Our data demonstrates that OSBP contributes to HEV replication. OSBP silencing led to a significant reduction in the replication of an HEV replicon. Our results show that the HEV helicase is co-localized with OSBP, interacts with OSBP, and blocks its preferential translocation to the Golgi in co-transfected cells. Further analysis indicates the interaction between these two proteins occurs possibly via their C-terminal lobes. These results suggest that HEV may recruit OSBP to assist viral replication.
The OSBP silencing did not affect the cell viability much but significantly inhibited HEV replication. The possible reason for the minimal effect on cell viability is that the cells reaching confluency might not rely on OSBP for maintenance or other ORPs complement the OSBP loss under cultured conditions. However, the OSBP silencing reduces HEV replication. OSBP silencing possibly affects HEV RNA replication as the HEV p6/luc replicon was used, and the luciferase is expressed from the subgenomic RNA. The lower luciferase yield indicates a lower level of the subgenomic RNA that is produced by HEV RdRp. The proviral role of OSBP in HEV replication was further confirmed by the trans-complementation of OSBP in the cells with OSBP silencing. This ectopic expression of OSBP restored the HEV replication, which also excludes the possibility of an off-target effect of the shRNA silencing.
Among the HEV proteins, the helicase was discovered to co-localize with OSBP and interact with OSBP. There are multiple motifs on helicase, most of which (Ia, III, IV, V, and VI) possess nucleic acid or NTP binding activities [20]. The computational modeling of OSBP interaction with helicase predicted that the C-terminal lobes of both helicase and OSBP interact. The co-IP result indicates that OSBP interacts with the C-terminal region of the helicase.
To further examine how the virus regulates the OSBP activity, we did transient expression of OSBP and the HEV helicase in HeLa cells and found that the helicase blocked the preferential translocation of OSBP to the Golgi, suggesting that HEV may recruit OSBP via the helicase. Host lipid homeostasis is often vital for virus infection, and many lipid-associated proteins are required for virus replication [47, 48]. For instance, apolipoprotein B, which is associated with the maturation and secretion of LDL, plays an essential role in HCV particle exocytosis [49]. Upon Coxsackievirus B3 (CVB3) infection, the viral 3A protein localizes to secretory organelle membranes. It recruits GBF1/Arf1, thereby enhancing the accumulation of PI4KIIIB, which further catalyzes the production of uncoated PI4P-enriched structures adjacent to ER exit sites [50].
During the infection by some viruses, OSBP is exploited to facilitate the trafficking of lipid components to promote viral replication. For example, OSBP is a downstream effector of phosphatidylinositol 4-kinase alpha (PI4KIIIα) for HCV replication, aiding in the cholesterol trafficking in the viral-induced membranous web [51, 52]. The depletion of OSBP decreases the replication of HCV. Interestingly, complete depletion of OSBP reduces both intracellular and extracellular HCV viral RNA. However, partial depletion of OSBP affects only the accumulation of extracellular HCV RNA, which suggests that OSBP should be involved in the assembly and release of infectious HCV virions [53]. In Aichi virus replication, OSBP is recruited to the Aichi virus replication complex, possibly for constructing the organelle membrane [54]. In other circumstances, the substrate of OSBP can combat virus infection. For instance, the existence of oxysterol can inhibit HBV replication in both DNA and protein levels in hepatocytes [55]. Our results suggest that HEV recruits OSBP to benefit the virus replication. Further research is needed to study the mechanism of the proviral role of OSBP in HEV replication.
HEV helicase possesses RNA 5'-triphosphatase, RNA unwinding, and NTPase activities, which are thought to be involved in viral RNA synthesis [19, 20]. In addition to these enzyme activities, the HEV helicase was found to interact with multiple cellular proteins identified via yeast two-hybrid analysis [56]. Some of these proteins are involved in metabolic and biological processes. Our data shows that the helicase interacts with OSBP and changes its preferential localization, which might affect cellular cholesterol homeostasis. A previous study indicates that HEV infection reduces cholesterol levels in A549 cells and that increasing cholesterol levels in cultured cells reduces HEV release [26]. HEV infection in patients reduces serum TG, total cholesterol, and LDL levels [26]. Therefore, the helicase interaction with OSBP and changing its preferential localization might contribute to the cholesterol modulation in HEV-infected cells and patients.
This study's limitations are that a luciferase-based HEV replicon was used and that the OSBP interaction with helicase was not performed in hepatocytes. Future studies using full-length HEV RNA transfection or HEV infection are needed to confirm the proviral role of OSBP and its interaction with pORF1 or helicase since both ORF2 and ORF3 products are involved in HEV assembly and release. The full-length HEV replicons with HA or V5-tagged ORF1 in the HVR [57, 58] appear to fit studies to determine the OSBP and pORF1 interaction.
In conclusion, this study shows that OSBP contributes to HEV replication and that the HEV helicase interacts with OSBP and blocks OSBP preferential translocation to the Golgi apparatus. The results shed light on the HEV recruitment of the lipid regulator OSBP and improve our understanding of the virus-cell interactions.