Initiation of Duck Hepatitis B Virus Infection Requires Cleavage by a Furin-Like Protease

Abstract

The entry mechanism of hepatitis B virus (HBV) has not been defined, and this impedes development of antiviral therapies aimed at an early step in the viral life cycle. HBV infection has both host and tissue specificities. For the related duck hepatitis B virus (DHBV), duck carboxypeptidase D (DCPD) has been proposed as the species-specific docking receptor, while glycine decarboxylase (DGD) may serve as a tissuespecific cofactor or secondary receptor. DGD binds to several truncated versions of the viral large envelope protein but not to the full-length protein, suggesting a need for proteolytic cleavage of the envelope protein by a furin-like proprotein convertase. In the present study, we found that transfected DCPD could confer DHBV binding to non-duck cell lines but that this was followed by rapid virus release from cells. Coexpression of furin led to DCPD cleavage and increased virus retention. Treatment of DHBV particles with endosome prepared from duck liver led to cleavage of the large envelope protein, and such viral preparation could generate a small amount of covalently closed circular DNA in LMH cells, a chicken hepatoma cell line resistant to DHBV infection. A furin inhibitor composed of decanoyl-RVKR-chloromethylketone blocked endosomal cleavage of the large envelope protein in vitro and suppressed DHBV infection of primary duck hepatocytes in vivo. These findings suggest that furin or a furin-like proprotein convertase facilitates DHBV infection by cleaving both the docking receptor and the viral large envelope protein.Hepatitis B virus (HBV) infection has host and tissue specificities. Only humans and other higher primates are susceptible. The liver is the primary target, followed by the kidney and the pancreas (19). The tissue-specific factors and host barriers against HBV infection are still not fully understood, although transcription factors required for HBV gene transcription are enriched in the liver (46). HBV is an enveloped DNA virus. It expresses large (L), middle (M), and small (S) envelope proteins through alternative in-frame translation initiation sites within the envelope gene. Inside the envelope is the core particle that encloses the viral genome and DNA polymerase. Following virus entry into hepatocytes, the viral genome of 3.2 kb is translocated to the nucleus, where it is converted from a partially double-stranded molecule into a covalently closed circle, or the ccc DNA. The ccc DNA has a minichromosome structure and serves as the template for transcription of viral messenger RNAs, which direct the translation of core, envelope, and polymerase proteins. One particular transcript, the pregenomic RNA, is packaged into newly assembled core protein particles together with polymerase, where it serves as the template for reverse transcription into the minus DNA strand. Further degradation of RNA template and initiation of plusstrand-DNA synthesis lead to virion formation and secretion (15).The late steps in the HBV life cycle, including transcription, translation, replication, and vi...

Nucleic Acid Polymers Inhibit Duck Hepatitis B Virus InfectionIn Vitro

“…The initial steps during DHBV infection in vitro involve virus binding to the hepatocyte surface via the pre-S region of the large envelope protein. Duck carboxypeptidase D and glycine decarboxylase have been proposed as virus receptor molecules, and uptake of virus is thought to occur by receptor-mediated endocytosis, with cleavage of the DHBV envelope protein by a furin-like protease (24) and virus entry into early endosomes (25). Although many details of the intracellular pathways involved have not yet been determined, it has been hypothesized that the virus envelope fuses with the endosome to allow delivery of the virus nucleocapsid to the cytoplasm.…”

From DCPD to NTCP: The long journey towards identifying a functional hepatitis B virus receptor

“… 16 We found that dCPD mediated DHBV binding/endocytosis is followed by rapid viral exit, which could be minimized by co-transfection of chicken furin. 17 dCPD, p120, and DHBV L protein colocalized in endosomal fraction, and endosomal enzymes could cleave the L protein on DHBV particles. 17 Incubation of chicken hepatoma cell line LMH with such cleaved DHBV particles generated small amount of cccDNA.…”

“… 17 dCPD, p120, and DHBV L protein colocalized in endosomal fraction, and endosomal enzymes could cleave the L protein on DHBV particles. 17 Incubation of chicken hepatoma cell line LMH with such cleaved DHBV particles generated small amount of cccDNA. Finally, a furin inhibitor capable of blocking L protein cleavage by purified endosomal fraction could also inhibit DHBV infection of duck hepatocytes.…”

Small non-coding RNAs encoded within the herpes simplex virus type 1 latency associated transcript (LAT) cooperate with the retinoic acid inducible gene I (RIG-I) to induce beta-interferon promoter activity and promote cell survival

“…The 2 kb LAT is not capped or poly-adenylated because it is a stable intron (Farrell et al, 1991; Krummenacher and Zabolotny, 1997). The LAT locus also encodes numerous micro-RNAs (miRNA) (Cui et al, 2006; Jurak et al, 2010; Umbach et al, 2008, 2009). Two small non-coding RNAs, 62 nt and 36 nt long, are expressed from the first 1.5 kb of LAT coding sequences (LAT sncRNA1 and sncRNA2) (Peng et al, 2008).…”

“…LAT sncRNA1 and sncRNA2 are not miRNAs because the mature miRNA band that migrates between 21 and 23 nucleotides is not detected, they lack certain structural features of miRNAs, and both sncRNAs have the potential to form complex secondary structures. It is unlikely that LAT sncRNA1 and sncRNA2 were detected using procedures described for the HSV-1 encoded LAT miRNAs (Jurak et al, 2010; Umbach et al, 2008) because RNA species migrating between 17 and 30 nucleotides were size selected and then deep-sequencing performed.…”