Mechanisms and Effects on HBV Replication of the Interaction between HBV Core Protein and Cellular Filamin B

Hepatitis B virus (HBV) infection is one of the major problems that threatens global health. There have been many studies on HBV, but the relationship between HBV and host factors is largely unexplored and more studies are needed to clarify these interactions. Filamin B is an actin-binding protein that acts as a cytoskeleton protein, and it is involved in cell development and several signaling pathways. In this study, we showed that filamin B interacted with HBV core protein, and the interaction promoted HBV replication. The interaction between filamin B and core protein was observed in HEK 293T, Huh7 and HepG2 cell lines by co-immunoprecipitation and co-localization immnofluoresence. Overexpression of filamin B increased the levels of HBV total RNAs and pre-genome RNA (pgRNA), and improved the secretion level of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). In contrast, filamin B knockdown inhibited HBV replication, decreased the level of HBV total RNAs and pgRNA, and reduced the secretion level of HBsAg and HBeAg. In addition, we found that filamin B and core protein may interact with each other via four blocks of argentine residues at the C-terminus of core protein. In conclusion, we identify filamin B as a novel host factor that can interact with core protein to promote HBV replication in hepatocytes. Our study provides new insights into the relationship between HBV and host factors and may provide new strategies for the treatment of HBV infection.     

Structural and functional analysis of the cerato-platanin-like protein Cpl1 suggests diverging functions in smut fungi

Plant-pathogenic fungi are causative agents of the majority of plant diseases and can lead to severe crop loss in infected populations. Fungal colonization is achieved by combining different strategies, such as avoiding and counteracting the plant immune system and manipulating the host metabolome. Of major importance are virulence factors secreted by fungi, which fulfil diverse functions to support the infection process. Most of these proteins are highly specialized, with structural and biochemical information often absent. Here, we present the atomic structures of the cerato-platanin-like protein Cpl1 from Ustilago maydis and its homologue Uvi2 from Ustilago hordei. Both proteins adopt a double-Ψβ-barrel architecture reminiscent of cerato-platanin proteins, a class so far not described in smut fungi. Our structure–function analysis shows that Cpl1 binds to soluble chitin fragments via two extended grooves at the dimer interface of the two monomer molecules. This carbohydrate-binding mode has not been observed previously and expands the repertoire of chitin-binding proteins. Cpl1 localizes to the cell wall of U. maydis and might synergize with cell wall-degrading and decorating proteins during maize infection. The architecture of Cpl1 harbouring four surface-exposed loop regions supports the idea that it might play a role in the spatial coordination of these proteins. While deletion of cpl1 has only mild effects on the virulence of U. maydis, a recent study showed that deletion of uvi2 strongly impairs U. hordei virulence. Our structural comparison between Cpl1 and Uvi2 reveals sequence variations in the loop regions that might explain a diverging function.     

Antisense oligodeoxynucleotides targeted against molecular chaperonin Hsp60 block human hepatitis B virus replication

The major role of hepatitis B virus polymerase (HBV pol) is polymerization of nucleotides, but it also participates in protein priming and the packaging of its own genome into capsids. Therefore, HBV pol may require many assistance factors for its roles. Previous reports have shown that Hsp60, a molecular chaperone, activates HBV pol both in vitro and ex vivo, such as inside insect cells. Moreover, HBV pol binds to Hsp60 in the HepG2 host cell line. In this report, we show that Hsp60 plays a role in the in vivo replication of HBV. Antisense oligodeoxynucleotides (A-ODNs) specifically directed against Hsp60 induced its down-regulation, severely reducing the level of replication-competent HBV without influencing cell proliferation and capsid assembly under these conditions. Furthermore, we found that Hsp60 did not encapsidate into nucleocapsids. Our results indicate that Hsp60 is important for HBV replication in vivo, presumably through activation of HBV pol before encapsidation of HBV pol into HBV core particle. In addition, A-ODNs specific for Hsp60 also inhibit replication of a mutant HBV strain that is resistant to the nucleoside analogue 3TC, which is the main drug used for HBV treatment, and we suggest that A-ODNs directed against Hsp60 are possible reagents as anti-HBV drugs. Conclusively, this report shows that the host factor, Hsp60, is essential for in vivo HBV replication and that mechanism of Hsp60 is probably through an activation of HBV pol by Hsp60.     

Interaction between nucleophosmin and HBV core protein increases HBV capsid assembly

Host factors are involved in Hepatitis B virus (HBV) genome replication and capsid formation during the viral life cycle. A host factor, nucleophosmin (B23), was found to bind to HBV core protein dimers, but its functional role has not been studied. This interaction promoted HBV capsid assembly and decreased the degree of capsid dissociation when subjected to denaturant treatments in vitro. In addition, inhibition of B23 reduced intracellular capsid formation resulting in a decrease of HBV production in HepG2.2.15 cells. These results provide important evidence that B23 acts on core capsid assembly via its interaction with HBV core dimers.     

乙肝病毒感染导致Mrell下调及基因组断裂

[目的]乙肝病毒的持续感染与肝细胞癌的发生密切相关.本文探讨了乙肝病毒感染后导致宿主蛋白Mrell的变化,并研究这种蛋白的变化可能导致肝癌发生的机制.[方法]本文通过Westernblot检测了乙肝病毒感染HL7702细胞及肝癌组织标本的Mrell蛋白的变化,并且通过RNA干涉的方法干扰了Mrell的表达,用连接介导PCR检测基因组的变化.[结果]本研究发现乙肝病毒感染细胞导致Mrell表达下调,肝癌组织也可以发现Mrell表达下调;下调Mrell表达可以导致细胞基因组的断裂增多.[结论]HBV感染导致Mrell表达下调导致基因组不稳定,这可能与HBV感染导致细胞恶性转化相关.     

Functional characterization of the interaction between human La and hepatitis B virus RNA

The La protein is a multifunctional RNA-binding protein and has also been suggested to be involved in the stabilization of hepatitis B virus (HBV) RNA. Here we demonstrate that antibodies against the human La protein specifically precipitate HBV RNA from HBV ribonucleoprotein-containing mammalian cell extracts, providing evidence for the association between human La and HBV RNA. Moreover, we report that the turnover of HBV RNA depends on structural features and less on the primary sequence of the La-binding site on the viral RNA. In addition we show that the interaction between human La and HBV RNA in vitro is modulated by accessory factor(s) in a phosphorylation-dependent manner. Taken together these data indicate that both structural features, the composition of La/HBV ribonucleoprotein particles as well as interacting cellular factors, are critical determinants in the regulation of the stability of the HBV RNA.     

Purification and properties of the Dictyostelium calpain-like protein,Cpl

Calpains are intracellular, cysteine proteases found in plants, animals, and fungi. There is emerging evidence that they are important mediators of cell adhesion and motility in animal cells. Because the cellular slime mold, Dictyostelium discoideum, is a genetically tractable model for cell adhesion and motility, we have investigated whether a calpain-like protein is expressed in this organism. Contig 13130 (Sanger Institute Dictyostelium sequencing project) was identified as a three-exon gene that encodes a calpain-like protein. Using a custom peptide antibody to assay for the presence of this putative protein, we identified Dictyostelium calpain-like protein (Cpl) and purified it to near homogeneity. Cpl is a 72278 Da cytosolic protein. Weak caseinolytic activity inhibitable by cysteine protease inhibitors was copurified with Cpl immunoreactivity, and purified Cpl appeared to undergo autoproteolysis upon transfer to inhibitor-free buffer. The major cleavage, generating a 51291 Da form, occurred after Pro 189. The Cpl domain structure resembles mammalian calpain 10, comprising an N-terminal catalytic domain followed by tandem calpain D-III domains. The putative catalytic domain appears to possess His and Gln active site residues, instead of the canonical His and Asn residues in calpains. The active site Cys has not yet been identified, and definitive proof of a proteolytic function awaits further study. Its phylogenetic distribution in D. discoideum and several protists suggests that the calpain D-III domain evolved early in eukaryotic cells.     

Hepatitis B virus X protein modulates upregulation of DHX9 to promote viral DNA replication

Hepatitis B virus (HBV) infection is a major cause of acute and chronic liver diseases. During the HBV life cycle, HBV hijacks various host factors to assist viral replication. In this research, we find that the HBV regulatory protein X (HBx) can induce the upregulation of DExH‐box RNA helicase 9 (DHX9) expression by repressing proteasome‐dependent degradation mediated by MDM2. Furthermore, we demonstrate that DHX9 contributes to viral DNA replication in dependence on its helicase activity and nuclear localization. In addition, the promotion of viral DNA replication by DHX9 is dependent on its interaction with Nup98. Our findings reveal that HBx‐mediated DHX9 upregulation is essential for HBV DNA replication.     

Hepatocystin/80K-H inhibits replication of hepatitis B virus through interaction with HBx protein in hepatoma cell

Hepatitis B virus (HBV) X protein (HBx) is a key player in HBV replication as well as HBV-induced hepatocellular carcinoma (HCC). However, the pathogenesis of HBV infection and the mechanisms of host–virus interactions are still elusive. In this study, a combination of affinity purification and mass spectrometry was applied to identify the host factors interacting with HBx in hepatoma cells. Thirteen proteins were identified as HBx binding partners. Among them, we first focused on determining the functional significance of the interaction between HBx and hepatocystin. A physical interaction between HBx and hepatocystin was confirmed by co-immunoprecipitation and Western blotting. Immunocytochemistry demonstrated that HBx and hepatocystin colocalized in the hepatoma cells. Domain mapping of both proteins revealed that the HBx C-terminus (amino acids 110–154) was responsible for binding to the mannose 6-phosphate receptor homology domain (amino acids, 419–525) of hepatocystin. Using translation and proteasome inhibitors, we found that hepatocystin overexpression accelerated HBx degradation via a ubiquitin-independent proteasome pathway. We demonstrated that this effect was mediated by an interaction between both proteins using a HBx deletion mutant. Hepatocystin overexpression significantly inhibited HBV DNA replication and expression of HBs antigen concomitant with HBx degradation. Using the hepatocystin mutant constructs that bind HBx, we also confirmed that hepatocystin inhibited HBx-dependent HBV replication. In conclusion, we demonstrated for the first time that hepatocystin functions as a chaperon-like molecule by accelerating HBx degradation, and thereby inhibits HBV replication. Our results suggest that inducing hepatocystin may provide a novel therapeutic approach to control HBV infection.     

Interleukin-1 and Tumor Necrosis Factor-α Trigger Restriction of Hepatitis B Virus Infection via a Cytidine Deaminase Activation-induced Cytidine Deaminase (AID)

Virus infection is restricted by intracellular immune responses in host cells, and this is typically modulated by stimulation of cytokines. The cytokines and host factors that determine the host cell restriction against hepatitis B virus (HBV) infection are not well understood. We screened 36 cytokines and chemokines to determine which were able to reduce the susceptibility of HepaRG cells to HBV infection. Here, we found that pretreatment with IL-1β and TNFα remarkably reduced the host cell susceptibility to HBV infection. This effect was mediated by activation of the NF-κB signaling pathway. A cytidine deaminase, activation-induced cytidine deaminase (AID), was up-regulated by both IL-1β and TNFα in a variety of hepatocyte cell lines and primary human hepatocytes. Another deaminase APOBEC3G was not induced by these proinflammatory cytokines. Knockdown of AID expression impaired the anti-HBV effect of IL-1β, and overexpression of AID antagonized HBV infection, suggesting that AID was one of the responsible factors for the anti-HBV activity of IL-1/TNFα. Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity. The antiviral effect of IL-1/TNFα was also observed on different HBV genotypes but not on hepatitis C virus. These results demonstrate that proinflammatory cytokines IL-1/TNFα trigger a novel antiviral mechanism involving AID to regulate host cell permissiveness to HBV infection.