Citation

  • Authors: Kicmal TM. et al.
  • Year: 2019
  • Journal: J Virol 93 e01054-19
  • Applications: in vitro / mRNA / jetMESSENGER
  • Cell type: HEK-293T
    Description: Human embryonic kidney Fibroblast
    Known as: HEK293T, 293T

Method

- RNA was purified as described from WT and VP3234R CVB3-infected cell supernatant. 293T cells were seeded at 7.5 × 10^3 cells per well in a 96-well plate in DMEM with 2% FBS. - The cells were treated for 96 h with 500 μM DFMO. - After DFMO treatment, the cells were transfected with the purified viral RNA using the jetMessenger transfection reagent (Polyplus). - Four hours after transfection, fresh medium was replenished on the cells to limit the cytotoxicity of the transfection reagent. - Cell supernatant was collected 32 h postinfection, and viral titers were determined via plaque assay.

Abstract

Polyamines are small polycationic molecules with flexible carbon chains that are found in all eukaryotic cells. Polyamines are involved in the regulation of many host processes and have been shown to be implicated in viral replication. Depletion of polyamine pools in cells treated with FDA-approved drugs restricts replication of diverse RNA viruses. Viruses can exploit host polyamines to facilitate nucleic acid packaging, transcription, and translation, but other mechanisms remain largely unknown. Picornaviruses, including Coxsackievirus B3 (CVB3), are sensitive to the depletion of polyamines and remain a significant public health threat. We employed CVB3 as a model system to investigate a potential proviral role for polyamines using a forward screen. Passaging CVB3 in polyamine-depleted cells generated a mutation in capsid protein VP3 at residue 234. We show that this mutation confers resistance to polyamine depletion. Through attachment assays, we demonstrate that polyamine depletion limits CVB3 attachment to susceptible cells, which is rescued by incubating virus with polyamines. Furthermore, the capsid mutant rescues this inhibition in polyamine-depleted cells. More divergent viruses also exhibited reduced attachment to polyamine-depleted cells, suggesting that polyamines may facilitate attachment of diverse RNA viruses. These studies inform additional mechanisms of action for polyamine-depleting pharmaceuticals, with implications for potential antiviral therapies.IMPORTANCE Enteroviruses are significant human pathogens that can cause severe disease. These viruses rely on polyamines, small positively charged molecules, for robust replication, and polyamine depletion limits infection in vitro and in vivo The mechanisms by which polyamines enhance enteroviral replication are unknown. Here, we describe how Coxsackievirus B3 (CVB3) utilizes polyamines to attach to susceptible cells and initiate infection. Using a forward genetic screen, we identified a mutation in a receptor-binding amino acid that promotes infection of polyamine-depleted cells. These data suggest that pharmacologically inhibiting polyamine biosynthesis may combat virus infection by preventing virus attachment to susceptible cells.

Pubmed