Sirtuin Inhibitors Are Broadly Antiviral against Arboviruses

Abstract

Arthropod-borne viruses are diverse pathogens and are often associated with human disease. These viruses span multiple genera, including flaviviruses, alphaviruses, and bunyaviruses. In a high-throughput drug screen, we found that tenovin-1 was antiviral against the flaviviruses Zika virus and dengue virus. Tenovin-1 is a sirtuin inhibitor, and here we found that inhibition of sirtuins, but not inhibition of the related histone deacetylases, is potently antiviral against diverse arboviruses. Sirtuin inhibitors block infection of arboviruses in multiple human cell types. We found that sirtuin inhibitors arrest infection downstream of entry but that they do so at an early step, preventing the accumulation of viral RNA and protein. However, sirtuin inhibitors had no impact on the replication of flaviviral replicons, suggesting a defect in the establishment of replication. Consistent with this, we found that sirtuin inhibitors impacted double-stranded RNA (dsRNA) accumulation during flaviviral infection. Since these viruses infect vector insects, we also tested whether sirtuin inhibitors impacted infection of adult flies and found that these inhibitors blocked infection; therefore, they target highly conserved facets of replication. Taken together, these results suggest that sirtuin inhibitors represent a new class of potent host-targeting antivirals.IMPORTANCE Arthropod-borne viruses are diverse pathogens and are associated with human disease. Through high-throughput drug screening, we found that sirtuin inhibitors are potently antiviral against diverse arboviruses, including flaviviruses such as West Nile virus, bunyaviruses such as Rift Valley fever virus, and alphaviruses such as chikungunya virus. Sirtuin inhibitors block infection of these viruses in multiple human cell types. Moreover, we found that sirtuin inhibitors arrest infection downstream of entry but that they do so at an early step, preventing the accumulation of viral RNA and protein. Since these viruses infect vector insects, we also tested whether sirtuin inhibitors impacted infection of adult flies and found that these inhibitors blocked infection; therefore, they target highly conserved facets of replication. Taken together, these results suggest that sirtuin inhibitors represent a new class of potent host-targeting antivirals.

Keywords: alphavirus; arbovirus; bunyavirus; flavivirus; sirtinol; sirtuin.

FIG 1

Tenovin-1 is broadly antiviral against arboviruses. (A) U2OS cells were infected with the indicated virus (MOI of 0.5, 24 hpi) and at 2.5 h postinoculation, vehicle control (DMSO ctrl) or 2 μM tenovin-1 was added. Total cellular RNA was collected and analyzed by RT-qPCR. Data represent means ± standard errors of the means (SEM), n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student's t test). vRNA, viral RNA. (B) TCID₅₀ analysis of WNV-Kunjin and RVFV MP12 in U2OS cells, comparing vehicle control (DMSO) to 2 μM tenovin-1, added at 2.5 hpi. Data represent means ± SEM. n = 3; **, P < 0.01 (Student's t test). (C) U2OS cells were infected with WNV-Kunjin (MOI of 0.5, 24 hpi) for 2.5 h prior to addition of tenovin-1 at the indicated concentration. Percent infection was quantified by automated microscopy; data representing means ± SEM of results from two experiments are shown.

FIG 2

Sirtuin inhibitors, but not other HDAC inhibitors, are antiviral against diverse arboviruses. U2OS cells (A, C, and E) or HBMECs (B, D, and F) were infected with the indicated virus (MOI of 0.5 [ZIKV MOI of 2.5], 24 h). At 2.5 hpi, vehicle control or (A and B) 20 mM NaPB or (C and D) 200 nM panobinostat or (E and F) 200 μM sirtinol was added. Total cellular RNA was collected and analyzed by RT-qPCR. Data represent means ± SEM. n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student's t test). (G and H) U2OS cells were infected with (G) WNV-Kunjin or (H) RVFV-MP12 (MOI of 0.5, 24 hpi) for 2.5 h prior to addition of sirtinol at the indicated concentration. Percent infection was quantified by automated microscopy; means ± SEM of results from two experiments are shown. (I) TCID₅₀ analysis of WNV-K and RVFV MP12 in U2OS cells was performed with the indicated compound added at 2.5 hpi. Data represent means ± SEM. n = 3; n.s., not significant; **, P < 0.01 (Student's t test).

FIG 3

Selective inhibition of SIRT1 or SIRT2 is not antiviral. (A and C) U2OS cells were infected with the indicated virus (MOI of 0.5 [ZIKV MOI 2.5], 24 h). At 2.5 hpi, vehicle or (A) 100 nM SIRT1 inhibitor EX527 or (C) 22 μM SIRT2 inhibitor AGK2 was added. Total cellular RNA was collected and analyzed by RT-qPCR. Data represent means ± SEM. n = 3; *, P < 0.05 (Student's t test). (B and D) U2OS cells were infected with WNV-Kunjin (MOI of 0.5, 24 hpi) for 2.5 h prior to addition of (B) EX527 or (D) AGK2 at the indicated concentration. Percent infection was quantified by automated microscopy; means ± SEM of results from three experiments are shown.

FIG 4

Sirtinol blocks viral infection at the organismal level in adult flies. (A) Adult flies were treated with vehicle or sirtinol and infected with either WNV-Kunjin or RVFV-MP12. At 7 dpi, groups of 15 flies were processed for RT-qPCR analysis. Data represent means ± SEM. n = 3; *, P < 0.05; ****, P < 0.0001 (Student's t test). (B) Experiments were performed as described for panel A; adult flies were treated with vehicle or sirtinol and infected with WNV-Kunjin. Groups of 5 flies were processed 7 dpi and subjected to plaque assay normalized to control. Data represent means ± SEM. n = 3. *, P < 0.05 (Student's t test). (C and D) Adult flies of the indicated genotypes were treated with vehicle or sirtinol and infected with WNV-Kunjin as described for panel A. At 7 dpi, groups of 15 flies were processed for RT-qPCR analysis. Data represent means ± SEM. n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way analysis of variance [ANOVA] with multiple comparisons).

FIG 5

Sirtinol inhibits postentry steps prior to release but not established infection. (A) U2OS cells were treated with vehicle (DMSO), ammonium chloride (NH₄Cl), or sirtinol at the indicated time relative to WNV-Kunjin (MOI of 0.5, 24 h). At 2.5 hpi, NH₄Cl was added to all wells to block viral spread. Total cellular RNA was collected and analyzed by RT-qPCR. Data represent means ± SEM. n = 3; ****, P < 0.0001 (Student's t test compared to control). (B) 293T cells were infected with WNV-Kunjin (MOI of 0.5, 24 h), or 293T cells harboring WNV-Kunjin replicons were treated with vehicle (DMSO) or sirtinol (200 μM) for 24 h, and total cellular RNA was analyzed by RT-qPCR. Data represent means ± SEM. n = 3; ***, P < 0.001 (Student's t test).

FIG 6

Sirtinol treatment arrests dsRNA replication. (A and B) U2OS cells were left uninfected or infected with WNV-Kunjin (MOI of 1), and vehicle (DMSO) or sirtinol (200 μl) was added at 2 hpi. Following fixation at the indicated time point postinfection, cells were stained for dsRNA replication intermediates (anti-J2, green) and total nuclei (DAPI [4′,6-diamidino-2-phenylindole], blue). (B) Images representative of results of three independent experiments; representative higher-magnification images from DMSO controls are shown. (C) Quantification of granules shown in panel A. Data represent means ± SEM. n = 3; *, P < 0.05; **, P < 0.01 (Student's t test).