Suppression of Interferon Stimulated Genes Enhance Viral Replication of Seasonal Influenza A Virus through the JAK/STAT pathway

doi:10.21203/rs.3.rs-1760102/v1

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Suppression of Interferon Stimulated Genes Enhance Viral Replication of Seasonal Influenza A Virus through the JAK/STAT pathway

https://doi.org/10.21203/rs.3.rs-1760102/v1

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Background

In a previous study, we described the diverse growth capabilities of circulating seasonal influenza A virus (IAV) with low to high viral copies in vitro. In this study, we analyzed the cause of differences in growth capability by evaluating Interferon-β and antiviral interferon-stimulated genes (ISGs) expression.

Methods

A549 cells (3.0x10⁵ cells) were inoculated with circulating seasonal IAV strains for 6 and 24 h. In cells inoculated for 6 h, IAV production was assessed by IAV-RNA copies in culture supernatant and cell pellets for gene expression evaluation. At 24 hours post-infection, cells were collected for IFN-β and ISG-15 protein expression. JAK/STAT pathway inhibitor, pyridone 6 (P6) was used to evaluate the influence of ISGs on the growth capability of seasonal IAV.

Results

A549 cells inoculated with seasonal IAV strains with high growth capability expressed less IFN-β and ISGs, whereas low growth capability strains expressed more IFN-β and ISGs. The use of a P6, a JAK/STAT inhibitor, suppressed the expression of ISGs and enhanced the viral copies of seasonal IAV strains with low growth capability. This result suggests that growth capability is regulated by ISG-mediated IFN-β production.

Conclusion

In this study, we provide a new insight into the mechanism of circulating seasonal IAV replication. Our study showed that the difference in the growth capability of circulating seasonal IAV strains is due to the regulation of antiviral ISGs. Inhibition of the JAK/STAT pathway permits seasonal IAV strains with low growth capability to enhance their viral copies by suppressing antiviral ISGs. Our results may contribute in developing new effective therapeutic strategies and reduce the risk of developing severe influenza disease.

Influenza A virus

growth capability

Interferon-β

Interferon-stimulated genes

The current circulating seasonal influenza A virus (IAV) subtypes H1N1 and H3N2 are the most common causes of pneumonia-related morbidity in developed countries, and mortality can be much higher during pandemics [1, 2]. The clinical severity of influenza virus infection can range from asymptomatic to severe symptomatic illness, and it has been shown that a higher viral load is associated with a more severe case of the disease and a high probability of virus transmission and spread [3, 4]. Moreover, one of the probable factors affecting viral load is the intrinsic viral growth capability of the virus [5].

Our previous studies demonstrated emerging pandemic viruses consisted of only highly replicative strains, but more-slowly replicating strains were observed soon in the following seasons [5, 6]. We hypothesized that the difference in growth capability could be due to the immune response of the human host, and understanding how viral load and host immune responses are related is critical for identifying disease-specific markers that may help predict disease prognosis.

In response to viral infection, the production of type I interferons (IFNs) (IFN-α/β) induces a signal that allows the binding of IFN-β to its receptor, followed by activation of the JAK/STAT pathway to initiate the transcription of hundreds of IFN-stimulated genes (ISGs) [7–9]. ISGs eliminate the virus and prevent its spread by promoting an antiviral state in neighboring cells, thus limiting the severity of the disease [10]. In the context of influenza virus infection, ISGs, specifically interferon-stimulated gene 15 (ISG-15), interferon-induced transmembrane protein 1 (IFITM1), and tripartite motif containing 22 (TRIM22), target genomic replication, viral entry, and inhibition of proteasomal degradation, respectively [11]. However, given the critical role of IFN-β and ISGs as the first line of defense against IAV infection, the virus has developed strategies to block IFN-induced JAK/STAT pathway at different levels using several viral factors through various mechanisms to increase their replication efficiency [7, 12]. Among IAV viral proteins, non-structural protein 1 (NS1) has been identified as the main type I IFN antagonistic factor [13].

Several groups have investigated the contribution of NS1 protein mutations to viral replication of IAV [14–17]. Manipulation of single amino acid substitution at position 45 (G to R; G45R) in a mouse-adapted laboratory strain of influenza (PR8) can inhibit the production of IFN, leading to enhanced viral replication and increased viral load [14]. In contrast, manipulation of single amino acid substitution at position 64 (I to T; I64T), decreases its ability to inhibit interferons, leading to decreased viral replication [15–17]. While the IAV NS1 protein is well studied for anti-type I IFN function, little is known about the antiviral ISGs response in circulating seasonal IAV strains and their possible contribution to growth capability. In this study, we confirmed that the inhibition of the JAK/STAT pathway permits the seasonal IAV strains with low growth capability to enhance their viral copies by suppressing ISG-15, IFITM1, and TRIM22. This finding suggests that the difference in the growth capability of seasonal IAV strains could be due to the regulation of antiviral ISGs.

Cells and viruses

A549 cells (human lung epithelial cells) were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM, FUJIFILM Wako, Japan) supplemented with 10% fetal bovine serum (FBS, Gibco) and penicillin-streptomycin at 37°C in an atmosphere of 5% CO2.

From 21 seasonal Influenza A virus culture positive nasal swab and nasopharyngeal aspirate samples collected from hospitals in Tottori prefecture; Tsuchie Internal Medicine and Pediatric Clinic, Sakaiminato, Department of Pediatrics, Tottori Prefectural Kousei Hospital, Kurayoshi, Kasagi Children’s Clinic for Health Service, Yonago, and Tanaka Pediatric Clinic, Tottori, Japan, 7 seasonal IAV representative strains were evaluated for IFN-β and ISGs expression. A reference strain (A/Puerto Rico/8/1934(H1N1), PR8) and seven epidemic strains (A/Tottori/ST215/2009(H1N1), 1; A/Tottori/ST1349/2014(H3N2), 2; A/Tottori/ST488/2014(H3N2), 3; A/Tottori/ST488/2013(H1N1), 4), A/Tottori/TT039/2011(H1N1) 5; A/Tottori/ST1705/2014(H3N2), 6) (A/Tottori/ST777/2011(H3N2), 7) were prepared and stored in a deep freezer (-80°C) until ready for use.

Virus infection of cells

A549 cells (3.0x10⁵) were washed twice with phosphate-buffered saline (PBS) and inoculated with seasonal IAV strains. Briefly, an aliquot (500 µL) of 10⁷ copies of virus was inoculated into the cells. After 1 h of inoculation, cells were washed twice with PBS and maintained in Dulbecco's modified Eagle’s complete medium supplemented with 5µg of trypsin (Difco™ Trypsin 250, Becton Dickinson, Japan) per ml, 0.2% heat-inactivated bovine serum albumin, 4 mM L-glutamine, 200 units of penicillin G per ml, and 100μg of streptomycin per ml, at 34°C in 5% CO2/95% air atmosphere. The cells were further incubated for 6 h and 24 h. Cells were inoculated for 6 h, and the culture supernatants were subjected to viral load and cell pellets for gene expression evaluation. At 24 h post-infection, the cells were collected for IFN-β and ISG-15 protein expression.

RNA extraction and Polymerase Chain Reaction

Influenza RNA in the culture supernatant (140 µL) was extracted using a QIAamp Viral RNA Mini Kit (QIAGEN, Japan) and subjected to quantitative reverse transcription PCR. The amount of influenza RNA was assayed using the Luna Universal One-Step qRT–PCR kit (New England Biolabs, Japan) and forward (MAT-F, 5‘-CTTCTAACCGAGGTCGAAACGTA-3’) and reverse (MAT-R, 5‘-GGTGACAGGATTGGTCTTGTCTTTA-3’) primers. The PCR signal was assessed by relating to a standard curve (encompassing 10² to 10⁹ copies/reaction of in vitro transcribed RNA transcript).

IFN-β and ISGs expression

Total RNA was isolated using RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer`s protocol. RNA quality was evaluated using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, USA). cDNAs were synthesized using Superscript III cDNA synthesis kit (Invitrogen, Japan) and quantification of gene expression was performed on a StepOne Real-Time PCR System (Applied Biosystems, USA) using the SYBR green method. Predesigned primers for expression analysis of IFN-β (sense: 5‘-AACTGCAACCTTTCGAAGCC-3’; antisense: 5‘-TGTCGCCTACTACCTGTTGTGC-3’), ISG-15 (sense:5‘-GAGAGGCAGCGAACTCATCT-3’; antisense: 5‘-CTTCAGCTCTGACACCGACA-3`), IFITM1 (sense:5‘-ACTCCGTGAAGTCTAGGGACA-3’; antisense: 5‘- TGTCACAGAGCCGAATACCAG-3’), and TRIM22 (sense: 5‘-GGGTGGACGTGATGCTGAA-3’; antisense:5‘-TCACTTGTCTCTGATCCACAGAAATA-3’). The data were normalized to 18S rRNA (sense: 5‘-GGAGCCTGCGGCTTAATTTG-3’; atisense5‘- CCACCCACGGAATCGAGAAA-3’) expression using the relative standard curve method [18].

IFN-β and ISG-15 protein production

Cell lysis buffer (Wako, Fujifilm, Japan) supplemented with protease inhibitor cocktail set I (Wako, Fujifilm, Japan) was used to prepare the cell lysate from 24 hours-inoculated with low growth capability strains for A549 cells. The concentrations of human IFN-β (DuoSet, R&D Systems、Japan) and ISG-15 (Circulex, MBL Life Sciences、Japan) were measured using an ELISA kit, according to the manufacturer’s instructions.

JAK inhibition

A549 cells were treated with 5μM pyridone 6 [2-(1,1-dimethylethyl)-9-fluoro-1,6-dihydro7H-benz[h]imidazo[4,5-f]isoquinolin-7-one (CMP6)] (Funakoshi frontiers in life science, Japan), a Janus-associated kinase inhibitor 1 (JAK-1) [19]. The JAK inhibitor was prepared as a 10mM stock concentration in DMSO. Following pretreatment of cells with 5μM JAK inhibitor and DMSO as a control for 24 h, the medium was removed, and the cells were rinsed with PBS before infection with low growth capability IAV strains. At 6 h post-infection, ISG-15, IFITM1, and TRIM22 expression was quantified from total RNA.

NS1 sequencing

Total RNA was extracted using a QIAamp Viral RNA Mini Kit (QIAGEN, Tokyo, Japan). Eight RNA segments of influenza A virus were simultaneously reverse transcribed and amplified using a SuperScript™III One Step RT-Polymerase Chain Reaction (PCR) System (Life Technologies, Tokyo, Japan) with the MBTuni-12/MBTuni-13 primer pair [5,20]. The first-round PCR products were subjected to a second PCR to amplify NS1 using A-NS-M13 primers (sense: 5`- TGTAAAACGACGGCCAGTAGCAAAAGCAGGGTGACAAAGACA-3`; antisense: 5`- CAGGAAACAGCTATGACCAGTAGAAACAAGGGTGTTTTTTAT-3`). The nucleotide sequences of the amplified NS1 were determined using a BigDye® Terminator v3.1 Cycle Sequencing Kit in accordance with the manufacturer’s instructions (Life Technologies) and requested FASMAC (Atsugi City, Japan) to analyze the data.

Statistical analysis

Statistical analyses for quantitative reverse transcription PCR (qRT-PCR) data were performed using a relative expression spreadsheet-based method [21]. Statistical analysis of viral copies was performed using the t-test with GraphPad Prism software (version 8.0). P values of ≤ 0.05 is given one asterisks (*).

IAV showed diverse growth capability in vitro

The 21 seasonal Influenza A virus culture positive nasal swab and nasopharyngeal aspirate samples collected from hospitals in Tottori prefecture showed a diverse growth capability in vitro using A549 human lung epithelial cells (Fig. 1A). Four strains with the highest viral copies (1, 2, 3, and 4) and three strains with the lowest viral copies (5, 6, and 7) were selected to evaluate the IFN-β, ISG-15, IFITM1, and TRIM22 at 6 h post-infection.

IFN-β and antiviral ISGs expression was increased in A549 cells inoculated with low growth capability IAV strains

To gain insight into the differences in the growth capability of seasonal IAV strains, gene expression of IFN-β and antiviral ISGs (ISG-15, IFITM1, and TRIM22) was determined using A549 cells. We obtained 4 and 3 seasonal IAV strains with high and low growth capabilities, respectively. Remarkably, in strains with low growth capability, the IFN-β and ISGs expression was parallel; the high expression of IFN-β upregulated the expression of antiviral ISGs. In contrast, the expression of IFN-β and ISGs in IAV with high growth capability was downregulated (Fig. 1B).

Ifn-β And Isg-15 Protein Production

Seasonal IAV with low growth capabilities showed increased expression of IFN-β. Moreover, among the antiviral ISGs, the ISG-15 has the highest expression. To determine whether the difference in growth capability was due to the expression of IFN-β and ISG-15 in A549 cells, the levels of protein expression were confirmed by ELISA 24 h post-infection. The results showed a significant difference in IFN-β and ISG-15 production when A549 cells were inoculated with low growth capability strains compared to high growth capability strains (Fig. 1C-D).

JAK/STAT pathway inhibition increased the susceptibility of A549 cells to seasonal IAV strains with low growth capability

To assess the role of ISGs (ISG-15, IFITM1, and TRIM22) in the growth capability of seasonal IAV, A549 cells were treated with 5µM pyridine 6 (P6), a pan-JAK inhibitor known to shut down the ISG expression [19, 22]. As expected, the expression of ISG-15, IFITM1, and TRIM22, were significantly reduced in P6-treated A549 cells (Fig. 2A-C). The suppression of viral ISGs in A549 cells using P6 resulted in a significantly increased viral copies (p < 0.05) at 6 h post-infection of seasonal IAV strains with low growth capability (Fig. 2D).

Ns1 Mutations Are Present In The Circulating Seasonal Iav Strains

Comparative analyses of the various NS1 point mutations of wild-type (WT) and mutant strains have been identified to influence IAV replication, which has provided links to the growth capability of the virus. Here, we checked the NS1 sequences of each seasonal influenza A strain and compared them with existing NS1 mutations that may influence the viral load (Table 1). Notably, all strains with high growth capability had G45R and K66E combination mutations, which is similar to the control strain PR8. Alternatively, strains with low growth capability have I64T and E55X combination mutations.

Table 1

NS1 mutations present in seasonal Influenza A virus that may influence the viral replication
Strain ID	Viral RNA, 6 h^a	NS1 mutations that may influence the viral replication of IAV
		Increase		Decrease
		G45R[14]	K66E[38]	I64T[15–17]	E55X^b[35, 39]
1	3.30 x10⁶	R	E	-	-
2	1.31 x10⁶	R	E	-	K
3	2.28 x10⁵	R	E	-	K
4	2.19 x10⁵	R	E	-	K
5	2.72x10⁴	R	-	T	N
6	1.67 x10⁴	-	-	T	H
7	1.89 x10³	-	-	T	H
^aAmount of IAV-RNA (copies/mL) detected in culture supernatant of A549 cells at 6 hours post-infection.
^bAmino acid X denotes any amino acid mutation

The growth capability of the influenza A virus is influenced by interactions between the virus and host regulation factors, and IFN-β and antiviral ISGs are the primary mediators of the host’s innate immune response. These molecules are induced following viral infection and establish an antiviral state in both infected and neighboring cells by stimulating the expression of antiviral genes, known as interferon-stimulated genes (ISGs) [23]. Since viral replication is necessary for disease pathogenesis in IAV infections, information on the correlation between viral load and antiviral ISGs can provide answers to the difference in the growth capability of seasonal IAV strains.

In this study, the differences in the growth capability of seasonal IAVs were dependent on the expression of IFN-β and antiviral ISGs (ISG-15, IFITM1, and TRIM22) in A549 cells. The control strain PR8, with a high growth capability was associated with low gene expression of IFN-β and antiviral ISGs (Fig. 1B). We found similar patterns of decreased cell gene expression in circulating seasonal IAV strains with high growth capability. Interestingly, strains with low growth capability have an inverse pattern of IFN-β and ISGs expression, which indicates that the growth capability is due to strain-dependent regulation of IFN-β and antiviral ISGs.

The interferon (IFN) family plays a major role in the host’s innate response by restricting the early stages of IAV infection [24]. However, the IAV possesses IFN-β antagonist activity, which can provide a favorable environment for replication [25]. The viral mechanisms involved in the suppression of the IFN-β response have been intensively investigated, and it has been postulated to be inhibited mainly by blocking IFN-β expression [12, 26–29]. In an in vitro study, it had been reported that A549 cells inoculated with IAVs expressed lower levels of IFN-β, indicating the effect of the viral factors in inhibiting the expression of IFN-β [26]. In contrast to strains with high growth capability, we found that circulating IAVs with low growth capability significantly upregulated the gene expression of IFN-β and viral ISGs.

It is well recognized that the inhibition of JAK/STAT pathway results in reduced ISG expression [30]. To establish the influence of antiviral ISGs on the growth capability of circulating seasonal IAVs, we inhibited the JAK/STAT pathway, which regulates the expression of ISG-15, IFITM1, and TRIM22. We found a significantly lower expression of antiviral ISGs after JAK/STAT inhibition and a significant increase in the viral copies of seasonal IAV strains with low growth capability. This finding suggests that the suppression of ISGs resulted in enhanced viral replication of seasonal IAV in A549 cells. A similar study using laboratory strain IAV also reported that the same pattern as JAK/STAT inhibition by a pan-JAK inhibitor reversed the antiviral state of ISGs and resulted in increased IAV replication in vitro [19]. Antiviral ISGs exert their antiviral function at every stage of the influenza virus replication cycle through different mechanisms, such as direct targeting of viruses, inhibiting translation, or blocking the release of virions [11, 31]. Among these ISGs, the ubiquitin-like protein ISG-15 is strongly induced by IFN-β and is one of the most potent host responses that can directly inhibit IAV replication [32]. ISG-15 targets the NS1 protein by conjugation, resulting in loss of its function, thereby inhibiting viral replication [33]. Alternatively, IFITM1, an interferon-induced transmembrane protein, is significantly upregulated by IFN-β stimulation. IFITM1 was proven to be more effective in resisting the influenza virus infection by blocking the fusion of the viral particle with the cellular host membrane during the uncoating step [34, 35]. TRIM22 expression is also upregulated by IFN-β in response to IAV infection and has been reported to be a marker of activation of the type I IFN system in A549 cells. The restriction mechanism of TRIM22 appears to directly target the nucleoprotein and cause its degradation, inhibition of IAV replication [36, 37].

In this study, we showed that the high expression of antiviral ISGs in seasonal IAV inoculated A549 cells could influence the regulation of viral replication, providing the existence of high and low growth capability strains. Although the NS1 mutations could be the reason for this difference (Table 1), these findings suggest that circulating seasonal IAV with low growth capability can be explained by the inability of the virus to prevent the inhibitory action of IFN-β and antiviral effects of ISGs. It has been reported that NS1 point mutations at positions 45 and 66 can enhance NS1 function to counteract the interferon response. In the present study, all high growth capability strains had G54R and K66E mutations that could probably increase the viral load [14, 38]. Remarkably, all low growth capability strains have I64T and E55X mutations that diminish the function of NS1 to counteract the effect of interferons, leading to decreased viral load [15–17, 35, 39].

In this study, we have demonstrated the influence of ISGs on the growth capability of seasonal IAV in vitro by suppressing the JAK/STAT pathway. Inhibition of ISGs expression increases the viral copies of seasonal IAV, thus, the existence of high and low growth capabilities may be caused by differences in IFN-β and ISGs responses. We also found that the seasonal IAV strains with high growth capability has G54R and K66E and low growth capability strains with I64T and E55X mutations. Several studies reported that NS1 mutations are involved in the growth capability of mouse-adapted laboratory strain of IAV [14–17, 35, 38–40], thus, the occurrence of these mutations might be one of crucial factors to cause differences in IFN-β and ISGs expression. This evidence provides a new insight into the mechanism of seasonal IAV replication that low growth capability and activation of IFN-β and JAK/STAT pathway by IAV infection could lead to excessive production of antiviral ISGs, resulting in decreased viral copies in vitro. This evidence may contribute in developing new effective therapeutic strategies and reduce the risk of developing severe influenza disease.

IAV

Influenza A virus

IFN-β

Interferon-beta

ISG

Interferon-stimulated genes:ISG-15:Interferon-stimulate gene 15

IFITM1

Interferon induced transmembrane protein 1

TRIM22

Tripartite Motif Containing 22

JAK/STAT

Janus kinase/signal transducer and activator of transcription

Pyridone 6.

Ethics approval and consent to participate

The present study was conducted with the approval and under the control of the Institutional Review Board of the Faculty of Medicine, Tottori University, Japan (no. 1981). All procedures performed in studies involving human participants were in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author contributions

Conceived and designed the experiments: AJrH, KK, and SeK; performed the experiments: AJrH and KK; Assisted the laboratory works: ATT, SoK、WYP, and YA. Analyzed the data: AJrH and KK; Wrote the paper: AJrH and KK; Comprehensive reading the manuscript: all authors. All authors read and approved the final manuscript.

Acknowledgements

We thank Dr. Hideaki Tsuchie, Tsuchie Internal Medicine and Pediatric Clinic, Sakaiminato, Dr. Takayoshi Okada, Department of Pediatrics, Tottori Prefectural Kousei Hospital, Kurayoshi, Dr. Masaaki Kasagi, Kasagi Children’s Clinic for Health Service, Yonago and Dr. Kiyoshi Tanaka, Tanaka Pediatric Clinic, Tottori for their support in collecting clinical specimens.

Availability of data and materials

Not applicable

Funding

Not applicable

Authors information

Alfredo Jr A. Hinay: [email protected]

Sosuke Kakee: [email protected]

Seiji Kageyama: [email protected]

Akeno Tsuneki-Tokunaga: [email protected]

Waldy Yudha Perdana: [email protected]

Yui Akena^: [email protected]

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No competing interests reported.

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Suppression of Interferon Stimulated Genes Enhance Viral Replication of Seasonal Influenza A Virus through the JAK/STAT pathway

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Background

Methods

Results

Ifn-β And Isg-15 Protein Production

Ns1 Mutations Are Present In The Circulating Seasonal Iav Strains

Discussion

Conclusion

List Of Abbreviations

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References

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