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. 2022 Mar 23;96(6):e0011922.
doi: 10.1128/jvi.00119-22. Epub 2022 Feb 16.

N-Acetyltransferase 8 Promotes Viral Replication by Increasing the Stability of Enterovirus 71 Nonstructural Proteins

Xiaohui Zhao  1 , Huijun Yuan  1 , Hang Yang  1 , Yan Liu  1 , Meng Xun  1 , Xiaozhen Li  1 , Tingting Fan  1 , Bo Wu  1 , Shangrui Guo  1 , Hongliang Wang  1   2
Affiliations

N-Acetyltransferase 8 Promotes Viral Replication by Increasing the Stability of Enterovirus 71 Nonstructural Proteins

Xiaohui Zhao et al. J Virol. .

Erratum in

Abstract

Enterovirus 71 (EV71) is deemed a reemergent pathogen, with recent outbreaks worldwide. EV71 infection causes hand, foot, and mouth disease (HFMD) and has been associated with severe cardiac and central nervous system complications and even death. Viruses need host factors to complete their life cycle; therefore, the identification of the host factors for EV71 infection is pivotal to new antiviral research. Emerging evidence has highlighted the importance of protein acetylation during infection by various human viruses. The endoplasmic reticulum (ER), as the prominent organelle of EV71 replication, also has a unique acetylation regulation mechanism. However, the pathogenesis of EV71 and its relationship with the ER-based acetylation machinery are not fully understood. In this study, we demonstrated for the first time that the ER-resident acetyltransferase N-acetyltransferase 8 (NAT8) is a host factor for EV71 infection. Inhibiting NAT8 with CRISPR or a small compound significantly suppressed EV71 infection in SK-N-SH cells. NAT8 promoted EV71 replication in an acetyltransferase-activity-dependent manner. Additionally, we found that NAT8 facilitates EV71 infection by interacting with EV71 2B, 3AB, and 3C proteins and increasing the stability of these proteins. These results uncovered a novel function of NAT8 and elucidated a new mechanism underlying the regulation of EV71 replication. IMPORTANCE EV71 is one of the most common pathogens causing HFMD in young children, and some patients experience severe or fatal neurological consequences. To ensure efficient replication, the virus must hijack multiple host factors for its own benefit. Here, we show that the ER-resident acetyltransferase NAT8 is a host factor for EV71 infection. EV71 fails to complete its infection in various cells in the absence of NAT8. We further show that NAT8 benefits EV71 replication in an acetyltransferase-activity-dependent manner. Finally, we show that NAT8 facilitates EV71 infection by interacting with EV71 2B, 3AB, and 3C proteins and increasing the stability of these proteins. These results uncovered a novel function of NAT8 in EV71 infection and elucidated a new mechanism underlying the regulation of EV71 replication.

Keywords: acetylation; enterovirus; viral replication.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
CRISPR screening identified NAT8 as a vital host factor for EV71 infection in SK-N-SH cells. (A) Schematic diagram of the genome-wide CRISPR/Cas9 screening. A library of CRISPR/Cas9 KO SK-N-SH cells were infected with EV71, and cells resistant to viral infection were selected. Genes enriched were then compared with those from uninfected cells. (B) SK-N-SH cells stably transduced with lentivirus encoding Cas9 and a sgRNA targeting the indicated gene were immunoblotted for the indicated proteins. (C) SK-N-SH cell pools as described in panel B were infected with EV71 at an MOI of 5, and CPE was examined by light microscopy at 24 hpi. Bar, 100 μm. (D) SK-N-SH cell pools as described in panel B were infected with EV71, and cell viability was determined with the CellTiter-Glo assay at 24 hpi. *, P < 0.05, compared to control sgRNA. (E) SK-N-SH cell pools as described in panel B were infected with EV71, and viral RNA was determined with RT-qPCR at 24 hpi. **, P < 0.01, compared to control sgRNA. (F) SK-N-SH cell pools as described in panel B were infected with EV71, and cells were then immunoblotted for VP1 and β-actin. (G) Control cells, NAT8 KO cells, and NAT8 KO cells transduced with sgRNA-resistant NAT8 were infected with EV71 and immunoblotted with the indicated antibodies. (H) Stably transduced cells as described in panel G were infected with EV71, and cell viability was determined 24 h later. **, P < 0.01 for NAT8 rescue, compared to no rescue.
FIG 2
FIG 2
NAT8 is required for EV71 replication. (A) Control or NAT8 KO cells were infected with NL-EV71 reporter virus, and luciferase activity was measured at the indicated time points postinfection. **, P < 0.01, compared to control sgRNA; NS, not significant. (B) NAT8 does not affect EV71 binding to the host cells. Viral RNA from the indicated SK-N-SH cells incubated with EV71 (MOI of 5) at 4°C for 1 h was determined by RT-qPCR. (C) NAT8 does not affect EV71 internalization. Viral RNA from the indicated SK-N-SH cells incubated with EV71 (MOI of 5) at 4°C for 1 h followed by 37°C for another 1 h was determined by RT-qPCR. (D) Control or NAT8 KO cells were transfected with EV71 SGR RNA, and luciferase activity was measured at the indicated time points postinfection. *, P < 0.05; **, P < 0.01, compared to control. (E) SK-N-SH cells infected with EV71 for 24 h were immunostained for dsRNA (red) and NAT8 (green). Nuclei were counterstained with DAPI (blue). Bar, 10 μm.
FIG 3
FIG 3
Acetyltransferase activity is required for NAT8 to support EV71 infection. (A) Control or NAT8 KO cells transduced with lentiviral vectors expressing wild-type or R149K mutant NAT8 were infected with EV71, and cell viability was determined at 24 hpi. **, P < 0.01 for NAT8 rescue, compared to no rescue; NS, not significant for R149K mutant rescue, compared to no rescue. (B) Cells as described in panel A were infected with EV71, and then they were immunoblotted with the indicated antibodies. (C) SK-N-SH cells infected with NL-EV71 were treated with the indicated concentration of compound 9 (C9), and luciferase activity was determined at 24 hpi. Cell viability was determined with the CellTiter-Glo assay. *, P < 0.05, compared to control; NS, not significant. (D) SK-N-SH cells infected with EV71 were treated with indicated concentrations of compound 9, and NAT8 and viral RNA were determined by RT-qPCR. *, P < 0.05, compared to control; NS, not significant. (E) Cells were treated as in panel D, and VP1 and NAT8 protein were determined by immunoblotting.
FIG 4
FIG 4
Compound 9 (C9) inhibits EV71 infection in different cell lines. (A to G) Upper, SH-SY5Y, HUVEC, Vero, RD, 293T, Caco2, and HepG2 cells were infected with NL-EV71 at an MOI of 1, 1, 1, 0.05, 0.1, 5, and 5, respectively; 4 h later, cells were treated with the indicated amount of compound 9. NL luciferase was determined at 24 hpi. Cell viability was measured with uninfected cells to indicate the cytotoxicity of compound 9. Lower, SH-SY5Y, HUVEC, Vero, RD, 293T, Caco2, and HepG2 cells were infected with EV71 at an MOI of 1, 1, 1, 0.05, 0.1, 5, and 5, respectively; 4 h later, cells were treated with the indicated amount of compound 9. VP1 expression was determined by immunoblotting at 24 hpi. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared to control; NS, not significant.
FIG 5
FIG 5
NAT8B and AT-1 are required for EV71 infection. (A) SK-N-SH cells stably transduced with lentivirus encoding Cas9 and a sgRNA targeting the indicated gene were immunoblotted for the indicated proteins. (B) SK-N-SH cells stably transduced with lentivirus encoding Cas9 and a sgRNA targeting the indicated gene were infected with EV71 and then immunoblotted for the indicated proteins. (C) SK-N-SH cell pools as described in panel B were infected with EV71 at an MOI of 5, and CPE was examined by light microscopy at 24 hpi. Bar, 100 μm. (D) SK-N-SH cell pools as described in panel B were infected with EV71, and cell viability was examined at 24 hpi. *, P < 0.05, compared to control sgRNA.
FIG 6
FIG 6
NAT8 interacts with the NS proteins. (A) 293T cells cotransfected with NAT8-Flag and plasmids encoding GFP-tagged 2B, 2C, 3AB, 3C, 3D, or OST48 were immunoprecipitated (IP) with anti-Flag antibody, and interactions between NAT8 and viral proteins were detected by Western blotting (immunoblot [IB]) analyses using anti-GFP antibody. (B) SK-N-SH cells overexpressing NAT8-Flag were infected with EV71 and immunoprecipitated with anti-Flag antibody, and interactions between NAT8 and 3AB or 3C protein were analyzed. (C) Colocalization of NAT8 and 2B, 3AB, and 3C in 293T cells. 293T cells cotransfected with NAT8-Flag and GFP-tagged 2B, 3AB, or 3C were immunostained with anti-Flag (red) and anti-GFP (green). Nuclei were counterstained with DAPI (blue). Bar, 10 μm. (D) Colocalization of NAT8 and 3AB or 3C in EV71-infected SK-N-SH cells. SK-N-SH cells overexpressing NAT8-Flag were infected with EV71 and immunostained with anti-Flag (green) and anti-3AB (upper, red) or anti-3C (lower, red). Nuclei were counterstained with DAPI (blue). Bar, 10 μm.
FIG 7
FIG 7
NAT8 regulates viral NS protein expression. (A) 293T cells were transfected with GFP-tagged 2B, 3AB, or 3C with increasing amounts of NAT8 expression vectors (0, 0.5, or 1 μg), and then the protein mount of NS proteins was examined by immunoblotting using an anti-GFP antibody. (B) 293T cells transfected with expression plasmids for GFP-tagged 2B, 3AB, or 3C were incubated in the presence of the NAT8 inhibitor compound 9 (C9) (0, 10, or 20 μM) for 24 h. The cell lysates were then immunoblotted with anti-GFP antibody.
FIG 8
FIG 8
NAT8 enhances the stability of NS proteins. (A) 293T cells transfected with a GFP-tagged 2B, 3AB, or 3C construct with or without NAT8-Flag coexpression were treated with cycloheximide (125 μM), and the amount of NS protein expression was determined with anti-GFP antibody at the indicated time points. (B) Control or NAT8-Flag-overexpressing SK-N-SH cells were infected with EV71; 24 h later, cells were treated with 125 μM cycloheximide, and the amounts of 3AB (left) or 3C (right) protein expression were determined at the indicated time points. (C) SK-N-SH cells were infected with EV71 for 4 h before they were treated with 0 or 10 μM compound 9; 24 h later, cells were treated with 125 μM cycloheximide, and the amounts of 3AB (left) or 3C (right) protein expression were determined at the indicated time points.
FIG 9
FIG 9
NAT8 stabilizes NS proteins by inhibiting autophagy-lysosome degradation. (A) Diagram of proteasome-mediated (upper) and autophagy-mediated (lower) protein degradation. The specific inhibitor (MG132 or BafA1) targeting each pathway is indicated. (B to D) 293T cells were transfected with expression plasmids for GFP-tagged 2B (B), 3AB (C), or 3C (D) and were incubated in the presence of the NAT8 inhibitor compound 9 (C9) (0, 10, or 20 μM) with MG132 (10 μM) or BafA1 (10 nM) treatment for 24 h. The cell lysates were then immunoblotted with anti-GFP antibody.
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