Neutral Sphingomyelinase 2-Dependent Secretory Pathway May Contribute to Extracellular Hepatitis A Virus RNA Levels

Hepatitis A virus (HAV) infection is a major cause of acute viral hepatitis worldwide. However, no effective antiviral therapy is currently available. HAV manipulates host secretory pathways, including the endosomal sorting complex required for transport (ESCRT)-dependent pathways, to facilitate viral propagation. Nevertheless, whether HAV also utilizes alternative secretory pathways remains unclear. Here, we provide evidence suggesting that an ESCRT-independent pathway may contribute to extracellular HAV RNA levels in Huh7 cells infected with HAV genotype IB (HM175-18f) and genotype IIIA (HA11-1299). Treatment with the ESCRT-independent pathway inhibitor GW4869 significantly reduced extracellular HAV RNA levels. GW4869 did not significantly affect HAV subgenomic RNA replication. These results suggest that the neutral sphingomyelinase 2 (nSMase2)-dependent ESCRT-independent pathway may contribute to extracellular HAV RNA levels rather than intracellular HAV RNA replication. Consistent with these findings, knockdown of sphingomyelin phosphodiesterase 3, which encodes SMase2, also significantly reduced extracellular HAV RNA levels. These findings suggest that an nSMase2-dependent ESCRT-independent extracellular vesicle pathway may contribute to extracellular HAV RNA levels.