Alpha interferon (IFN-α) induces the transfer of resistance to hepatitis B virus (HBV) from liver nonparenchymal cells (LNPCs) to hepatocytes via exosomes. However, little is known about the entry machinery and pathway involved in the transmission of IFN-α-induced antiviral activity. In this study, we found that macrophage exosomes uniquely depend on T cell immunoglobulin and mucin receptor 1 (TIM-1), a hepatitis A virus (HAV) receptor, to enter hepatocytes for delivering IFN-α-induced anti-HBV activity. Moreover, two primary endocytic routes for virus infection, clathrin-mediated endocytosis (CME) and macropinocytosis, collaborate to permit exosome entry and anti-HBV activity transfer. Subsequently, lysobisphosphatidic acid (LBPA), an anionic lipid closely related to endosome penetration of virus, facilitates membrane fusion of exosomes in late endosomes/multivesicular bodies (LEs/MVBs) and the accompanying exosomal cargo uncoating. Together, our findings provide comprehensive insights into the transmission route of macrophage exosomes to efficiently deliver IFN-α-induced antiviral substances and highlight the similarities between the entry mechanisms of exosomes and virus.IMPORTANCE Our previous study showed that LNPC-derived exosomes could transmit IFN-α-induced antiviral activity to HBV replicating hepatocytes, but the concrete transmission mechanisms, which include exosome entry and exosomal cargo release, remain unclear. In this study, we found that virus entry machinery and pathway were also applied to exosome-mediated cell-to-cell antiviral activity transfer. Macrophage-derived exosomes distinctively exploit hepatitis A virus receptor for access to hepatocytes. Later, CME and macropinocytosis are utilized by exosomes, followed by exosome-endosome fusion for efficient transfer of IFN-α-induced anti-HBV activity. We believe that understanding the cellular entry pathway of exosomes will be beneficial to designing exosomes as efficient vehicles for antiviral therapy.

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Myeloid differentiation primary response protein 88 (MyD88), which can be induced by alpha interferon (IFN-␣), has an antiviral activity against the hepatitis B virus (HBV). The mechanism of this antiviral activity remains poorly understood. Here, we report that MyD88 inhibited HBV replication in HepG2.2.15 cells and in a mouse model. The knockdown of MyD88 expression weakened the IFN-␣-induced inhibition of HBV replication. Furthermore, MyD88 posttranscriptionally reduced the levels of viral RNA. Remarkably, MyD88 accelerated the decay of viral pregenomic RNA in the cytoplasm. Mapping analysis showed that the RNA sequence located in the 5-proximal region of the pregenomic RNA was critical for the decay. In addition, MyD88 inhibited the nuclear export of pre-S/S RNAs via the posttranscriptional regulatory element (PRE). The retained pre-S/S RNAs were shown to degrade in the nucleus. Finally, we found that MyD88 inhibited the expression of polypyrimidine tract-binding protein (PTB), a key nuclear export factor for PRE-containing RNA. Taken together, our results define a novel antiviral mechanism against HBV mediated by MyD88.Hepatitis B virus (HBV) is a noncytopathic, enveloped virus with a circular, double-stranded DNA genome. It causes both acute and chronic infection of the human liver. Although a highly effective preventive vaccine is now available, HBV infection remains a major health problem worldwide. It is estimated that chronic HBV infection affects 350 to 400 million people globally, about a quarter of whom will eventually develop severe liver diseases, including liver cirrhosis, liver failure, and hepatocellular carcinoma (HCC) (4).Current antiviral therapies involve the use of nucleoside analogs and alpha interferon (IFN-␣) (28). IFN-␣, a type I interferon, engages the IFN-␣ receptor complex to activate the Jak/Stat pathway and trigger the transcription of a diverse set of genes, referred to as IFN-stimulated genes (ISGs) (2, 40). In total, the gene products of ISGs establish an antiviral response in target cells (2, 40). IFN-␣ inhibits HBV replication through a variety of mechanisms. It was reported previously that IFN-␣ can suppress viral gene expression, prevent the formation of viral RNA-containing core particles, and reduce the accumulation of viral replicative intermediates (11,35,37,(46)(47)(48). Importantly, the precise antiviral mechanism of IFN-␣ and the biological functions of many ISGs have not been fully elucidated.Myeloid differentiation primary response protein 88 (MyD88) is a key adaptor in the signaling cascade of the innate immune response (22). We and others have shown that MyD88 expression can be induced by IFN-␣ and that MyD88 has an antiviral activity against HBV in hepatoma cells that is mediated by nuclear factor B (NF-B) activation (12, 25, 51, 52). To counteract its inhibition, the HBV polymerase dampens the activation of the MyD88 promoter by blocking the nuclear translocation of Stat1, thereby reducing IFN-␣-inducible MyD88 expression (50), further suggesting a critical rol...