Coxsackievirus B3 Responds to Polyamine Depletion via Enhancement of 2A and 3C Protease Activity

Abstract

Polyamines are small positively-charged molecules abundant in eukaryotic cells that are crucial to RNA virus replication. In eukaryotic cells, polyamines facilitate processes such as transcription, translation, and DNA replication, and viruses similarly rely on polyamines to facilitate transcription and translation. Whether polyamines function at additional stages in viral replication remains poorly understood. Picornaviruses, including Coxsackievirus B3 (CVB3), are sensitive to polyamine depletion both in vitro and in vivo; however, precisely how polyamine function in picornavirus infection has not been described. Here, we describe CVB3 mutants that arise with passage in polyamine-depleted conditions. We observe mutations in the 2A and 3C proteases, and we find that these mutant proteases confer resistance to polyamine depletion. Using a split luciferase reporter system to measure protease activity, we determined that polyamines facilitate viral protease activity. We further observe that the 2A and 3C protease mutations enhance reporter protease activity in polyamine-depleted conditions. Finally, we find that these mutations promote cleavage of cellular eIF4G during infection of polyamine-depleted cells. In sum, our results suggest that polyamines are crucial to protease function during picornavirus infection. Further, these data highlight viral proteases as potential antiviral targets and highlight how CVB3 may overcome polyamine-depleting antiviral therapies.

Keywords: Coxsackievirus B3; polyamines; protease.

Figure 1

CVB3 gains resistance to polyamine depletion over passage with difluoromethylornithine. (A) Vero-E6 cells were left untreated or treated with 500 μM DFMO for four days prior to infection with CVB3 at MOI 0.1. Virus was collected at 24 hpi and used to inoculate the next passage. Viral titers were determined by plaque assay for the passages as shown. (B) CVB3 passaged five times over Vero-E6 cells, either treated with 500 μM DFMO or untreated, were used to infect Vero cells treated with increasing doses of DFMO for 24 hpi. Viral titers were determined by plaque assay. Error bars represent ± 1 SEM.

Figure 2

CVB3 mutations in 2A and 3C proteases confer resistance to polyamine depletion. (A) Vero cells were left untreated or treated with 500 μM DFMO for four days prior to infection with CVB3 and 2A and 3C protease mutants. Samples were collected every 24 h and titered via plaque assay. (B) Vero cells were treated with increasing doses of DFMO, from 100 μM to 1 mM, for four days prior to infection with wildtype CVB3 or protease mutants. Viral titers were determined by plaque assay at 48 hpi. (C) Viral titers from (A) were used to calculate the percent replication in DFMO, by dividing the titer of the virus after infection of DFMO-treated cells by the titer of the virus after infection of untreated cells at 48 hpi. (D) Vero cells were left untreated or treated with 100 μM DENSpm for 16 h prior to infection with wildtype and mutant CVB3 at MOI 0.1. Viral titers were determined by plaque assay at 24 hpi. (E) Percent replication was determined as in (C) but using titers from (D). (F) Thin-layer chromatograms resolving the polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) after treatment with DFMO (above) and DENSpm (below). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 using Student’s t-test (n ≥ 3), comparing treated samples to untreated controls. Error bars represent ± 1 SEM.

Figure 3

CVB3 stability, fitness, and specific infectivity are not altered with protease mutation. Vero cells were infected with wildtype and mutant CVB3 for 24 h, at which time (A) viral titers were determined by plaque assay and (B) viral genomes in cell supernatant were determined by qPCR. (C) The ratio of genomes-to-PFU was calculated by dividing the relative genomes in (B) by the titer in (A). (D) Plaque sizes of virus mutants were determined after a two-day plaque assay. (E) WT and mutant CVB3 were used to infect untreated Vero cells for five passages at which time viral RNA was reverse transcribed, PCR amplified and sequenced. Highlighted nucleotide indicates mutant.

Figure 4

CVB3 protease activity is modulated by cellular polyamine levels. (A) Veros were left untreated or treated with increasing doses of DFMO for four days or increasing doses of DENSpm for 16 h prior to infection with wildtype CVB3. Total cellular protein was collected at 24 hpi and analyzed via western blot for eIF4G and β-actin. (B) Split luciferase protease activity reporter systems were cloned as shown and co-transfected with (C) 2A and (D) 3C into 293T cells left untreated or treated with increasing doses of DFMO. Firefly luciferase activity was measured 24 h later and normalized to renilla luciferase transfection efficiency control and subsequently normalized to untreated cell transfection. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 using Student’s t test (n ≥ 3), comparing treated samples to untreated controls. Error bars represent ± 1 SEM.

Figure 5

CVB3 protease mutations enhance proteolytic activity in polyamine-depleted cells. (A) 293T cells were treated with 500 μM DFMO or left untreated and transfected with empty vector, wildtype 2A, and mutant 2A. Protease activity was measured via dual luciferase assay at 24 h. (B) 293T cells were treated and transfected as in (A) but using wildtype and mutant 3C constructs. (C) Vero cells were left untreated or treated with 500 μM DFMO for four days prior to infection with wildtype and protease mutant CVB3. At 24 hpi, total cellular protein was collected and analyzed for eIF4G and GAPDH. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, or not significant (NS) using Student’s t test (n ≥ 3), comparing treated samples to untreated controls. Error bars represent ± 1 SEM.