Inducible CYP4F12 enhances Hepatitis C virus infection via association with viral nonstructural protein 5B.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) functions as an RNA-dependent RNA polymerase in the HCV replication complex derived from the endoplasmic reticulum in hepatic cells. In this study, NS5B was used as bait in a yeast two-hybrid assay to screen a human liver cDNA library. We confirmed that CYP4F12, a member of the cytochrome P450 superfamily, interacted with NS5B. Furthermore, overexpression of CYP4F12 facilitated HCV replication. In contrast, knockdown of CYP4F12 by specific shRNA decreased HCV replication and viral protein expression. Moreover, our results demonstrated that HCV infection increased the binding of the transcription factor SREBP1 to the CYP4F12 promoter and activated the promoter activity, which indicated that HCV infection increased the expression of CYP4F12 through the SREBP1 pathway. Our results showed that HCV infection induced expression of CYP4F12 protein, which bound to the HCV replication complex to facilitate viral replication.

Molecular identification of two new self-incompatible alleles (S-alleles) in Chinese pear (Pyrus bretschneideri).

Self-incompatibility (SI) is an important intraspecific reproductive barrier in flowering plants. To identify the S-alleles of Chinese pear species (Pyrus pyrifolia, P. bretschneideri, P. ussuriensis and P. sinkiangenis etc.), S-RNase-specific PCR amplification, sequence analyses and field pollination tests were performed using two cultivars 'Jingxiang' and 'Esu' of P. bretschneideri as materials. Two new S-RNase genes were identified from the two cultivars. They were 1,122 bp and 1,058 bp in length, and designated as S37-RNase (GenBank accession no. DQ839238) and S38-RNase (GenBank accession no. DQ839239). By comparison of their deduced amino acid sequences with those of S1-to S36-alleles of Oriental pear, it can be seen that both the two new S-alleles had their conserved regions C1 and C2, but their hypervariable regions (HV) were quite different from those of the others. S37 showed a higher similarity (96%) to S38 in the amino acid sequences deduced from them, whereas both of them displayed the highest similarity (98%) to S15 and the lowest (63%) to S32. The two S-alleles had introns of 786 bp and 723 bp, respectively, similar in size to that of S15 (777 bp). Finally, the S-genotypes of 'Jinxiang' and 'Esu' were unambiguously determined as S34S37 and S15S38, respectively.

Development of a more efficient hepatitis B virus vaccine by targeting hepatitis B virus preS to dendritic cells.

BACKGROUND: Conventional hepatitis B virus (HBV) vaccines fail to induce protective antibody titers in 5-10% of immune-competent vaccinees. Therefore, there remains an urgent need to develop a safe and effective HBV vaccine. METHODS: In this study, we developed an effective and economical method for producing the HBV vaccine by using the high binding capacity of biotin-streptavidin. The preS antigen of HBV was fused with the core streptavidin (cSA) moiety (preS-cSA) and highly expressed in Escherichia coli. We investigated whether the preS-cSA protein could target dendritic cells (DCs) by binding a biotinylated antibody against the DC receptor CD205 (biotin-αCD205). Moreover, we evaluated the preS-cSA/biotin-αCD205 complex as a candidate vaccine by detecting the humoral and cellular immune responses elicited by this vaccine. RESULTS: Our data show that the preS-cSA/biotin-αCD205 complex targeted DCs and induced high anti-HBV antibody titers of IgG2a, IgG1, and IgG in vivo. Furthermore, vaccination with the preS-cSA/biotin-αCD205 complex prevented HBV infection in female mice. More interestingly, this novel vaccine exerted a therapeutic role in mice with HBV infection. CONCLUSIONS: Taken together, our results reveal that the preS-cSA/biotin-αCD205 vaccine induces effective immunological protection against HBV, and is a promising candidate for preventing HBV infections.

Soluble interleukin-6 receptor is elevated during influenza A virus infection and mediates the IL-6 and IL-32 inflammatory cytokine burst.

Influenza A virus (IAV) infection is a major worldwide public health problem. However, the factors involved in mediating the inflammatory response to this infection and their relationships remain poorly understood. Here, we show that IAV infection stimulates the expression of the soluble IL-6 receptor (sIL-6R), a multifunctional protein involved in IL-6 signaling. Interestingly, sIL-6R expression upregulated the levels of its own ligand, IL-6 and those of the pro-inflammatory cytokine IL-32. shRNA-mediated knockdown of sIL-6R suppressed IL-6 and IL-32, indicating that this regulation is dependent on sIL-6R during IAV infection. Furthermore, our results demonstrate that IL-32 participates in a negative feedback loop that inhibits sIL-6R while upregulating IL-6 expression during IAV infection. Therefore, we show that sIL-6R is a critical cellular factor involved in the acute inflammatory response to viral infection.

MicroRNA-548 down-regulates host antiviral response via direct targeting of IFN-λ1.

Interferon (IFN)-mediated pathways are a crucial part of the cellular response against viral infection. Type III IFNs, which include IFN-λ1, 2 and 3, mediate antiviral responses similar to Type I IFNs via a distinct receptor complex. IFN-λ1 is more effective than the other two members. Transcription of IFN-λ1 requires activation of IRF3/7 and nuclear factor-kappa B (NF-κB), similar to the transcriptional mechanism of Type I IFNs. Using reporter assays, we discovered that viral infection induced both IFN-λ1 promoter activity and that of the 3'-untranslated region (UTR), indicating that IFN-λ1 expression is also regulated at the post-transcriptional level. After analysis with microRNA (miRNA) prediction programs and 3'UTR targeting site assays, the miRNA-548 family, including miR-548b-5p, miR-548c-5p, miR-548i, miR-548j, and miR-548n, was identified to target the 3'UTR of IFN-λ1. Further study demonstrated that miRNA-548 mimics down-regulated the expression of IFN-λ1. In contrast, their inhibitors, the complementary RNAs, enhanced the expression of IFN-λ1 and IFN-stimulated genes. Furthermore, miRNA-548 mimics promoted infection by enterovirus-71 (EV71) and vesicular stomatitis virus (VSV), whereas their inhibitors significantly suppressed the replication of EV71 and VSV. Endogenous miRNA-548 levels were suppressed during viral infection. In conclusion, our results suggest that miRNA-548 regulates host antiviral response via direct targeting of IFN-λ1, which may offer a potential candidate for antiviral therapy.

HCV NS4B induces apoptosis through the mitochondrial death pathway.

The hepatitis C virus (HCV) NS4B protein is known to induce the formation of a membranous web that is thought to be the site of viral RNA replication. However, the exact functions of NS4B remain poorly characterized. In this study, we found that NS4B induced apoptosis in 293T cells and Huh7 cells, as confirmed by Hoechst staining, DNA fragmentation, and annexin V/PI assays. Furthermore, protein immunoblot analysis demonstrated that NS4B triggered the cleavage of caspase 3, caspase 7, and poly(ADP-ribose) polymerase (PARP). Further studies revealed that NS4B induced the activation of caspase 9, the reduction of mitochondrial membrane potential and the release of cytochrome c from the mitochondria. However, NS4B expression did not trigger XBP1 mRNA splicing and increase the expression of binding immunoglobulin protein (BiP, or GRP78) and C/EBP homologous protein (CHOP), which serves as the indicators of ER stress. Taken together, our results suggest that HCV NS4B induces apoptosis through the mitochondrial death pathway.