Reduced sphingosine kinase 1 activity in dengue virus type-2 infected cells can be mediated by the 3′ untranslated region of dengue virus type-2 RNA

Abstract

Sphingosine kinase 1 (SphK1) is a lipid kinase with important roles including regulation of cell survival. We have previously shown reduced SphK1 activity in cells with an established dengue virus type-2 (DENV-2) infection. In this study, we examined the effect of alterations in SphK1 activity on DENV-2 replication and cell death and determined the mechanisms of the reduction in SphK1 activity. Chemical inhibition or overexpression of SphK1 after established DENV-2 infection had no effect on infectious DENV-2 production, although inhibition of SphK1 resulted in enhanced DENV-2-induced cell death. Reduced SphK1 activity was observed in multiple cell types, regardless of the ability of DENV-2 infection to be cytopathic, and was mediated by a post-translational mechanism. Unlike bovine viral diarrhea virus, where SphK1 activity is decreased by the NS3 protein, SphK1 activity was not affected by DENV-2 NS3 but, instead, was reduced by expression of the terminal 396 bases of the 39 UTR of DENV-2 RNA. We have previously shown that eukaryotic elongation factor 1A (eEF1A) is a direct activator of SphK1 and here DENV-2 RNA co-localized and coprecipitated with eEF1A from infected cells. We propose that the reduction in SphK1 activity late in DENV-2-infected cells is a consequence of DENV-2 out-competing SphK1 for eEF1A binding and hijacking cellular eEF1A for its own replication strategy, rather than a specific host or virus-induced change in SphK1 to modulate viral replication. Nonetheless, reduced SphK1 activity may have important consequences for survival or death of the infected cell.

Reduction in sphingosine kinase 1 influences the susceptibility to dengue virus infection by altering antiviral responses

“…1d). This suggests that in our infection system and similar to our prior observation, where increasing SK1 after an infection was established had no effect on DENV replication (Carr et al, 2013a), increased levels of exogenous SK1 prior to DENV challenge also has no significant impact on DENV infection.…”

“…The above studies linking high levels of SK with enhancement of viral infection are in contrast to our own observations of a reduction in SK1 activity in response to dengue virus (DENV) infection (Wati et al, 2011), as also reported for the related flavivirus bovine viral diarrhea virus (Yamane et al, 2009). Close analysis of the time-course of the reduction in SK1 in DENV-infected cells demonstrates a correlation with high levels of the DENV 39 UTR, which can compete for the SK1 activator eEF1A (Carr et al, 2013a;Leclercq et al, 2008Leclercq et al, , 2011, which is utilized by flaviviruses to assist replication (Blackwell & Brinton, 1997;Davis et al, 2007). Additionally, our previous data (Carr et al, 2013a;Calvert et al, 2015), as well as that of Yamane et al (2009), suggest an increase in SK1 activity in the early hours following infection.…”

“…and observed a decrease in SK1 activity at 30 h p.i. (Carr et al, 2013a), a more detailed analysis of changes in SK1 activity within the first 24 h of infection did not support any further early DENV-induced changes in endogenous SK1.…”

“…We have previously defined the time-course for changes in SK1 activity in DENVinfected cells from 2 to 48 h pi and shown a late reduction in SK1 activity in DENV-infected cells (w30 h p.i.) (Carr et al, 2013a;Wati et al, 2011). In the study herein, we have more closely examined the first 24 h following DENV infection, but failed to demonstrate any additional changes in SK1 activity.…”

“…Close analysis of the time-course of the reduction in SK1 in DENV-infected cells demonstrates a correlation with high levels of the DENV 39 UTR, which can compete for the SK1 activator eEF1A (Carr et al, 2013a;Leclercq et al, 2008Leclercq et al, , 2011, which is utilized by flaviviruses to assist replication (Blackwell & Brinton, 1997;Davis et al, 2007). Additionally, our previous data (Carr et al, 2013a;Calvert et al, 2015), as well as that of Yamane et al (2009), suggest an increase in SK1 activity in the early hours following infection. Thus the roles of SK1 in viral infection may vary in relation to the time post-infection (p.i.…”