Critical role of TRAF3 in the Toll-like receptor-dependent and -independent antiviral response

Type I interferon (IFN) production is a critical component of the innate defence against viral infections. Viral products induce strong type I IFN responses through the activation of Toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as protein kinase R (PKR). Here we demonstrate that cells lacking TRAF3, a member of the TNF receptor-associated factor family, are defective in type I IFN responses activated by several different TLRs. Furthermore, we show that TRAF3 associates with the TLR adaptors TRIF and IRAK1, as well as downstream IRF3/7 kinases TBK1 and IKK-epsilon, suggesting that TRAF3 serves as a critical link between TLR adaptors and downstream regulatory kinases important for IRF activation. In addition to TLR stimulation, we also show that TRAF3-deficient fibroblasts are defective in their type I IFN response to direct infection with vesicular stomatitis virus, indicating that TRAF3 is also an important component of TLR-independent viral recognition pathways. Our data demonstrate that TRAF3 is a major regulator of type I IFN production and the innate antiviral response.     

Defective interfering particles with covalently linked [+/-]RNA induce interferon.

Defective interfering (DI) particles of vesicular stomatitis virus which contain covalently linked complementary [+]message and [-]anti-message RNA as a single-stranded ribonucleoprotein complex within the particle, are extremely efficient inducers of interferon. A single particle can induce a quantum yield of interferon. A single molecule of double-stranded RNA presumed to form, at least in part, on entry into the cell is thought to induce interferon synthesis. Conventional [-]RNA DI particles with the same polypeptide composition as [+/-]RNA DI particles fail to induce interferon.     

Science Letters: Transient expression of chicken alpha interferon gene in lettuce

We investigated the possibility of producing chicken alpha interferon （ChlFN-α） in transgenic plants. The cDNA encoding ChlFN-α was introduced into lettuce （Lactuca sativa L.） plants by using an agro-infiltration transient expression system. The ChlFN-a gene was correctly transcribed and translated in the lettuce plants according to RT-PCR and ELISA assays. Recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus （VSV） replication on chicken embryonic fibroblast （CEF）. The results demonstrate that biologically active avian cytokine with potential pharmaceutical applications could be expressed in transgenic lettuce plants and that it is possible to generate interferon protein in forage plants for preventing infectious diseases of poultry.     

Virus-specific effects of interferon in embryonal carcinoma cells

Embryonal carcinoma (EC) cells are susceptible to infection by a variety of viruses, but do not become resistant to infection by Semliki Forest virus or vesicular stomatitis virus (VSV) on treatment with interferon. These observations have led to the conclusion that interferon does not induce an antiviral state in EC cells. We report here, however, that EC cells treated with interferon become resistant to infection by two picornaviruses and two ts mutants of VSV, whereas they remain sensitive to wild-type VSV, Sindbis and influenza virus infectin. These results suggest that a partial antiviral state is induced in EC cells by interferon and that the induced antiviral protein(s) interferes with the replication of specific viruses. A significant common feature of these viruses is their replication through structures containing double-stranded RNA (dsRNA).     

2'-O methylation of the viral mRNA cap evades host restriction by IFIT family members

Cellular messenger RNA (mRNA) of higher eukaryotes and many viral RNAs are methylated at the N-7 and 2'-O positions of the 5' guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability, the function of 2'-O methylation has remained uncertain since its discovery 35 years ago. Here we show that a West Nile virus (WNV) mutant (E218A) that lacks 2'-O MTase activity was attenuated in wild-type primary cells and mice but was pathogenic in the absence of type I interferon (IFN) signalling. 2'-O methylation of viral RNA did not affect IFN induction in WNV-infected fibroblasts but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISGs) implicated in regulation of protein translation. Poxvirus and coronavirus mutants that lacked 2'-O MTase activity similarly showed enhanced sensitivity to the antiviral actions of IFN and, specifically, IFIT proteins. Our results demonstrate that the 2'-O methylation of the 5' cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression, and suggest an evolutionary explanation for 2'-O methylation of cellular mRNA: to distinguish self from non-self RNA. Differential methylation of cytoplasmic RNA probably serves as an example for pattern recognition and restriction of propagation of foreign viral RNA in host cells.     

Constitutive expression of (2'-5') oligo A synthetase confers resistance to picornavirus infection

Study of the mechanisms by which interferon (IFN) treatment of cells induces resistance to virus infections has been complicated by the multiple biochemical changes induced. Over 20 proteins are increased by IFN, including the double-stranded (ds) RNA-activated protein kinase, (2'-5') oligo A synthetase, surface proteins such as the major histocompatibility complex (MHC) proteins, and various proteins with unknown functions. The availability of cloned complementary DNAs for several IFN-induced proteins now allows us to probe their roles in IFN action. For instance, the murine Mx protein has been shown to confer resistance, to influenza virus. We studied chinese hamster ovary (CHO) cell clones expressing high constitutive levels of (2'-5') A synthetase as a result of transfection with the cDNA encoding the enzyme form which has a relative molecular mass (Mr) of 40K. Elevated enzyme correlates directly with resistance to infection by a picornavirus such as Mengo, but does not make the cells resistant to vesicular stomatitis virus (VSV).     

An interferon-induced cellular enzyme is incorporated into virions

The mechanisms by which interferon inhibits viral growth are only partially understood. Several enzymatic activities increase in cells shortly after treatment with interferon. One of these enzymes, oligo-isoadenylate synthetase, synthesizes (2'-5') isoadenylate oligomers which strongly stimulate the activity of a cellular ribonuclease, RNase F (ref. 7). Interferon also significantly increases the activity of a protein kinase which phosphorylates the initiation factor eIF-2 and can inhibit in vitro protein synthesis. Such interferon-induced enzymes, which affect RNA and protein metabolism, might be responsible for many of its effects on viruses. Indeed, inhibition of viral protein and RNA synthesis appears to have a major role in the antiviral state. We have now investigated possible interactions of the two enzymes with viral constituents during the course of infection and found that in two different membrane-coated RNA viruses, vesicular stomatitis virus (VSV) and Moloney murine leukaemia virus (M-MuLV), there is an accumulation of the 2'-5') oligo-isoadenylate synthetase (E) in the virions. Most of the enzyme is bound to the virion ribonucleoprotein core. The incorporation of E into the virions suggests a direct involvement of the enzyme in regulation of virus functions.     

Translation in vitro of vesicular stomatitis virus mRNA lacking 5'-terminal 7-methylguanosine.

The 5'-terminus 7-methylguanosine of vesicular stomatitis virus mRNA is not essential for translation of the mRNA. The 7-methylguanosine seems to have a mediating rather than an obligatory role in ribosome binding by the mRNA and in mRNA translation.     

Interferon inhibits transformation by murine sarcoma viruses before integration of provirus

Neoplastic transformation by C-type retroviruses requires synthesis of a DNA copy (the provirus) of the RNA genome and its integration into the host cell DNA. We have previously shown that interferon (IFN) can stably prevent transformation of murine fibroblasts by the Kirsten strain of murine sarcoma virus (KiMSV), a murine leukaemia virus (MLV). A series of cell clones (IFN clones), isolated in the presence of IFN (10(4) U ml-1) from cultures of NIH-3T3 cells which had been treated with IFN, and then infected with KiMSV (KiMLV) in conditions where every cell was infected, were shown to be phenotypically untransformed. These untransformed cells did not produce virus or contain rescuable KiMSV. However, cells isolated using an identical procedure, but in the absence of IFN, were uniformly transformed and all produced KiMSV (KiMLV) or contained rescuable KiMSV. It was concluded that IFN either prevents synthesis or integration of the provirus, or else that in the presence of IFN the provirus is integrated such that it is not expressed. We now show that five representative clones contain no detectable KiMSV proviral DNA, and also that the initial stages of infection by KiMSV (KiMLV) are inhibited by IFN treatment. IFN seems to act before integration, preventing either the synthesis or the integration of proviral DNA.     

Endogenous viral genes are non-essential in the chicken.

DNA sequences homologous to the genomes of type C retroviruses are widespread among vertebrates. Ten genetic loci containing endogenous viral DNA sequences have been documented in the white Leghorn chicken alone. Six of these genetic loci are associated with the production of virus or of viral proteins in embryonic fibroblasts (refs 2--4, and S.M.A., L, B. Crittenden and E.G.B., in preparation) and one of the loci may be expressed in the erythroblasts of 5-day-old embryos. The abiquitous presence of endogenous viral genes among vertebrate species and the association of their expression with development of the haematopoietic system in the mouse have led to the proposal that these genes are involved in ontogeny. In addition, the genes may be implicated in oncogenesis as in the case of the AKR mouse in which a high incidence of spontaneous leukaemia is associated with the expression of endogenous murine laukaemia virus genomes. We report here the production of a fertile rooster which lacks avian leukosis virus-related endogenous viral genes and which seems to be completely normal and healthy. Thus, endogenous viral genes are apparently not essential for the normal development of the chicken. An endogenous virus-free state has also been reported for three species of jungle fowl and for the B-type viral genes of the mouse.     

Preliminary study on human fibroblasts as feeder layer for human embryonic stem cells culture in vitro

To avoid the direct contact with mouse cells and possible heterogeneous pathogen in future application ,we need to replace mouse embryonic fibroblasts with human fibroblasts as the feeder layer to maintain human embryonic stem cells growth in the undifferentiated state,We Success-fully use human fibroblasts derved from aborted fetus and adult prepuces as feeder layer to maintain human embryonic stem cells growth ,During the passage and growth on this feeder layer,the human embryonic stem cells can keep their undifferentiated state.     

A synthetic glycolipid prevents autoimmune encephalomyelitis by inducing TH2 bias of natural killer T cells

Experimental autoimmune encephalomyelitis (EAE) is a prototype autoimmune disease mediated by type 1 helper T (TH1) cells and under the control of regulatory cells. Here we report that a synthetic glycolipid ligand for CD1d-restricted natural killer T (NKT) cells expressing the semi-invariant T-cell receptor (Valpha14+) is preventive against EAE. The ligand is an analogue of alpha-galactosylceramide (alpha-GC), a prototype NKT cell ligand, with a truncated sphingosine chain. alpha-GC causes NKT cells to produce both interferon (IFN)-gamma and interleukin (IL)-4 (refs 4, 5). However, this new ligand can induce a predominant production of IL-4 by the NKT cells. A single injection of this glycolipid, but not of alpha-GC, consistently induced TH2 bias of autoimmune T cells by causing NKT cells to produce IL-4, leading to suppression of EAE. The lack of polymorphism of CD1d and cross-reactive response of mouse and human NKT cells to the same ligand indicates that targeting NKT cells with this ligand may be an attractive means for intervening in human autoimmune diseases such as multiple sclerosis.     

A diverse range of gene products are effectors of the type I interferon antiviral response

The type I interferon response protects cells against invading viral pathogens. The cellular factors that mediate this defence are the products of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified since their discovery more than 25 years ago, only a few have been characterized with respect to antiviral activity. For most ISG products, little is known about their antiviral potential, their target specificity and their mechanisms of action. Using an overexpression screening approach, here we show that different viruses are targeted by unique sets of ISGs. We find that each viral species is susceptible to multiple antiviral genes, which together encompass a range of inhibitory activities. To conduct the screen, more than 380 human ISGs were tested for their ability to inhibit the replication of several important human and animal viruses, including hepatitis C virus, yellow fever virus, West Nile virus, chikungunya virus, Venezuelan equine encephalitis virus and human immunodeficiency virus type-1. Broadly acting effectors included IRF1, C6orf150 (also known as MB21D1), HPSE, RIG-I (also known as DDX58), MDA5 (also known as IFIH1) and IFITM3, whereas more targeted antiviral specificity was observed with DDX60, IFI44L, IFI6, IFITM2, MAP3K14, MOV10, NAMPT (also known as PBEF1), OASL, RTP4, TREX1 and UNC84B (also known as SUN2). Combined expression of pairs of ISGs showed additive antiviral effects similar to those of moderate type I interferon doses. Mechanistic studies uncovered a common theme of translational inhibition for numerous effectors. Several ISGs, including ADAR, FAM46C, LY6E and MCOLN2, enhanced the replication of certain viruses, highlighting another layer of complexity in the highly pleiotropic type I interferon system.     

IRF7表达与系统性红斑狼疮的相关性

探讨了干扰素调节因子7(IRF7)的表达与中国汉族人群系统性红斑狼疮(SLE)的相关性.运用实时荧光定量聚合酶链式反应的方法分别测定疾病患者与正常对照组外周血IRF7mRNA的表达水平,并与血清中干扰素水平、干扰素积分以及SLEDAI积分作相关性分析.结果发现,患者外周血IRF7mRNA的表达水平较正常对照组的明显增高,且其与血清中干扰素水平、干扰素积分以及SLEDAI积分均呈正相关.由此可知,IRF7表达的增高可能促使干扰素通路异常激活,从而导致系统性红斑狼疮的发生.     

Construction, expression and recognition of an H-2 molecule lacking its carboxyl terminus

A mouse major histocompatibility antigen (H-2) gene, encoding a novel H-2Ld molecule lacking its intracytoplasmic domain, has been constructed and introduced into mouse L-cells. The novel H-2 molecule is found on the surface of the transfected cells at the same level as L-cells transfected with the native H-2Ld gene. Allo- and influenza-specific cytotoxic T lymphocytes can recognize the truncated H-2 gene product nearly as efficiently as the normal H-2Ld gene product. However, vesicular stomatitis virus-specific cytotoxic T lymphocytes recognize the truncated H-2Ld molecule less efficiently than the complete H-2Ld product. The rate of capping of the truncated H-2Ld molecule was investigated and found to be the same as that of the complete H-2Ld gene product.