Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B

Abstract

It has been proposed that the hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent screening of replicon cells with small interfering RNA identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence, and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled with bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRMs), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B-binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of the membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRMs, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infection in vitro and in vivo. Our findings provide new insights into HCV host cofactors. IMPORTANCEThe hepatitis C virus (HCV) protein NS4B can induce alteration of the endoplasmic reticulum and the formation of a membranous web structure, which provides a platform for the HCV replication complex. The molecular mechanism by which NS4B induces the membranous HCV replication compartment is not understood. We screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis, followed by screening with small interfering RNA. We identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. PREB is induced by HCV infection and recruited into the replication complex by interaction with NS4B. Recruited PREB promotes HCV RNA replication by participating in the formation of the membranous HCV replication compartment. To our knowledge, the effect of NS4B-binding protein on the formation of the membranous HCV replication compartment is newly described in this report. Our findings are expected to provide new insights into HCV host cofactors. H epatitis C virus (HCV) infects about 2% of the world's population and leads to several diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). HCV is the most common reason for patients requiring a liver transplant in developed countries and is thus a significant health burden worldwide (1). To date,...

Japanese Encephalitis Virus Induces Apoptosis and Encephalitis by Activating the PERK Pathway

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“…Cell lines, viruses, and mice. Baby hamster kidney (BHK) cells were used for virus titer measurement by 50% tissue culture infective dose (TCID 50 ) assays (16). Neuro-2a cells or BALB/c mice were infected with the P3 strain of JEV as indicated.…”

“…Reverse transcription-PCR and immunoblot analysis. Total RNA was extracted using TRIzol reagent and reverse transcription-PCR (RT-PCR) was performed as described previously (16). Specific primers for JEV NS1 and GADPH were used for RT-PCR.…”

“…Indirect immunofluorescence and apoptosis analysis. Indirect immunofluorescence was performed as described previously (16). Briefly, cells were fixed in acetone-methanol and incubated with the primary antibodies.…”

Protein Interactions during the Flavivirus and Hepacivirus Life Cycle

“…Similarly, prolactin regulatory element binding, a protein regulating anterograde ER-Golgi transport, was found to interact with HCV NS4B and relocalize to the viral RC upon interaction (129). Silencing of prolactin regulatory element binding decreased the size of membrane webs and reduced the number of double-and multimembrane vesicles, the site of HCV replication.…”