Novel Ionophores Active against La Crosse Virus Identified through Rapid Antiviral Screening

Abstract

Bunyaviruses are significant human pathogens, causing diseases ranging from hemorrhagic fevers to encephalitis. Among these viruses, La Crosse virus (LACV), a member of the California serogroup, circulates in the eastern and midwestern United States. While LACV infection is often asymptomatic, dozens of cases of encephalitis are reported yearly. Unfortunately, no antivirals have been approved to treat LACV infection. Here, we developed a method to rapidly test potential antivirals against LACV infection. From this screen, we identified several potential antiviral molecules, including known antivirals. Additionally, we identified many novel antivirals that exhibited antiviral activity without affecting cellular viability. Valinomycin, a potassium ionophore, was among our top targets. We found that valinomycin exhibited potent anti-LACV activity in multiple cell types in a dose-dependent manner. Valinomycin did not affect particle stability or infectivity, suggesting that it may preclude virus replication by altering cellular potassium ions, a known determinant of LACV entry. We extended these results to other ionophores and found that the antiviral activity of valinomycin extended to other viral families, including bunyaviruses (Rift Valley fever virus, Keystone virus), enteroviruses (coxsackievirus, rhinovirus), flavirivuses (Zika virus), and coronaviruses (human coronavirus 229E [HCoV-229E] and Middle East respiratory syndrome CoV [MERS-CoV]). In all viral infections, we observed significant reductions in virus titer in valinomycin-treated cells. In sum, we demonstrate the importance of potassium ions to virus infection, suggesting a potential therapeutic target to disrupt virus replication.

Keywords: La Crosse virus; antivirals; bunyaviruses; ionophores.

FIG 1

Rapid screening for LACV antivirals. Schematic of the screen performed in these studies. (A) Potential antivirals were added to cells 2 h prior to infection with La Crosse virus at an MOI of 0.01. At 48 hpi, cells were fixed and stained with crystal violet stain, which was then quantified, with darker-staining cells surviving drug treatment and virus infection. (B) Quantification of antiviral activity. Each dot represents a single compound analyzed in our assay. (C) The quantification of antiviral activity as represented in panel B was compared to control cells that were not treated (sample Abs/NT Abs) to obtain the relative antiviral activity. The top hits in this screen are indicated. (D to G) Huh7 cells were pretreated for 2 h with increasing doses of (D) valinomycin, (E) lagistase, (F) lapachol, and (G) superacyl and subsequently infected. Viral titers were determined at 24 hpi. The dotted line indicates titers from untreated samples.

FIG 2

Valinomycin is antiviral. Huh7 cells were treated with increasing doses of valinomycin for 2 h prior to LACV infection. (A) At 48 hpi, cells were stained with crystal violet and quantified. (B) Viral titers were measured with a plaque assay. The dashed line indicates the titer of untreated controls. Lines indicate the IC₅₀ value. (C) Viability was measured using a fluorescence intracellular ATP assay. Lines indicate the CC₅₀ value. (D) Vero cells were treated and infected as described for the Huh7 cells. Viral titers were determined at 48 hpi. Lines indicate the IC₅₀ value. *, P < 0.05; **, P < 0.01; ***, P < 0.0001 using two-tailed Student’s t test; n = 3. Error bars represent one standard error of the mean.

FIG 3

Valinomycin is antiviral over multiple rounds of infection. Huh7 cells were treated with 2 μM valinomycin for 2 h prior to infection at an MOI of 0.01. (A) Cellular supernatant was collected at the times indicated, and viral titers were determined with a plaque assay. (B) Viral RNA was purified from infected cell supernatant at 48 hpi and quantified by qPCR using primers specific to each genome segment. (C and D) Huh7 cells were treated with (C) 2 μM and (D) 5 μM valinomycin at various times prior to and after infection. Virus titers were determined with a plaque assay at 24 hpi. *, P < 0.05; **, P < 0.01 using two-tailed Student’s t test; n = 2 or 3. Error bars represent one standard error of the mean.

FIG 4

Valinomycin does not directly reduce particle infectivity. (A) Stock LACV was incubated with 2 μM valinomycin at 37°C for the times indicated before virus titer was determined with a plaque assay. (B) Stock virus was incubated with increasing doses of valinomycin at 37°C for 24 h prior to determination of the titer. (C) Viral genomes from panel B were quantified from purified RNA and compared to viral titers to calculate the relative viral genomes compared to infectious virus (PFU). No significant differences were observed. (D) Huh7 cells were treated with 2 μM valinomycin and subsequently washed with PBS and replenished with fresh medium as indicated. Fresh valinomycin was added as indicated following a PBS wash and medium replenishment. (E and F) Cells were similarly treated with (E) 2 μM or (F) 5 μM valinomycin and infected for 30 min before analysis of attached viral genomes, normalizing to input. (G) A similar analysis was also performed at 4 hpi. *, P < 0.05. NS, not significant using two-tailed Student’s t test; n ≥ 2. Error bars represent one standard error of the mean.

FIG 5

Ionophores are selectively antiviral. (A) Structures of valinomycin, nonactin, calcium ionophore I, nigericin, and sodium ionophore III. (B) Huh7 cells were treated with increasing doses of the iophores for 2 h prior to LACV infection. Virus titers were determined at 48 hpi with a plaque assay. The dotted line indicates titers from untreated (NT) control cells. n = 3. Error bars represent one standard error of the mean.

FIG 6

Valinomycin is broadly antiviral. (A to D) Huh7 cells were treated with increasing doses of valinomycin for 2 h prior to infection at an MOI of 0.1 with (A) bunyaviruses KEYV and RVFV (strain MP12) for 48 h, (B) flavivirus ZIKV for 48 h, (C) enteroviruses HRV2 and CVB3 for 24 h, and (D) coronaviruses HCoV-229E and MERS-CoV for 24 h. *, P < 0.05; **, P < 0.01; ***, P < 0.001 using two-tailed Student’s t test; n ≥ 2. Error bars represent one standard error of the mean.