Inhibitor of Sarco/Endoplasmic Reticulum Calcium-ATPase Impairs Multiple Steps of Paramyxovirus Replication

Abstract

Sarco/endoplasmic reticulum calcium-ATPase (SERCA) is a membrane-bound cytosolic enzyme which is known to regulate the uptake of calcium into the sarco/endoplasmic reticulum. Herein, we demonstrate for the first time that SERCA can also regulate virus replication. Treatment of Vero cells with SERCA-specific inhibitor (Thapsigargin) at a concentration that is nontoxic to the cells significantly reduced Peste des petits ruminants virus (PPRV) and Newcastle disease virus (NDV) replication. Conversely, overexpression of SERCA rescued the inhibitory effect of Thapsigargin on virus replication. PPRV and NDV infection induced SERCA expression in Vero cells, which could be blocked by Thapsigargin. Besides inducing enhanced formation of cytoplasmic foci, Thapsigargin was shown to block viral entry into the target cells as well as synthesis of viral proteins. Furthermore, NDV was shown to acquire significant resistance to Thapsigargin upon long-term passage (P) in Vero cells. As compared to the P0 and P70-Control, the fusion (F) protein of P70-Thapsigargin virus exhibited a unique mutation at amino acid residue 104 (E104K), whereas no Thapsigargin-associated mutations were observed in HN gene. To the best of our knowledge, this is the first report describing the virus-supportive role of SERCA and a rare report suggesting that viruses may acquire resistance even in the presence of an inhibitor that targets a cellular factor.

Keywords: SERCA; antiviral drug resistance; host-targeting antiviral agents; paramyxovirus; virus replication.

Figure 1

Antiviral efficacy of Thapsigargin. Cytotoxicity (MTT assay): five-fold serial dilutions of Thapsigargin (50–0.00064 μM) or equivalent volume of vehicle control (0.05% DMSO) were incubated with cultured Vero/MDBK cells for 96 h, and percentage of cell viability was measured by MTT assay. Cell viability of Vero (A) and MDBK (B) cells is shown. Antiviral efficacy: Vero/MDBK cells were infected with respective viruses at MOI = 0.1 in the presence of indicated concentration of Thapsigargin or vehicle control (0.05% DMSO). Progeny virus particles released in the supernatants were quantified by plaque assay. Antiviral efficacy of Thapsigargin against NDV (C), PPRV (D), BHV-1 (E), and BPXV (F) is shown. Virucidal activity: 10-fold dilutions of the Thapsigargin or DMSO were mixed with ~10⁶ pfu of the indicated viruses and incubated for 1.5 h at 37°C. Thereafter, the residual viral infectivity was determined by plaque assay in Vero (NDV, PPRV, and BPXV) or MDBK (BHV-1) cells (G). Effect of Thapsigargin on replication of NDV in embryonated chicken eggs. Embryonated SPF chicken eggs, in triplicates, were inoculated with indicated concentration of Thapsigargin via allantoic route. At 96 h post-Thapsigargin inoculation, eggs were visualized for viability of the embryos. LD₅₀ was determined by the Reed-Muench method (H). To analyze the effect of Thapsigargin on NDV replication in ovo, embryonated SPF chicken eggs, in triplicates, were infected with NDV via allantoic route, along with administration of Thapsigargin (10 ng/egg). At 6 and 96 hpi, the allantoic fluid was examined for NDV yield by HA (I). Error bars indicate SD. Pair-wise statistical comparisons were performed using Student’s t test (** = p < 0.01, *** = p < 0.001). NS represents no statistical significance.

Figure 2

SERCA facilitates paramyxovirus replication. Evaluation of the expression of recombinant SERCA2: HeLa cells were transfected with either an empty vector or with a construct expressing SERCA2-HA. At 48 h-post transfection, cell lysates were probed for expression of HA in Western blot analysis (A). Effect of overexpression of SERCA on paramyxovirus replication: HeLa/goat kidney cells were transfected with SERCA-expressing plasmid (pCR3-SERCA2) or control plasmid (pCR3, empty vector). At 48 h post-transfection, cells were infected with virus (NDV to HeLa cells and PPRV to goat kidney cells) at MOI of 1. Virus released in the supernatant at 24 hpi (NDV) (B) or 96 hpi (PPRV) (C) was quantified by plaque assay. Error bars indicate SD. Pair-wise statistical comparisons were performed using Student’s t test (** = p < 0.01).

Figure 3

Paramyxovirus infections induce SERCA expression. (A) PPRV infection induces SERCA2 expression: Vero cells were infected with PPRV at MOI 5, and the cell lysates were prepared at indicated time points. The levels of SERCA2 (upper panel) or house-keeping control protein (GAPDH) (lower panel) were examined by Western blot analysis. (B) Thapsigargin inhibits NDV-induced SERCA2 expression in Vero cells: Vero cells were infected with NDV at MOI of 5 for 1 h followed by washing with PBS and addition of fresh medium containing Thapsigargin or vehicle control (0.05% DMSO). Cell lysates were prepared at 16 hpi to probe SERCA2 and GAPDH by Western blot analysis. Relative fold-change in the levels of viral/cellular proteins was determined by ImageJ (NIH).

Figure 4

Time-of-addition assay. Confluent monolayers of Vero cells were infected, in triplicate, with PPRV or NDV at MOI of 5, washed six times with PBS and fresh medium with either 0.5 μM Thapsigargin or 0.05% DMSO were added at indicated times. Supernatants were collected at 12 (NDV) or 48 hpi (PPRV) and quantified by plaque assay. Time-of-addition assay for NDV (A) and PPRV (B) is shown.

Figure 5

Viral entry. Vero cell monolayers were prechilled to 4°C and infected with the respective viruses at MOI of 5 in Thapsigargin-free medium for 1 h at 4°C to permit the attachment, followed by washing and addition of fresh medium containing Thapsigargin or vehicle control. Entry was allowed to proceed at 37°C for 1 h after which the cells were washed again with PBS to remove any extracellular viruses and incubated with cell culture medium without any inhibitor. The progeny virus particles released in the cell culture supernatants in the treated and untreated cells were titrated by plaque assay. Entry assay for NDV (A) and PPRV (B) is shown. Vero cells were pretreated with Thapsigargin or DMSO for 1 h followed by washing with PBS and infection with NDV/PPRV at MOI of 5. NDV (C) and PPRV (D) released in the supernatant were quantified by plaque assay. Error bars indicate SD. Pair-wise statistical comparisons were performed using Student’s t test (** = p < 0.01, *** = p < 0.001, NS = nonsignificant difference).

Figure 6

RNA and protein synthesis. Vero cells were either mock-infected or infected with the indicated viruses at MOI of 10 for 3 h followed by washing with PBS and addition of 0.5 μM Thapsigargin or vehicle control (0.05% DMSO). The cells were scrapped at 9 and 20 hpi, respectively, for NDV and PPRV to prepare the cell lysate. Viral RNA was quantified by qRT-PCR. Ct values were normalized with β-actin house-keeping control gene, and relative fold-change was calculated by ΔΔCt method. Relative fold-change in RNA copy number of NDV (A) and PPRV (B) is shown. The levels of viral proteins were analyzed by Western blot analysis. The levels of viral proteins in NDV (C) and PPRV (D) infected cells are shown. NS = No significant difference.

Figure 7

Thapsigargin treatment induces enhanced formation of cytoplasmic foci. Vero cells at ~20% confluence were infected with NDV or PPRV at MOI of 10. At 3 (NDV) or 10 hpi (PPRV), cells were incubated with either 0.5 μM Thapsigargin or 0.05% DMSO. At 10 (NDV) or 36 hpi (PPRV), cells were fixed and subjected for immunofluorescence assay for localization of the viral proteins in the virus-infected cells. Virus was stained with FITC (green), whereas SERCA was stained with rhodamine conjugate. DAPI was used as nuclear stain. Subcellular localization of NDV (A) and PPRV (B) is shown. Virus-infected cells (FITC) were usually of two types – with or without punctuate structures. To quantitate the subcellular localization, 100 cells (under several different fields) were randomly counted. The percentage of the cells with or without punctuate structures in Thapsigargin-treated and control-treated is shown for NDV (C) and PPRV (D).

Figure 8

Selection of Thapsigargin-resistant viral mutants. Vero cells were infected with NDV or PPRV at MOI of 0.01 and grown in the presence of either 0.25 μM of Thapsigargin or vehicle control (0.05% DMSO). The progeny virus particles released in the supernatant were harvested either at 72–120 hpi or when ~75% cells exhibited CPE. Seventy (70) such sequential passages were made. (A) The levels of NDV inhibition at various passage levels in Thapsigargin-treated and untreated cells are shown. (B) Sensitivity of P70-Thapsigargin NDV to Thapsigargin: Vero cells, in triplicate, were infected with P0, P70-Thapsigargin, or P70-Control passaged viruses at MOI of 0.1 in the presence of either 0.5 μM Thapsigargin or 0.05% DMSO, and the progeny virus particles released in the supernatant at 24 hpi were quantified. (C) Relative growth (fold reduction) of P0, P70-Thapsigargin, and P70-Control viruses in the presence of Thapsigargin. (D,E) Sensitivity of plaque purified (P70) viruses to Thapsigargin: P70-Thapsigargin or P70-Control viruses were plaque purified [(four plaques each of P70-Control (C1, C2, C3, and C4) and P70-Thapsigargin (T1, T2, T3, and T4)] and amplified in Vero cells. To evaluate the sensitivity to Thapsigargin, plaque purified viruses were infected at MOI of 0.1, and virus yield was measured in the presence of 0.5 μM Thapsigargin or 0.05% DMSO. Error bars indicate SD. Pair-wise statistical comparisons were performed using Student’s t test (*** = p < 0.001).

Figure 9

Nucleotide sequencing of passaged virus. RNA was extracted from P70-Control and P70-Thapsigargin viruses as well as plaque purified viruses (two plaques each of P70-Control and P70-Thapsigargin) followed by amplification and sequence analysis of F gene. Nucleotide sequences were translated into amino acid sequences and compared with P0 as well as WT sequences retrieved from GenBank (n = 18). Amino acid mutations associated with Thapsigargin resistance as well as those acquired simply due to sequential cell culture passage (present in both P70-Control and P70-Thapsigargin) are shown.