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. 2015 Apr;89(7):3671-82.
doi: 10.1128/JVI.03610-14. Epub 2015 Jan 14.

A host susceptibility gene, DR1, facilitates influenza A virus replication by suppressing host innate immunity and enhancing viral RNA replication

Shih-Feng Hsu  1 , Wen-Chi Su  2 , King-Song Jeng  3 , Michael M C Lai  4
Affiliations

A host susceptibility gene, DR1, facilitates influenza A virus replication by suppressing host innate immunity and enhancing viral RNA replication

Shih-Feng Hsu et al. J Virol. 2015 Apr.

Abstract

Influenza A virus (IAV) depends on cellular factors to complete its replication cycle; thus, investigation of the factors utilized by IAV may facilitate antiviral drug development. To this end, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNA interference (RNAi) screen. Knockdown (KD) of DR1 resulted in reductions of viral RNA and protein production, demonstrating that DR1 acts as a positive host factor in IAV replication. Genome-wide transcriptomic analysis showed that there was a strong induction of interferon-stimulated gene (ISG) expression after prolonged DR1 KD. We found that beta interferon (IFN-β) was induced by DR1 KD, thereby activating the JAK-STAT pathway to turn on ISG expression, which led to a strong inhibition of IAV replication. This result suggests that DR1 in normal cells suppresses IFN induction, probably to prevent undesired cytokine production, but that this suppression may create a milieu that favors IAV replication once cells are infected. Furthermore, biochemical assays of viral RNA replication showed that DR1 KD suppressed viral RNA replication. We also showed that DR1 associated with all three subunits of the viral RNA-dependent RNA polymerase (RdRp) complex, indicating that DR1 may interact with individual components of the viral RdRp complex to enhance viral RNA replication. Thus, DR1 may be considered a novel host susceptibility gene for IAV replication via a dual mechanism, not only suppressing the host defense to indirectly favor IAV replication but also directly facilitating viral RNA replication.

Importance: Investigations of virus-host interactions involved in influenza A virus (IAV) replication are important for understanding viral pathogenesis and host defenses, which may manipulate influenza virus infection or prevent the emergence of drug resistance caused by a high error rate during viral RNA replication. For this purpose, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNAi screen as a positive regulator in IAV replication. In the current studies, we showed that DR1 suppressed the gene expression of a large set of host innate immunity genes, which indirectly facilitated IAV replication in the event of IAV infection. Besides this scenario, DR1 also directly enhanced the viral RdRp activity, likely through associating with individual components of the viral RdRp complex. Thus, DR1 represents a novel host susceptibility gene for IAV replication via multiple functions, not only suppressing the host defense but also enhancing viral RNA replication. DR1 may be a potential target for drug development against influenza virus infection.

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Figures

FIG 1
FIG 1
DR1 acts as a positive regulator of IAV replication. A549 cells were transduced with the indicated lentiviruses for 10 days to generate stable cell lines and then infected with IAV for 6 h at an MOI of 1 for qRT-PCR assay or Western blotting. (A and B) The stable cells harboring shDR1-1, shDR1-2, or the shLacZ control were infected with IAV, harvested, and then subjected to qRT-PCR assays or Western blotting by using the indicated antibodies. Cellular DR1 mRNA and NP vRNA were measured and normalized to GAPDH mRNA, and the value for the control cells was set to 1. Data represent means ± standard deviations (SD) (n ≥ 3; *, P ≤ 0.05 by Student's t test). (C) For overexpression assay, overexpressed Myc-tagged DR1 (DR1-Myc OE) or control (vector only) cells at 8 dpt were infected with IAV, harvested, and then subjected to Western blotting. The band intensities of NP and actin were quantified, and the NP/actin ratios are shown below the blots. (D) The stable DR1 KD and shLacZ control A549 cells were infected with IAV at an MOI of 0.01. At 24 or 48 hpi, the supernatants were titrated by plaque assay to determine the virus titers. Data represent means ± SD (n ≥ 3; *, P ≤ 0.05 by Student's t test). (E) For the wobble mutant rescue experiment, the DR1 KD (shDR1-1) and shLacZ control cells were further transduced with a lentivirus overexpressing a DR1 wobble mutant (DR1-Myc-wobble) or the control (vector only) for an additional 8 days before being infected with IAV, and the cell lysates were subjected to Western blotting.
FIG 2
FIG 2
The expression levels of DR1 KD-induced signal transducers in host innate immunity were analyzed by qRT-PCR assays in the absence of IAV infection. Cellular signal transducer mRNAs were measured and normalized to GAPDH mRNA, with the value for each gene in the control cells set to 1. Data represent means ± SD (n ≥ 3; *, P ≤ 0.05 by Student's t test).
FIG 3
FIG 3
DR1 KD effects on IFNB1 induction and ISG expression at different postransduction times in the absence of IAV infection. DR1 KD, ITCH KD, and shLacZ control A549 cells at 4, 7, or 10 dpt were infected with IAV for 6 h at an MOI of 1 for qRT-PCR assays or Western blotting. (A and B) Cellular DR1, ITCH, or IFNB1 mRNA levels were determined and normalized to the GAPDH mRNA level. The value for shLacZ control cells under each condition was set to 1. Data represent means ± SD (n ≥ 3; *, P ≤ 0.05 by Student's t test). (C) DR1 KD and shLacZ control HEK293T cells at 4 or 10 dpt were cotransfected with pIFN-FLuc and pRL-TK. At 24 h posttransfection, the total cell lysates were subjected to luciferase activity assay. The value for the control cells was set to 1. Data represent means ± SD (n ≥ 3; *, P ≤ 0.05 by Student's t test). (D) Cell lysates obtained under each condition were subjected to Western blotting by using the indicated antibodies. (E) ISG mRNA levels under each condition were determined and normalized to those of GAPDH mRNA. The value for shLacZ control cells under each condition was set to 1. Data represent means ± SD (n ≥ 3; *, P ≤ 0.05 by Student's t test).
FIG 4
FIG 4
DR1 KD effects on IAV replication with early or prolonged DR1 KD. (A and B) DR1 KD and shLacZ control A549 cells at 4 or 10 dpt were infected with IAV for 6 h at an MOI of 1 for qRT-PCR assays or Western blotting by using the indicated antibodies. NP vRNA levels were determined and normalized to the GAPDH mRNA level. The value for shLacZ control cells under each condition was set to 1. Data represent means ± SD (n ≥ 3; *, P ≤ 0.05 by Student's t test). (C and D) DR1 KD and shLacZ control A549 cells at 4 or 10 dpt were infected with IAV at an MOI of 10 (at 37°C) for the indicated times, harvested, and then subjected to Western blotting using the indicated antibodies. The band intensities for M1 and actin were quantified, and the M1/actin ratios are shown below the blots.
FIG 5
FIG 5
Abolishment of DR1 KD-associated ISG induction restored IAV replication. A549 cells were transduced with shRNAs as indicated at the top and then infected with IAV at an MOI of 1 for 6 h. Cells harboring both shLacZ and shLuc served as a negative control. The cell lysates were subjected to Western blotting using the indicated antibodies. The band intensities for NP and actin were quantified, and the NP/actin ratios are shown below the blots.
FIG 6
FIG 6
Early DR1 KD affects viral RNA transcription and replication. (A) DR1 KD and shLacZ control A549 cells at 4 dpt were infected with IAV at an MOI of 5 (at 37°C) for 15 or 25 min, harvested, and then subjected to subcellular fractionation of total RNA. The NP vRNA in different fractions was measured by qRT-PCR and normalized to U6 snRNA or GAPDH mRNA for the nuclear fraction or the cytoplasmic fraction, respectively. The percentage of vRNA was the ratio of the amount of vRNA from each fraction to the amount of vRNA from whole cells. Data represent the means for two independent experiments. (B and C) DR1 KD and shLacZ control A549 cells at 4 dpt were infected with IAV at an MOI of 1 for the indicated times, harvested, and then subjected to qRT-PCR assays. Data represent means ± SD (n ≥ 3; *, P ≤ 0.05 by Student's t test). (D) DR1 KD and shLacZ control HEK293T cells at 4 dpt were transfected with PB1, PB2, PA, and NP mRNAs, RLuc-mRNA, and FLuc-vRNA (3P/NP), or only with FLuc-vRNA and RLuc-mRNA, as negative controls (mock). At 24 h posttransfection, the total cell lysates were subjected to luciferase activity assay. The value for the control cells transfected with 3P/NP was set to 1. Data represent means ± SD (n ≥ 3; *, P ≤ 0.05 bt Student's t test).
FIG 7
FIG 7
In vitro and in vivo pulldown assays of DR1 with distinct components of the viral RdRp complex. (A) HEK293T cells were cotransfected with the indicated plasmids expressing individual HA-tagged viral proteins and Myc-tagged DR1. Cell lysates were harvested at 48 h posttransfection and immunoprecipitated (IP) by using anti-HA agarose. The resultant products were subjected to Western blotting. The input cell lysates are shown in the left panel. (B) HEK293T cells were transfected with or without Myc-tagged DR1 (DR1-Myc) for 48 h before infection with or without IAV at an MOI of 5 for 10 h. The cell lysates were immunoprecipitated by using protein G-agarose conjugated with the indicated antibodies. The resultant products were subjected to Western blotting by using the indicated antibodies. The input cell lysates are shown in the left panel.
FIG 8
FIG 8
Proposed model of DR1 functions in IAV replication. DR1 may involve the following dual mechanism in IAV replication: (i) DR1 modulates the expression of cellular genes involved in IFN production and/or action to suppress host innate immunity, providing a beneficial environment to favor IAV replication; and (ii) DR1 facilitates the viral RdRp activity through interacting with the viral RdRp complex and enhancing either the catalytic activity of the viral polymerase or the supply of precursor pre-mRNA for cap-snatching function. As such, DR1 represents a new class of host susceptibility genes for IAV replication. The question marks indicate issues yet to be investigated.
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References

    1. Fouchier RA, Munster V, Wallensten A, Bestebroer TM, Herfst S, Smith D, Rimmelzwaan GF, Olsen B, Osterhaus AD. 2005. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 79:2814–2822. doi:10.1128/JVI.79.5.2814-2822.2005. - DOI - PMC - PubMed
    1. Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. 1992. Evolution and ecology of influenza A viruses. Microbiol Rev 56:152–179. - PMC - PubMed
    1. Morens DM, Taubenberger JK, Fauci AS. 2009. The persistent legacy of the 1918 influenza virus. N Engl J Med 361:225–229. doi:10.1056/NEJMp0904819. - DOI - PMC - PubMed
    1. Carr CM, Kim PS. 1994. Flu virus invasion: halfway there. Science 266:234–236. doi:10.1126/science.7939658. - DOI - PubMed
    1. Su WC, Chen YC, Tseng CH, Hsu PW, Tung KF, Jeng KS, Lai MM. 2013. Pooled RNAi screen identifies ubiquitin ligase Itch as crucial for influenza A virus release from the endosome during virus entry. Proc Natl Acad Sci U S A 110:17516–17521. doi:10.1073/pnas.1312374110. - DOI - PMC - PubMed

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