Reticulon-3 modulates the incorporation of replication competent hepatitis C virus molecules for release inside infectious exosomes

Abstract

Background Cell released microvesicles specifically, exosomes, play an important role in mediating immunologic escape, treatment resistance, and disease persistence of Hepatitis C virus (HCV) infection. Reports on the molecular compositions of exosomes released by cells under diverse conditions, especially during viral infections, suggest that their cargo contents are not randomly loaded. However, the precise molecular mechanisms directing the selective cargo sorting and loading inside infectious viral exosomes remains elusive. Aim To decipher the role of Reticulon 3 (RTN3) in the selective molecular cargo sorting and loading inside infectious viral exosomes during HCV infection. Methods We used Huh7 cells–JFH1 HCV infection and HCV Full-Length (FL) replicon systems. Additionally, we analyzed human liver and serum exosome samples from healthy and treatment naïve HCV infected individuals. Our experiments made use of molecular biology and immunology techniques. Results HCV infection (JFH1-Huh7 or HCV-FL replicon cells) was associated with increased RTN3L&S isoforms expression in cells and cell released exosomes. Accordingly, increased expression of RTN3L&S was observed in liver and serum exosome samples of HCV infected individuals compared to healthy controls. RNA-ChIP analysis revealed that RTN3L&S interacted with dsHCV RNA. Lentiviral CRISPR/Cas9 mediated knockdown (KD) of RTN3 and plasmid overexpression (OE) of wild type, C- and N-terminal deletion mutants of RTN3L&S in HCV- infected Huh7 cells differentially impacted the cellular release of infectious viral exosomes. RTN3L&S KD significantly decreased, while RTN3S OE significantly increased the number of Huh7 cell-released infectious exosomes. The C-terminal domain of RTN3 interacted with and modulated the loading of dsHCV RNA inside infectious exosomes. Antiviral treatment of HCV infected Huh7 cells reduced virus-induced RTN3L&S expression and attenuated the release of infectious exosomes. Conclusion RTN3 constitutes a novel regulator and a potential therapeutic target that mediates the specific loading of infectious viral exosomes.

[Retracted] Identifying COVID‐19‐Specific Transcriptomic Biomarkers with Machine Learning Methods

“…Therefore, such gene could be summarized in this rule for the identification of normal controls. Similarly, RTN1 has been shown to be associated with macrophage-mediated immune suppressants, different from the immune activators in the previously discussed rules [78], thereby validating the predictions on normal control. Resources 6.8).…”

Harnessing the Therapeutic Potential of Exosomes: A Novel Strategy for Anticancer and Antiviral Therapy

“…As earlier mentioned, these nanovesicles can express several macromolecules including surface proteins that can facilitate the uptake of viral nucleic acids. In a recent study, it was revealed that exosomes significantly expressed the intracellular membrane protein, Reticulon 3 (RTN3), which modulated their uptake of replication-competent hepatitis C virus RNA and some viral nonstructural proteins [ 47 ]. This highlights the ability of exosomes to successfully incorporate infectious viral nucleic acids, serve as reservoirs for persistent infection or favor viral pathogenesis.…”

Advances in ER-Phagy and Its Diseases Relevance

“…The RHD of RTN3L may be able to mediate ER membrane bending and subsequent serving from the network to form fragments during ER-phagy. It has also been reported that the RHD of RTN3L promotes the formation and efflux of Hepatitis C Virus (HCV) exosomes [ 43 ]. And LIRs of RTN3L also have other functions besides interacting with LC3/GABARAP.…”

“…The membrane bending ability of RTNs presumably provides the flexibility of ER membrane to facilitate the viral escape [ 116 , 122 ]. Similarly, RTN3L can induce the bending of the ER membrane and promote the formation and excretion of exosomes that contain HCV [ 43 ]. Under viral infection conditions, RTNs are hijacked by the virus to help their escape and the physiological functions of RTNs in ER-phagy may be impaired.…”