RESUMO
The IFN immune system plays an essential role in protecting the host against most viral infections. In order to explore the interactions between the IFN pathway and human respiratory syncytial virus (RSV) infection, and to identify potential IFN-stimulated genes (ISGs) that may be involved in suppressing the replication of RSV, we utilized an IFN pathway-specific microarray to study the effects of RSV infection on the IFN pathway in HeLa cells. We showed that RSV infection enhanced the expression of a series of ISGs, including oligoadenylate synthetase 2, IFITM1 (IFN-induced transmembrane protein 1) and myxovirus resistance 2. Our results also showed that the IFITM proteins potently inhibited RSV infection mainly by interfering with both virus entry and the subsequent replication steps, but not the attachment process. The antiviral effect of IFITM3 was not affected by ubiquitination modification. Furthermore, knocking down the endogenous and IFN-induced expression of IFITM1 and 3 facilitated RSV infection. Expression of the IFITM proteins was found to delay the phosphorylation of IFN regulatory factor 3 through interfering with the detection of viral RNA by MDA5 (melanoma differentiation-associated gene 5) and RIG-I (retinoic acid-inducible gene I). These results demonstrated that the restriction of RSV infection by the IFITM proteins was achieved through the inhibition of virus entry and replication, and they provided further insight for exploring the mechanism of IFITM-protein-mediated virus restriction.
Assuntos
Antígenos de Diferenciação/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sincicial Respiratório Humano/metabolismo , Antígenos de Diferenciação/genética , Antivirais/metabolismo , Linhagem Celular Tumoral , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Células HeLa , Humanos , Helicase IFIH1 Induzida por Interferon , Interferons/genética , Interferons/metabolismo , Proteínas de Membrana/genética , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Fosforilação/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Infecções por Vírus Respiratório Sincicial/genética , Ubiquitinação/genética , Internalização do Vírus , Replicação Viral/genéticaRESUMO
Chronic hepatitis B virus infection is a major cause of hepatic fibrosis, leading to liver cirrhosis and hepatocellular carcinoma. Hepatitis B virus e antigen (HBeAg) is an accessory protein of HBV, not required for viral replication but important for natural infection in vivo. Hepatic stellate cells (HSCs) are the major producers of excessive extracellular matrix during liver fibrogenesis. Therefore, we examined the influence of HBeAg on HSCs. The rat HSC line HSC-T6 was transfected with HBeAg plasmids, and expression of α-smooth muscle actin, collagen I, transforming growth factor-ß1 (TGF-ß), and tissue inhibitors of metalloproteinase 1 (TIMP-1) was investigated by quantitative real-time PCR. The proliferation of HSCs was determined by MTS analysis. HBeAg transduction induced up-regulation of these fibrogenic genes and proliferation of HSCs. We found that HBeAg induced TGF-ß secretion in HSCs, and the activation of HSCs was prevented by a neutralizing anti-TGF-ß antibody. Depletion and addition of HBeAg protein in conditioned medium from HSC-T6 cells transduced with HBeAg indicated that HBeAg directly induced the activation and proliferation of rat primary HSCs. Taken together, HBeAg induces the activation and proliferation of HSCs, mainly mediated by TGF-ß, and HBeAg protein purified from cell medium can directly activate HSCs.
Assuntos
Células Estreladas do Fígado/imunologia , Células Estreladas do Fígado/virologia , Antígenos E da Hepatite B/fisiologia , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/patogenicidade , Animais , Linhagem Celular , Regulação da Expressão Gênica/imunologia , Células Estreladas do Fígado/metabolismo , Antígenos E da Hepatite B/genética , Antígenos E da Hepatite B/isolamento & purificação , Vírus da Hepatite B/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Ratos , Transfecção , Fator de Crescimento Transformador beta/fisiologiaRESUMO
We reported previously that heat shock protein gp96 and its N-terminal fragment were able to stimulate CTL expansion specific for a HBV peptide (SYVNTNMGL) in BALB/c mice. Here we characterized the adjuvant effects of gp96 on human HLA-A2 restricted T cells. Full-length gp96 isolated from healthy human liver and recombinant fragments both from prokaryotic cells and eukaryotic cells were analyzed for their ability to stimulate maturation of human dendritic cells. It was found that in vitro these proteins were capable of maturating human monocyte-derived dendritic cells (MDDC) isolated from healthy donors as well as from HBV-positive, hepatocellular carcinoma (HCC) patients. In HLA-A2.1/Kb transgenic mice, gp96 and the recombinant fragments were found to augment CTL response specific for the HBcAg(18-27) FLPSDFFPSV peptide of hepatitis B virus.