A Yeast Suppressor Screen Used To Identify Mammalian SIRT1 as a Proviral Factor for Middle East Respiratory Syndrome Coronavirus Replication

Abstract

Viral proteins must intimately interact with the host cell machinery during virus replication. Here, we used the yeast Saccharomyces cerevisiae as a system to identify novel functional interactions between viral proteins and eukaryotic cells. Our work demonstrates that when the Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a accessory gene is expressed in yeast it causes a slow-growth phenotype. ORF4a has been characterized as an interferon antagonist in mammalian cells, and yet yeast lack an interferon system, suggesting further interactions between ORF4a and eukaryotic cells. Using the slow-growth phenotype as a reporter of ORF4a function, we utilized the yeast knockout library collection to perform a suppressor screen where we identified the YDL042C/SIR2 yeast gene as a suppressor of ORF4a function. The mammalian homologue of SIR2 is SIRT1, an NAD-dependent histone deacetylase. We found that when SIRT1 was inhibited by either chemical or genetic manipulation, there was reduced MERS-CoV replication, suggesting that SIRT1 is a proviral factor for MERS-CoV. Moreover, ORF4a inhibited SIRT1-mediated modulation of NF-κB signaling, demonstrating a functional link between ORF4a and SIRT1 in mammalian cells. Overall, the data presented here demonstrate the utility of yeast studies for identifying genetic interactions between viral proteins and eukaryotic cells. We also demonstrate for the first time that SIRT1 is a proviral factor for MERS-CoV replication and that ORF4a has a role in modulating its activity in cells.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) initially emerged in 2012 and has since been responsible for over 2,300 infections, with a case fatality ratio of approximately 35%. We have used the highly characterized model system of Saccharomyces cerevisiae to investigate novel functional interactions between viral proteins and eukaryotic cells that may provide new avenues for antiviral intervention. We identify a functional link between the MERS-CoV ORF4a proteins and the YDL042C/SIR2 yeast gene. The mammalian homologue of SIR2 is SIRT1, an NAD-dependent histone deacetylase. We demonstrate for the first time that SIRT1 is a proviral factor for MERS-CoV replication and that ORF4a has a role in modulating its activity in mammalian cells.

Keywords: MERS-CoV; ORF4a; SIRT1; host-virus interaction; suppressor screen; virus-host interaction; yeast.

FIG 1

MERS-CoV ORF4a causes a slow-growth phenotype in yeast and a suppressor screen pulls out YDL042C/SIR2. (A) Yeast cells transformed with a galactose (Gal)-inducible plasmids to express MERS-CoV proteins were cultured for 2 days in media containing 2% raffinose to reach saturation. Yeast cells were diluted and cultured for 48 h in 2% Gal media at 30°C on a plate reader. Every 30 min, the plate was shaken for 30 s and the OD₆₀₀ was read. The growth curve has OD₆₀₀ plotted against time. Data are from one representative experiment performed using two separate colonies of yeast. Error bars represent deviations between results from the two colonies. (B) Yeast cells transformed with a Gal-inducible plasmid to express MERS-CoV ORF4a or a vector control were cultured for 2 days in media containing 2% raffinose to reach saturation. The OD₆₀₀ was measured, yeast cells were diluted to an OD₆₀₀ of 0.1 in 2% raffinose media, and then a 1:5 dilution series was made. This dilution series of the yeast was then replica plated on agar plates containing 2% glucose (Glu) or 2% Gal. (C) As described for panels A and B, vector control or ORF4a plasmid yeast cells were grown for 2 days in 2% raffinose to reach saturation. Yeast cells were diluted and cultured for 48 h in 2% Gal media at 30°C on a plate reader. Every 30 min, the plate was shaken for 30 s and the OD₆₀₀ was read. The growth curve has OD₆₀₀ plotted against time. Data are from three independent experiments, with error bars showing standard deviations. (D) The Gal-inducible ORF4a plasmid was transformed into a pool of the yeast knockout (YKO) library collection and plated onto agar plates containing Glu or Gal. A suppressor screen was performed by picking large colonies from the ORF4a YKO library on Gal plates and analyzing growth rates for colonies that no longer had the slow-growth phenotype caused by expression of ORF4a. The most commonly detected gene deletion was YDL042C/SIR2. (E) A representative graph for one of the large colonies picked from a Gal plate that was later determined to be ΔYDL042C/SIR2. (F) Western blot to demonstrate that the suppressor phenotype observed in ΔYDL042C/SIR2 colonies was not a result of a lack of ORF4a expression. (G) A known ΔYDL042C/SIR2 strain was picked from an arrayed collection of the YKO library and transformed with Gal-inducible ORF4a plasmid. Data show growth curves of these transformed yeast cells from 3 independent experiments (error bars represent standard deviations). (H) Western blot to confirm that ORF4a was still expressed in the transformed ΔYDL042C/SIR2 cells. (I) Control experiments testing whether ΔYDL042C/SIR2 yeast cells do or do not grow faster than wild-type yeast. SARS-CoV–ORF3b and an empty vector were transformed into yeast that were subsequently grown for 48 h in 2% raffinose media prior to growth to produce growth curves as described above.

FIG 2

Localization of ORF4a and SIRT1 in Huh7 cells. (A) Huh7 cells grown on coverslips were subjected to immunofluorescence staining with anti-SIRT1 antibody (visualized with anti-mouse Texas Red). (B) Huh7 cells grown on coverslips were transfected with a plasmid expressing ORF4a-GFP and were subsequently subjected to immunofluorescence labeling for SIRT1 (visualized with anti-mouse Texas Red). Arrows point toward nuclear and arrowheads point toward cytosolic ORF4a localizations. (C) Huh7 cells were plated to coverslips 1 day prior to infection with MERS-CoV at an MOI of 1 (uninfected cells were grown as a control). Infection was allowed to proceed for 18 h prior to fixation and immunofluorescence labeling for SIRT1 (visualized with anti-mouse Texas Red) and ORF4a (visualized with anti-rabbit FITC). Arrows point toward nuclear and arrowheads point toward cytosolic ORF4a localizations. In all experiments, nuclei were labeled with DAPI. Scale bars = 50 μm.

FIG 3

ORF4a and SIRT1 do not directly interact in mammalian cells. (A) Whole-cell lysates from untransfected (Unt.) Huh7 cells or cells transfected with wild-type SIRT1 or H363Y mutant SIRT1-Flag-tagged plasmids were subjected to Western blotting with anti-SIRT1 or anti-Flag antibodies. Short and long exposures were performed to allow visualization of endogenous SIRT1 as indicated. (B) Huh7 cells grown on coverslips were cotransfected with ORF4a-GFP and SIRT1-Flag plasmid prior to being subjected to immunofluorescence labeling with anti-Flag antibody (visualized with anti-mouse Texas Red). Nuclei were labeled with DAPI. Scale bar = 50 μm. (C) Huh7 cells transfected with ORF4a-GFP or with wild-type or H363Y SIRT1-Flag or subjected to cotransfection were lysed and subjected to immunoprecipitation (IP) using anti-GFP antibody. Whole-cell lysate and nonspecific (NS), flowthrough (FT), and IP fractions were all analyzed (see Materials and Methods). Membranes were subjected to Western blotting with anti-GFP antibody and anti-SIRT1 antibody. “HC” denotes the IgG heavy chain.

FIG 4

Sirtuin-modulating drugs impact MERS-CoV infection. (A) Huh7 cells were treated with the indicated concentrations of resveratrol at −2, 0, or +2 h relative to infection with MERS-CoV at an MOI of 0.1. Cells were incubated for 24 h and supernatant collected. Virus TCID₅₀ per milliliter in the supernatant was determined on Vero cells. Ethanol (EtOH) was the vehicle control for drug treatment. (B) As described for panel A, but cells were treated with EX527. For both panel A and panel B, data are from three independent experiments, with error bars representing standard deviations. (C) Cell viability was determined by CellTiter-Glo assay. Huh7 cells were treated with the indicated concentration of drug for 24 h prior to performance of the CellTiter-Glo assay to determine viability over a time period equivalent to that used in the MERS-CoV infection experiments. Error bars represent standard deviations of results of comparisons between 6 treatment wells from an experiment representing 3.

FIG 5

SIRT1 knockdown inhibits MERS-CoV replication. (A) Huh7 cells were transfected with one of two SIRT1-targeting siRNA sequences (siSIRT1-1 or siSIRT1-2) or scrambled siRNA as control. Cells were lysed and subjected to Western blotting for SIRT1. Results of long and short exposures of the blot are shown. (B) Cell viability of siRNA-transfected Huh7 cells was determined by CellTiter-Glo assay. Cells were plated 24 h prior to the assay to assess viability at a time that matched the time at which infections were performed. Lum., luminescence; A.U., arbitrary units. (C) siRNA-transfected cells were infected 24 h after plating with MERS-CoV was performed at an MOI of 0.1. Cells were incubated for 24 h prior to supernatant collection. Virus titer in the supernatant was determined by TCID₅₀ assay on Vero cells. Data are from 3 independent experiments, with error bars representing standard deviations. **, P < 0.01 (by Student’s t test). (D) Huh7 cells were transfected with wild-type or H363Y mutant SIRT1 plasmids and infected with MERS-CoV at an MOI of 0.1. Virus titer in supernatant was determined as described for panel C. (E) siRNA-transfected cells were infected with MERS-CoV at an MOI of 0.1 or 1 and additionally treated with 200 μM resveratrol or ethanol (EtOH) vehicle control (added at the same time as the virus). Virus titer in the supernatant was determined as described for panels C and D. ND = not detected. MOI 1 was additionally used to allow detection of virus in the double treated cells.

FIG 6

CRISPRi-mediated knockdown of SIRT1 inhibits MERS-CoV replication. (A) Two populations of Huh7 cells stably transfected with doxycycline (DOX)-inducible dCas9 were further transfected with two different lentiviral preparations containing a sgRNA sequence targeting SIRT1 to produce two independent populations of stable Huh7-dCas9-sgRNA cells. Each of the two populations was divided into two groups and either treated with DOX or grown in normal culture media for 7 days. Cells were lysed and samples subjected to Western blotting for SIRT1. (B) The stable Huh7-dCas9-sgRNA cells were grown for 7 days with or without DOX treatment and infected with MERS-CoV at an MOI of 0.1. For all cell groups, media were changed to normal growth media at the time of infection and samples were collected 24 h later to determine titer by TCID₅₀ assay on Vero cells. Data are from three independent infections, with error bars representing standard deviations. ****, P < 0.001 (by Student’s t test comparing DOX-treated and untreated samples). (C) The two populations of stable dCas9-Huh7-sgRNA cells were cultured as described for panel B and infected with MERS-CoV at an MOI of 0.1 or 1 and simultaneously treated with 200 μM resveratrol or ethanol (EtOH) vehicle control. Virus titer in supernatant was determined as described for panel B. Data are from a single infection; error bars represent standard deviations in calculations of TCID₅₀ per milliliter from three samples.

FIG 7

MERS-CoV ORF4a alters SIRT1 function in mammalian cells. (A) HEK293T cells were transfected with a κB-luciferase reporter plasmid and combinations of ORF4a-GFP, wild-type (WT) or H363Y mutant SIRT1 as labeled, and a GFP expressing plasmid to balance DNA levels (see Materials and Methods). Cells were lysed, and whole-cell lysates were Western blotted for SIRT1, GFP, and tubulin as a loading control. (B) As described for panel A, cells were transfected with various combinations of plasmids and then treated with TNF-α for 6 h prior to performing a firefly luciferase assay to assess production of luciferase from the κB promoter. Wild-type SIRT1 transfection significantly (P = 0.0132) enhanced luciferase production, as did H363Y SIRT1 (P = 0.0015), compared to cells with only the κB-luciferase plasmid (Luc. only). ORF4a significantly inhibited luciferase production (P = <0.0001) compared to the Luc.-only control. There was no significant difference when ORF4a and SIRT1 WT or H363Y were coexpressed in cells. Data are from analyses of three independent wells, with significance determined using one-way analysis of variance (ANOVA) with Bonferroni’s multiple-comparison test in Prism software. (C) Huh7 cells were transfected as described for panel A. Data from the Luc. only control and ORF4a data are the same as described for panel A; data are separated into two graphs for clarity. Cells were treated with resveratrol (Res) or EX527 (EX) at the indicated concentrations (in micromoles) for 2 h prior to addition of TNF-α and further incubation for 6 h. Luciferase production was determined as described for panel A. Drug treatment results are compared to those determined for the Luc. only cells (black annotation), and the results determined for ORF4a and drug are compared to those determined for ORF4a (red annotations) for significance testing (as described for panel B). Data are from three independent wells. (D) Control samples to assess background luminescence. Data are from experiments performed with the κB-luciferase reporter plasmid transfected along with a GFP expression plasmid or represent GFP expression only (no luc.) or no transfection. All samples were subjected to TNF-α treatment, with the exception of the no-TNF-α samples.