RNAi screen reveals a role of SPHK2 in dengue virus–mediated apoptosis in hepatic cell lines

Abstract

Hepatic dysfunction is a feature of dengue virus (DENV) infection. Hepatic biopsy specimens obtained from fatal cases of DENV infection show apoptosis, which relates to the pathogenesis of DENV infection. However, how DENV induced liver injury is not fully understood. In this study, we aim to identify the factors that influence cell death by employing an apoptosis-related siRNA library screening. Our results show the effect of 558 gene silencing on caspase 3-mediated apoptosis in DENV-infected Huh7 cells. The majority of genes that contributed to apoptosis were the apoptosis-related kinase enzymes. Tumor necrosis factor superfamily member 12 (TNFSF12), and sphingosine kinase 2 (SPHK2), were selected as the candidate genes to further validate their influences on DENV-induced apoptosis. Transfection of siRNA targeting SPHK2 but not TNFSF12 genes reduced apoptosis determined by Annexin V/PI staining. Knockdown of SPHK2 did not reduce caspase 8 activity; however, did significantly reduce caspase 9 activity, suggesting its involvement of SPHK2 in the intrinsic pathway of apoptosis. Treatment of ABC294649, an inhibitor of SPHK2, reduced the caspase 3 activity, suggesting the involvement of its kinase activity in apoptosis. Knockdown of SPHK2 significantly reduced caspase 3 activity not only in DENV-infected Huh7 cells but also in DENV-infected HepG2 cells. Our results were consistent across all of the four serotypes of DENV infection, which supports the pro-apoptotic role of SPHK2 in DENV-infected liver cells.

Renal Mesangial Cells Isolated from Sphingosine Kinase 2 Transgenic Mice Show Reduced Proliferation and are More Sensitive to Stress-Induced Apoptosis

“…In this view, a recent study on dengue virus-induced liver injury showed that SK-2 is a critical mediator of dengue virus-induced apoptosis of hepatic cells. Downregulating SK-2 by siRNA in various hepatic cell lines, reduced dengue virus-induced caspase-9 and the intrinsic pathway of apoptosis without affecting the extrinsic pathway [39]. This study revealed the same apoptotic pathway to be affected by SK-2 as found in our study on renal mesangial cells.…”

Role of MAPK/MNK1 signaling in virus replication

“…RNAi has enabled systematic exploration of host genes involved in virus replication and virulence, thereby allowing identification of novel drug targets (Radoshitzky et al, 2016). Since the last decade, a large number of genome-scale RNAi screens have been conducted, which identified cellular factors required for WNV (Krishnan et al, 2008), DENV (Morchang et al, 2017;Sessions et al, 2009), HIV-1 (Zhou et al, 2008), HCV Lupberger et al, 2015;Tai et al, 2009), IAV (Han et al, 2018;Karlas et al, 2010), JUNV (Lavanya et al, 2013), coronavirus (Staff, 2015), poxvirus (Kilcher et al, 2014), herpesvirus (Griffiths et al, 2013) and polyomavirus (Zhao and Imperiale, 2017) replication. siRNA screens reveal both proviral and antiviral cellular factors and therefore help in understanding the precise nature of virus-host interactions .…”

Divergent Role of Sphingosine 1-Phosphate in Liver Health and Disease

“…On the one hand, tumour necrosis factor alpha (TNF-α) stimulation of DENV-infected cells during productive infection leads to enhanced death by caspase-3-mediated apoptosis, which is accompanied by a reduced SphK1 activity [ 98 , 99 ]. On the other hand, the activation of SphK2 contributes to DENV-provoked apoptosis in hepatocytes [ 100 ]. Pharmacological inhibition of SphK2 or downregulation via siRNA reduced caspase-3 as well as caspase-9 activity in DENV-infected cells, indicating a pro-apoptotic role of SphK2 via the intrinsic pathway.…”