1. Autophagy reduces Semliki Forest virus replication in neuronal cells
Robert J Stott1&3, Emily Hopkins2, Nathifa Moyo2, Ian Hurley1, Neil Blake2, James Stewart2, Tom Wileman3 & Sareen E Galbraith1 1. School of Rehabilitation and Health Sciences, Faculty of Health and Social Sciences, Leeds Beckett University, Leeds, LS1 3HE 2. Institute of Infection and Global Health, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, L3 5RF 3. Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich, NR4 7TJ
Introduction
Semliki Forest virus (SFV) is a single stranded positive sense RNA virus from the alphavirus genus. Activation of autophagy during alphavirus infection of non-neuronal cells has been shown to promote viral replication, inhibit apoptosis and allow evasion of the innate immune response (Heaton & Randall, 2010; Jin et al., 2013; McLean et al., 2011). Autophagosomes have been reported to accumulate in SFV infected non-neuronal cells (Eng et al., 2012). Autophagy also enhances the replication of Chikungunya virus, another alphavirus, during infection of non-neuronal cells (Delphine et al., 2013). It has however previously been reported that autophagy provides a protective effect in virus infected neuronal cells (Overdahl et al., 2010; Shoji-Kawata et al., 2013).
This study aims to examine the role of autophagy in viral infection of neuronal cells.
Autophagy reduces viral RNA genome levels
Real-time PCR analysis also showed that autophagy induced through starvation significantly reduced the levels of viral RNA genome after 15 hours post-infection (Fig 1). The cells with autophagy induced were also observed to survive longer than control cells.
Autophagy reduces infectious virus released *
Fig 2: Semliki Forest virus titres in human neuroblastoma is significantly reduced at time points post-infection (denoted with *) when autophagy is induced through starvation (A) or with Torin-1 (B) compared with control neurons
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Fig 1: Relative fold change of intracellular Semliki Forest virus RNA levels is significantly higher after 15 hours post-infection in cells undergoing autophagy through starvation (* denotes significance of <0.05; ** denotes significance of <0.001)
Plaque assay quantification of infectious extracellular virus showed that neuronal cells with autophagy induced, through starvation (Fig 2A) or through incubation with Torin-1 (Fig 2B), release significantly lower levels of extracellular infectious virus when compared with control cells at time points later than 12 hours post infection.
Methods
SFV growth curve assay
Human neuroblastoma cells were seeded onto 6-well plates. Autophagy was induced with 3uM Torin-1 and through starvation by incubation with HBSS for 4h. Cells were infected with virus at an MOI of 0.1 and samples harvested at 2, 4, 12, 24 and 48 h post infection. Virus released was quantified by plaque assay using HeLa cells
Real-time PCR
Samples were harvested from SFV infected and mock infected cells at 2, 4, 15, 27 and 47 h post infection. RNA was extracted and cDNA synthesised. Real-Time PCR was carried out with primers from the E1 gene and a GAPDH reference gene and the relative fold change was calculated.
Conclusions
Autophagy in non-neuronal cells increases positive-sense ssRNA viral titres
Autophagy in human neurons results in decreased viral titres
Induction of autophagy also results in a decrease of the SFV RNA genome
Autophagy in neuronal cells has a protective effect against viral infection
References
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