Emerging complexity and new roles for the RIG-I-like receptors in innate antiviral immunity

Innate immunity is critical for the control of virus infection and operates to restrict viral susceptibility and direct antiviral immunity for protection from acute or chronic viral-associated diseases including cancer. RIG-I like receptors (RLRs) are cytosolic RNA helicases that function as pathogen recognition receptors to detect RNA pathogen associated molecular patterns (PAMPs) of virus infection. The RLRs include RIG-I, MDA5, and LGP2. They function to recognize and bind to PAMP motifs within viral RNA in a process that directs the RLR to trigger downstream signaling cascades that induce innate immunity that controls viral replication and spread. Products of RLR signaling also serve to modulate the adaptive immune response to infection. Recent studies have additionally connected RLRs to signaling cascades that impart inflammatory and apoptotic responses to virus infection. Viral evasion of RLR signaling supports viral outgrowth and pathogenesis, including the onset of viral-associated cancer.     

Both E6 and E7 oncoproteins of human papillomavirus 16 inhibit IL-18-induced IFN-gamma production in human peripheral blood mononuclear and NK cells.

Cervical carcinoma is the predominant cancer among malignancies in women throughout the world, and human papillomavirus (HPV) 16 is the most common agent linked to human cervical carcinoma. The present study was performed to investigate the mechanisms of immune escape in HPV-induced cervical cancer cells. The presence of HPV oncoproteins E6 and E7 in the extracellular fluids of HPV-containing cervical cancer cell lines SiHa and CaSki was demonstrated by ELISA. The effect of HPV 16 oncoproteins E6 and E7 on the production of IFN-gamma by IL-18 was assessed. E6 and E7 proteins reduced IL-18-induced IFN-gamma production in both primary PBMCs and the NK0 cell line. FACS analysis revealed that the viral oncoproteins reduced the binding of IL-18 to its cellular surface receptors on NK0 cells, whereas there was no effect of oncoproteins on IL-1 binding to its surface IL-1 receptors on D10S, a subclone of the murine Th cell D10.G4.1. In vitro pull-down assays also revealed that the viral oncoproteins and IL-18 bound to IL-18R alpha-chain competitively. These results suggest that the extracellular HPV 16 E6 and E7 proteins may inhibit IL-18-induced IFN-gamma production locally in HPV lesions through inhibition of IL-18 binding to its alpha-chain receptor. Down-modulation of IL-18-induced immune responses by HPV oncoproteins may contribute to viral pathogenesis or carcinogenesis.     

水稻免疫机制研究进展

植物先天免疫主要由两部分组成：一类是通过细胞膜上的病原菌分子模式识别受体识别病原微生物表面存在的分子特征激发的免疫反应（PTI）;另一类是专化性的抗病R蛋白识别病原微生物的效应蛋白,从而激发下游的病原菌小种特异性的防卫反应过程（ETI）．随着水稻抗病信号途径中越来越多的抗病基因以及关键的调控基因被克隆和功能鉴定,同时多种水稻病原菌效应蛋白的发现,水稻抗病机理的研究也越来越深入．本文阐述了水稻的PTI,ETI及其下游参与免疫信号转导的关键性组分,从而形成一个初步的水稻免疫调控网络．     

Innate sensing of self and non-self RNAs by Toll-like receptors

Toll-like receptors (TLRs) have an important role in innate immunity in mammals by recognizing conserved microbial components that are known as pathogen-associated molecular patterns (PAMPs). Although the majority of these receptors sense pathogen components on the cell surface, a subset of them (TLR3, TLR7, TLR8 and TLR9) senses viral and bacterial nucleic acids in endosomal compartments. Of considerable interest is the recent finding that TLR7 and TLR8 can also recognize small interfering RNA (siRNA), which is the main effector in RNA interference. This immune activation by siRNAs can be abrogated by the 2'-ribose modification of uridines. Here, we discuss the recent developments that have expanded the understanding of self-non-self discrimination of RNAs by the innate immune system, and consider future directions for therapeutic applications of these findings.     

Innate immunity to respiratory viruses

Pattern recognition receptors are critically involved in the development of innate and adaptive antiviral immunity. Innate immune activation by viruses may occur via cell surface, intracellular and cytosolic pattern recognition receptors. These receptors sense viral components and may activate unique downstream pathways to generate antiviral immunity. In this article, we summarize the pattern recognition receptors that recognize major human respiratory viral pathogens, including influenza virus, respiratory syncytial virus and adenovirus. We also provide an overview of the current knowledge of regulation of type I interferons and inflammatory cytokines in viral infection.     

The JAK-STAT Transcriptional Regulator,STAT-5, Activates the ATM DNA Damage Pathway to Induce HPV 31 Genome Amplification upon Epithelial Differentiation

High-risk human papillomavirus (HPV) must evade innate immune surveillance to establish persistent infections and to amplify viral genomes upon differentiation. Members of the JAK-STAT family are important regulators of the innate immune response and HPV proteins downregulate expression of STAT-1 to allow for stable maintenance of viral episomes. STAT-5 is another member of this pathway that modulates the inflammatory response and plays an important role in controlling cell cycle progression in response to cytokines and growth factors. Our studies show that HPV E7 activates STAT-5 phosphorylation without altering total protein levels. Inhibition of STAT-5 phosphorylation by the drug pimozide abolishes viral genome amplification and late gene expression in differentiating keratinocytes. In contrast, treatment of undifferentiated cells that stably maintain episomes has no effect on viral replication. Knockdown studies show that the STAT-5β isoform is mainly responsible for this activity and that this is mediated through the ATM DNA damage response. A downstream target of STAT-5, the peroxisome proliferator-activated receptor γ (PPARγ) contributes to the effects on members of the ATM pathway. Overall, these findings identify an important new regulatory mechanism by which the innate immune regulator, STAT-5, promotes HPV viral replication through activation of the ATM DNA damage response.     

Immunogenicity of Bivalent Human Papillomavirus DNA Vaccine Using Human Endogenous Retrovirus Envelope-Coated Baculoviral Vectors in Mice and Pigs

Human papillomavirus is known to be the major pathogen of cervical cancer. Here, we report the efficacy of a bivalent human papillomavirus type 16 and 18 DNA vaccine system following repeated dosing in mice and pigs using a recombinant baculovirus bearing human endogenous retrovirus envelope protein (AcHERV) as a vector. The intramuscular administration of AcHERV-based HPV16L1 and HPV18L1 DNA vaccines induced antigen-specific serum IgG, vaginal IgA, and neutralizing antibodies to levels comparable to those achieved using the commercially marketed vaccine Cervarix. Similar to Cervarix, AcHERV-based bivalent vaccinations completely blocked subsequent vaginal challenge with HPV type-specific pseudovirions. However, AcHERV-based bivalent vaccinations induced significantly higher cell-mediated immune responses than Cervarix, promoting 4.5- (HPV16L1) and 3.9-(HPV18L1) fold higher interferon-γ production in splenocytes upon stimulation with antigen type-specific pseudovirions. Repeated dosing did not affect the immunogenicity of AcHERV DNA vaccines. Three sequential immunizations with AcHERV-HP18L1 DNA vaccine followed by three repeated dosing with AcHERV-HP16L1 over 11 weeks induced an initial production of anti-HPV18L1 antibody followed by subsequent induction of anti-HPV16L1 antibody. Finally, AcHERV-based bivalent DNA vaccination induced antigen-specific serum IgG immune responses in pigs. These results support the further development of AcHERV as a bivalent human papillomavirus DNA vaccine system for use in preventing the viral infection as well as treating the infected women by inducing both humoral and cell-mediated immune responses. Moreover, the possibility of repeated dosing indicates the utility of AcHERV system for reusable vectors of other viral pathogen vaccines.     

Toll-Like Receptor-3 Is Dispensable for the Innate MicroRNA Response to West Nile Virus (WNV)

The innate immune response to West Nile virus (WNV) infection involves recognition through toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), leading to establishment of an antiviral state. MiRNAs (miRNAs) have been shown to be reliable biomarkers of TLR activation. Here, we sought to evaluate the contribution of TLR3 and miRNAs to the host response to WNV infection. We first analyzed HEK293-NULL and HEK293-TLR3 cells for changes in the innate immune response to infection. The presence of TLR3 did not seem to affect WNV load, infectivity or phosphorylation of IRF3. Analysis of experimentally validated NFκB-responsive genes revealed a WNV-induced signature largely independent of TLR3. Since miRNAs are involved in viral pathogenesis and the innate response to infection, we sought to identify changes in miRNA expression upon infection in the presence or absence of TLR3. MiRNA profiling revealed 70 miRNAs induced following WNV infection in a TLR3-independent manner. Further analysis of predicted gene targets of WNV signature miRNAs revealed genes highly associated with pathways regulating cell death, viral pathogenesis and immune cell trafficking.     

Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity

Alpha/beta interferon immune defenses are essential for resistance to viruses and can be triggered through the actions of the cytoplasmic helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Signaling by each is initiated by the recognition of viral products such as RNA and occurs through downstream interaction with the IPS-1 adaptor protein. We directly compared the innate immune signaling requirements of representative viruses of the Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Reoviridae for RIG-I, MDA5, and interferon promoter-stimulating factor 1 (IPS-1). In cultured fibroblasts, IPS-1 was essential for innate immune signaling of downstream interferon regulatory factor 3 activation and interferon-stimulated gene expression, but the requirements for RIG-I and MDA5 were variable. Each was individually dispensable for signaling triggered by reovirus and dengue virus, whereas RIG-I was essential for signaling by influenza A virus, influenza B virus, and human respiratory syncytial virus. Functional genomics analyses identified cellular genes triggered during influenza A virus infection whose expression was strictly dependent on RIG-I and which are involved in processes of innate or adaptive immunity, apoptosis, cytokine signaling, and inflammation associated with the host response to contemporary and pandemic strains of influenza virus. These results define IPS-1-dependent signaling as an essential feature of host immunity to RNA virus infection. Our observations further demonstrate differential and redundant roles for RIG-I and MDA5 in pathogen recognition and innate immune signaling that may reflect unique and shared biologic properties of RNA viruses whose differential triggering and control of gene expression may impact pathogenesis and infection.     

Quantitative keratinocyte assay detects two biological activities of human papillomavirus DNA and identifies viral types associated with cervical carcinoma.

Keratinocytes electroporated with human papillomavirus (HPV) DNA (HPV-6, 11, 16 and 18) exhibited an increased cellular proliferation which was quantitated as microcolony and macrocolony formation. However, only macrocolonies induced by HPV-16 or HPV-18 DNA (the two viral types most commonly found in human cervical carcinomas) gave rise to proliferating, poorly-stratified colonies when grown in the presence of serum and calcium. Hydrocortisone increased the frequency of these differentiation-resistant colonies, and studies showed that they were immortalized, contained one copy of viral DNA per cell, expressed three discrete species of viral RNA and synthesized the viral E7 protein. HPV-induced cellular proliferation and altered differentiation are therefore separable events and may represent the activity of different viral genes.     

Identification and Validation of Human Papillomavirus Encoded microRNAs

We report here identification and validation of the first papillomavirus encoded microRNAs expressed in human cervical lesions and cell lines. We established small RNA libraries from ten human papillomavirus associated cervical lesions including cancer and two human papillomavirus harboring cell lines. These libraries were sequenced using SOLiD 4 technology. We used the sequencing data to predict putative viral microRNAs and discovered nine putative papillomavirus encoded microRNAs. Validation was performed for five candidates, four of which were successfully validated by qPCR from cervical tissue samples and cell lines: two were encoded by HPV 16, one by HPV 38 and one by HPV 68. The expression of HPV 16 microRNAs was further confirmed by in situ hybridization, and colocalization with p16INK4A was established. Prediction of cellular target genes of HPV 16 encoded microRNAs suggests that they may play a role in cell cycle, immune functions, cell adhesion and migration, development, and cancer. Two putative viral target sites for the two validated HPV 16 miRNAs were mapped to the E5 gene, one in the E1 gene, two in the L1 gene and one in the LCR region. This is the first report to show that papillomaviruses encode their own microRNA species. Importantly, microRNAs were found in libraries established from human cervical disease and carcinoma cell lines, and their expression was confirmed in additional tissue samples. To our knowledge, this is also the first paper to use in situ hybridization to show the expression of a viral microRNA in human tissue.