Inhibition of Translation Initiation by Protein 169: A Vaccinia Virus Strategy to Suppress Innate and Adaptive Immunity and Alter Virus Virulence.

Vaccinia virus (VACV) is the prototypic orthopoxvirus and the vaccine used to eradicate smallpox. Here we show that VACV strain Western Reserve protein 169 is a cytoplasmic polypeptide expressed early during infection that is excluded from virus factories and inhibits the initiation of cap-dependent and cap-independent translation. Ectopic expression of protein 169 causes the accumulation of 80S ribosomes, a reduction of polysomes, and inhibition of protein expression deriving from activation of multiple innate immune signaling pathways. A virus lacking 169 (vΔ169) replicates and spreads normally in cell culture but is more virulent than parental and revertant control viruses in intranasal and intradermal murine models of infection. Intranasal infection by vΔ169 caused increased pro-inflammatory cytokines and chemokines, infiltration of pulmonary leukocytes, and lung weight. These alterations in innate immunity resulted in a stronger CD8+ T-cell memory response and better protection against virus challenge. This work illustrates how inhibition of host protein synthesis can be a strategy for virus suppression of innate and adaptive immunity.

Inhibition of the RNA polymerase III-mediated dsDNA-sensing pathway of innate immunity by vaccinia virus protein E3.

The vaccinia virus E3 protein is an important intracellular modulator of innate immunity that can be split into distinct halves. The C terminus contains a well defined dsRNA-binding domain, whereas the N terminus contains a Z-DNA-binding domain, and both domains are required for virulence. In this study, we investigated whether the E3 Z-DNA-binding domain functions by sequestering cytoplasmic dsDNA thereby preventing the induction of type I interferon (IFN). In line with this hypothesis, expression of E3 ablated both IFN-beta expression and NF-kappaB activity in response to the dsDNA, poly(dA-dT). However, surprisingly, the ability of E3 to block poly(dA-dT) signalling was independent of the N terminus, whereas the dsRNA-binding domain was essential, suggesting that the Z-DNA-binding domain does not bind immunostimulatory dsDNA. This was confirmed by the failure of E3 to co-precipitate with biotinylated dsDNA, whereas the recruitment of several cytoplasmic DNA-binding proteins could be detected. Recently, AT-rich dsDNA was reported to be transcribed into 5'-triphosphate poly(A-U) RNA by RNA polymerase III, which then activates retinoic acid-inducible gene I (RIG-I). Consistent with this, RNA from poly(dA-dT) transfected cells induced IFN-beta and expression of the E3 dsRNA-binding domain was sufficient to ablate this response. Given the well documented function of the E3 dsRNA-binding domain we propose that E3 blocks signalling in response to poly(dA-dT) by binding to transcribed poly(A-U) RNA preventing RIG-I activation. This report describes a DNA virus-encoded inhibitor of the RNA polymerase III-dsDNA-sensing pathway and extends our knowledge of E3 as a modulator of innate immunity.

Epstein-Barr virus-encoded EBNA1 inhibits the canonical NF-kappaB pathway in carcinoma cells by inhibiting IKK phosphorylation.

BACKGROUND: The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all EBV-associated tumours, including undifferentiated nasopharyngeal carcinoma (NPC), where it is indispensable for viral replication, genome maintenance and viral gene expression. EBNA1's transcription factor-like functions also extend to influencing the expression of cellular genes involved in pathways commonly dysregulated during oncogenesis, including elevation of AP-1 activity in NPC cell lines resulting in enhancement of angiogenesis in vitro. In this study we sought to extend these observations by examining the role of EBNA1 upon another pathway commonly deregulated during carcinogenesis; namely NF-kappaB. RESULTS: In this report we demonstrate that EBNA1 inhibits the canonical NF-kappaB pathway in carcinoma lines by inhibiting the phosphorylation of IKKalpha/beta. In agreement with this observation we find a reduction in the phosphorylation of IkappaBalpha and reduced phosphorylation and nuclear translocation of p65, resulting in a reduction in the amount of p65 in nuclear NF-kappaB complexes. Similar effects were also found in carcinoma lines infected with recombinant EBV and in the EBV-positive NPC-derived cell line C666-1. Inhibition of NF-kappaB was dependent upon regions of EBNA1 essential for gene transactivation whilst the interaction with the deubiquitinating enzyme, USP7, was entirely dispensable. Furthermore, in agreement with EBNA1 inhibiting p65 NF-kappaB we demonstrate that p65 was exclusively cytoplasmic in 11 out of 11 NPC tumours studied. CONCLUSIONS: Inhibition of p65 NF-kappaB in murine and human epidermis results in tissue hyperplasia and the development of squamous cell carcinoma. In line with this, p65 knockout fibroblasts have a transformed phenotype. Inhibition of p65 NF-kappaB by EBNA1 may therefore contribute to the development of NPC by inducing tissue hyperplasia. Furthermore, inhibition of NF-kappaB is employed by viruses as an immune evasion strategy which is also closely linked to oncogenesis during persistent viral infection. Our findings therefore further implicate EBNA1 in playing an important role in the pathogenesis of NPC.

Epstein-Barr virus-encoded EBNA1 modulates the AP-1 transcription factor pathway in nasopharyngeal carcinoma cells and enhances angiogenesis in vitro.

The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumours, including nasopharyngeal carcinoma (NPC), where it plays an essential role in EBV genome maintenance, replication and transcription. Previous studies suggest that EBNA1 may have additional effects relevant to oncogenesis, including enhancement of cell survival, raising the possibility that EBNA1 may influence cellular gene expression. We have recently demonstrated by gene expression microarray profiling in an NPC cell model that EBNA1 influences the expression of a range of cellular genes, including those involved in transcription, translation and cell signalling. Here, we report for the first time that EBNA1 enhances activity of the AP-1 transcription factor in NPC cells and demonstrate that this is achieved by EBNA1 binding to the promoters of c-Jun and ATF2, enhancing their expression. In addition, we demonstrate elevated expression of the AP-1 targets interleukin 8, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1alpha in response to EBNA1 expression, which enhances microtubule formation in an in vitro angiogenesis assay. Furthermore, we confirm elevation of VEGF and the phosphorylated isoforms of c-Jun and ATF2 in NPC biopsies. These findings implicate EBNA1 in the angiogenic process and suggest that this viral protein might directly contribute to the development and aggressively metastatic nature of NPC.