Hepatitis Delta Virus Alters the Autophagy Process To Promote Its Genome Replication.

A substantial number of viruses have been demonstrated to subvert autophagy to promote their own replication. Recent publications have reported the proviral effect of autophagy induction on hepatitis B virus (HBV) replication. Hepatitis delta virus (HDV) is a defective virus and an occasional obligate satellite of HBV. However, no previous work has studied the relationship between autophagy and HDV. In this article, we analyze the impact of HBV and HDV replication on autophagy as well as the involvement of the autophagy machinery in the HDV life cycle when produced alone and in combination with HBV. We prove that HBxAg and HBsAg can induce early steps of autophagy but ultimately block flux. It is worth noting that the two isoforms of the HDV protein, the small HDAg (S-HDAg) and large HDAg (L-HDAg) isoforms, can also efficiently promote autophagosome accumulation and disturb autophagic flux. Using CRISPR-Cas9 technology to generate specific knockouts, we demonstrate that the autophagy machinery, specifically the proteins implicated in the elongation step (ATG7, ATG5, and LC3), is important for the release of HBV without affecting the level of intracellular HBV genomes. Surprisingly, the knockout of ATG5 and ATG7 decreased the intracellular HDV RNA level in both Huh7 and HepG2.2.15 cells without an additional effect on HDV secretion. Therefore, we conclude that HBV and HDV have evolved to utilize the autophagy machinery so as to assist at different steps of their life cycle.IMPORTANCE Hepatitis delta virus is a defective RNA virus that requires hepatitis B virus envelope proteins (HBsAg) to fulfill its life cycle. Thus, HDV can only infect individuals at the same time as HBV (coinfection) or superinfect individuals who are already chronic carriers of HBV. The presence of HDV in the liver accelerates the progression of infection to fibrosis and to hepatic cancer. Since current treatments against HBV are ineffective against HDV, it is of paramount importance to study the interaction between HBV, HDV, and host factors. This will help unravel new targets whereby a therapy that is capable of simultaneously impeding both viruses could be developed. In this research paper, we evidence that the autophagy machinery promotes the replication of HBV and HDV at different steps of their life cycle. Notwithstanding their contribution to HBV release, autophagy proteins seem to assist HDV intracellular replication but not its secretion.

Exosomes as Conduits: Facilitating Hepatitis B Virus-Independent Hepatitis D Virus Transmission and Propagation in Hepatocytes.

A number of research studies, including ours, have spotlighted exosomes as critical facilitators of viral dissemination. While hepatitis B virus (HBV) transmission through exosomes has been studied, the focus on its satellite virus, the hepatitis delta virus (HDV), has been unexplored in this context. HDV, although being a defective virus, can replicate its genome autonomously within hepatocytes, independently of HBV. Investigations on Huh7 cells revealed an intriguing phenomenon: the HDV proteins, S-HDAg and L-HDAg, are transmitted between cells without a complete viral structure. Detailed analysis further revealed that the expression of these proteins not only bolstered exosome secretion but also ensured their enrichment within these vesicles. Our experimental approach utilized transfection of various plasmids to examine the role of HDV RNA and proteins in the process. One salient finding was the differential propagation of the HDV proteins S-HDAg and L-HDAg, suggesting intricate molecular mechanisms behind their transmission. Notably, the purity of our exosome preparations was monitored using markers such as TSG101 and CD81. Importantly, these exosomes were found to carry both HDV RNA and proteins, highlighting their role in HDV dissemination. This novel study underscores the role of exosomes in mediating the transmission of HDV components between hepatocytes independent of HBV. These revelations about the exosomal pathway of HDV transmission provide a foundation for the development of innovative therapeutic strategies against HDV infections.

HepG2BD: A Novel and Versatile Cell Line with Inducible HDV Replication and Constitutive HBV Expression.

Individuals chronically infected with hepatitis B virus (HBV) and hepatitis Delta virus (HDV) present an increased risk of developing cirrhosis and hepatocellular carcinoma in comparison to HBV mono-infected individuals. Although HDV only replicates in individuals coinfected or superinfected with HBV, there is currently no in vitro model that can stably express both viruses simultaneously, mimicking the chronic infections seen in HBV/HDV patients. Here, we present the HepG2BD cell line as a novel in vitro culture system for long-term replication of HBV and HDV. HepG2BD cells derive from HepG2.2.15 cells in which a 2 kb HDV cDNA sequence was inserted into the adeno-associated virus safe harbor integration site 1 (AAVS1) using CRISPR-Cas9. A Tet-Off promoter was placed 5' of the genomic HDV sequence for reliable initiation/repression of viral replication and secretion. HBV and HDV replication were then thoroughly characterized. Of note, non-dividing cells adopt a hepatocyte-like morphology associated with an increased production of both HDV and HBV virions. Finally, HDV seems to negatively interfere with HBV in this model system. Altogether, HepG2BD cells will be instrumental to evaluate, in vitro, the fundamental HBV-HDV interplay during simultaneous chronic replication as well as for antivirals screening targeting both viruses.

Revealing Drug Self-Associations into Nano-Entities.

The aqueous properties of the drugs Sorafenib, Lapatinib, Gefitinib, Fulvestrant, and Clofazimine were explored to monitor their tendency to self-associate. A combination of nuclear magnetic resonance, dynamics light scattering, and electron and confocal microscopies found that they tended to form large nano-entities having distinct types and sizes and were capable of entering cells. The combination of strategies employed serves to detect and reveal nano-entities along with their three-state equilibria and behaviors in buffers, media, and cells.

LC3B is not recruited along with the autophagy elongation complex (ATG5-12/16L1) at HCV replication site and is dispensable for viral replication.

Hepatitis C virus (HCV) infection is known to induce autophagosome accumulation as observed by the typical punctate cytoplasmic distribution of LC3B-II in infected cells. Previously, we showed that viral RNA-dependent RNA polymerase (NS5B) interacts with ATG5, a major component of the autophagy elongation complex that is involved in the formation of double-membrane vesicles (DMV), and demonstrated that the autophagy elongation complex (ATG5-12/16L1) but not LC3B is required for proper membranous web formation. In this study, the colocalization and in situ interaction of all HCV replicase components with the constituent of the autophagy elongation complex and LC3B were analyzed. The results clearly show the recruitment of the elongation complex to the site of viral replication. Using in situ proximity ligation assay, we show that ATG5, but not ATG16L1, interacts with several HCV replicase components suggesting that the recruitment is directed via the ATG5-12 conjugate. Interestingly, no E3-like conjugation activity of ATG5-12/16L1 can be detected at the at HCV replication site since LC3B-II is not found along with the elongation complex at the site of viral replication. In agreement with this result, no sign of in situ interaction of LC3B with the replicase components is observed. Finally, using dominant negative forms of ATG proteins, we demonstrate that ATG5-12 conjugate, but not LC3-II formation, is critical for viral replication. Altogether, these findings suggest that although HCV needs the elongation complex for its replication, it has developed a mechanism to avoid canonical LC3-II accumulation at viral replication site.