings suggest that let-7c binding to the 3′-UTR of M1 may serve as a therapeutic target for the prophylaxis and control of influenza virus infections.