Vaccinia Virus E3 Protein Research Articles

Small Hero with Great Powers: Vaccinia Virus E3 Protein and Evasion of the Type I IFN Response.

Poxviridae have developed a plethora of strategies to evade innate and adaptive immunity. In this review, we focused on the vaccinia virus E3 protein, encoded by the E3L gene. E3 is present within the Chordopoxvirinae subfamily (with the exception of the avipoxviruses and molluscum contagiosum virus) and displays pleiotropic effects on the innate immune system. Initial studies identified E3 as a double-stranded RNA (dsRNA)-binding protein (through its C terminus), able to inhibit the activation of protein kinase dependent on RNA (PKR) and the 2′5′-oligoadenylate synthetase (OAS)/RNase L pathway, rendering E3 a protein counteracting the type I interferon (IFN) system. In recent years, N-terminal mutants of E3 unable to bind to Z-form nucleic acids have been shown to induce the cellular death pathway necroptosis. This pathway was dependent on host IFN-inducible Z-DNA-binding protein 1 (ZBP1); full-length E3 is able to inhibit ZBP1-mediated necroptosis. Binding to what was identified as Z-RNA has emerged as a novel mechanism of counteracting the type I IFN system and has broadened our understanding of innate immunity against viral infections. This article gives an overview of the studies leading to our understanding of the vaccinia virus E3 protein function and its involvement in viral pathogenesis. Furthermore, a short summary of other viral systems is provided.

Correction for Eduardo-Correia et al., ISG15 Is Counteracted by Vaccinia Virus E3 Protein and Controls the Proinflammatory Response against Viral Infection.

Correction for Eduardo-Correia et al., ISG15 Is Counteracted by Vaccinia Virus E3 Protein and Controls the Proinflammatory Response against Viral Infection.

Mutational analysis of vaccinia virus E3 protein: the biological functions do not correlate with its biochemical capacity to bind double-stranded RNA.

Vaccinia E3 protein has the biochemical capacity of binding to double-stranded RNA (dsRNA). The best characterized biological functions of the E3 protein include its host range function, suppression of cytokine expression, and inhibition of interferon (IFN)-induced antiviral activity. Currently, the role of the dsRNA binding capacity in the biological functions of the E3 protein is not clear. To further understand the mechanism of the E3 protein biological functions, we performed alanine scanning of the entire dsRNA binding domain of the E3 protein to examine the link between its biochemical capacity of dsRNA binding and biological functions. Of the 115 mutants examined, 20 were defective in dsRNA binding. Although the majority of the mutants defective in dsRNA binding also showed defective replication in HeLa cells, nine mutants (I105A, Y125A, E138A, F148A, F159A, K171A, L182A, L183A, and I187/188A) retained the host range function to various degrees. Further examination of a set of representative E3L mutants showed that residues essential for dsRNA binding are not essential for the biological functions of E3 protein, such as inhibition of protein kinase R (PKR) activation, suppression of cytokine expression, and apoptosis. Thus, data described in this communication strongly indicate the E3 protein performs its biological functions via a novel mechanism which does not correlate with its dsRNA binding activity. dsRNAs produced during virus replication are important pathogen-associated molecular patterns (PAMPs) for inducing antiviral immune responses. One of the strategies used by many viruses to counteract such antiviral immune responses is achieved by producing dsRNA binding proteins, such as poxvirus E3 family proteins, influenza virus NS1, and Ebola virus V35 proteins. The most widely accepted model for the biological functions of this class of viral dsRNA binding proteins is that they bind to and sequester viral dsRNA PAMPs; thus, they suppress the related antiviral immune responses. However, no direct experimental data confirm such a model. In this study of vaccinia E3 protein, we found that the biological functions of the E3 protein are not necessarily linked to its biochemical capacity of dsRNA binding. Thus, our data strongly point to a new concept of virus modulation of cellular antiviral responses triggered by dsRNA PAMPs.

ISG15 Is Counteracted by Vaccinia Virus E3 Protein and Controls the Proinflammatory Response against Viral Infection

Conjugation of ISG15 inhibits replication of several viruses. Here, using an expression system for assaying human and mouse ISG15 conjugations (ISGylations), we have demonstrated that vaccinia virus E3 protein binds and antagonizes human and mouse ISG15 modification. To study ISGylation importance in poxvirus infection, we used a mouse model that expresses deconjugating proteases. Our results indicate that ISGylation restricts in vitro replication of the vaccinia virus VVΔE3L mutant but unconjugated ISG15 is crucial to counteract the inflammatory response produced after VVΔE3L infection.

Regulation of vaccinia virus E3 protein by small ubiquitin-like modifier proteins.

The vaccinia virus (VACV) E3 protein is essential for virulence and has antiapoptotic activity and the ability to impair the host innate immune response. Here we demonstrate that E3 interacts with SUMO1 through a small ubiquitin-like modifier (SUMO)-interacting motif (SIM). SIM integrity is required for maintaining the stability of the viral protein and for the covalent conjugation of E3 to SUMO1 or SUMO2, a modification that has a negative effect on the E3 transcriptional transactivation of the p53-upregulated modulator of apoptosis (PUMA) and APAF-1 genes. We also demonstrate that E3 is ubiquitinated, a modification that does not destabilize the wild-type protein but triggers the degradation of an E3-ΔSIM mutant. This report constitutes the first demonstration of the important roles that both SUMO and ubiquitin play in the regulation of the VACV protein E3.

Comparative Analysis of Poxvirus Orthologues of the Vaccinia Virus E3 Protein: Modulation of Protein Kinase R Activity, Cytokine Responses, and Virus Pathogenicity

Poxviruses are important human and animal pathogens that have evolved elaborate strategies for antagonizing host innate and adaptive immunity. The E3 protein of vaccinia virus, the prototypic member of the orthopoxviruses, functions as an inhibitor of innate immune signaling and is essential for vaccinia virus replication in vivo and in many human cell culture systems. However, the function of orthologues of E3 expressed by poxviruses of other genera with different host specificity remains largely unknown. In the present study, we characterized the E3 orthologues from sheeppox virus, yaba monkey tumor virus, swinepox virus, and myxoma virus for their ability to modulate protein kinase R (PKR) function, cytokine responses and virus pathogenicity. We found that the E3 orthologues of myxoma virus and swinepox virus could suppress PKR activation and interferon (IFN)-induced antiviral activities and restore the host range function of E3 in HeLa cells. In contrast, the E3 orthologues from sheeppox virus and yaba monkey tumor virus were unable to inhibit PKR activation. While the sheeppox orthologue was unable to restore the host range function of E3, the yaba monkey tumor virus orthologue partially restored E3-deficient vaccinia virus replication in HeLa cells, correlated with its ability to suppress IFN-induced antiviral activities. Moreover, poxvirus E3 orthologues show varying ability to inhibit the induction of antiviral and proinflammatory cytokines. Despite these in vitro results, none of the E3 orthologues tested was capable of restoring pathogenicity to E3-deficient vaccinia virus in vivo.

RNA helicase retinoic acid-inducible gene I as a sensor of Hantaan virus replication

Hantaan virus (HTNV) causes severe human disease. The HTNV genome consists of three ssRNA segments of negative polarity that are complexed with viral nucleocapsid (N) protein. How the human innate immune system detects HTNV is unclear. RNA helicase retinoic acid-inducible gene I (RIG-I) does not sense genomic HTNV RNA. So far it has not been analysed whether pathogen-associated molecular patterns generated during the HTNV replication trigger RIG-I-mediated innate responses. Indeed, we found that knock-down of RIG-I in A549 cells, an alveolar epithelial cell line, increases HTNV replication and prevents induction of 2',5'-oligoadenylate synthetase, an interferon-stimulated gene. Moreover, overexpression of wild-type or constitutive active RIG-I in Huh7.5 cells lacking a functional RIG-I diminished HTNV virion production. Intriguingly, reporter assays revealed that in vitro-transcribed HTNV N RNA and expression of the HTNV N ORF triggers RIG-I signalling. This effect was completely blocked by the RNA-binding domain of vaccinia virus E3 protein, suggesting that dsRNA-like secondary structures of HTNV N RNA stimulate RIG-I. Finally, transfection of HTNV N RNA into A549 cells resulted in a 2 log-reduction of viral titres upon challenge with virus. Our study is the first demonstration that RIG-I mediates antiviral innate responses induced by HTNV N RNA during HTNV replication and interferes with HTNV growth.

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-β expression and NF-κB 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-β 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.

Translation Control by Protein Kinase R Restricts Minute Virus of Mice Infection: Role in Parvovirus Oncolysis

The relevance of translational control in the gene expression and oncotropism of the autonomous parvoviruses was investigated with MVMp, the prototype strain of minute virus of mice (MVM), infecting normal and transformed rodent and human cells of different tissue origins. Mouse embryo fibroblasts (MEFs) and NIH 3T3 fibroblasts were resistant to MVMp infection, but 3T3 fibroblasts derived from double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) knockout mice (PKR(o/o)) behaved in a manner that was highly permissive to productive MVMp replication. NIH 3T3 resistance correlated with significant phosphorylation of eukaryotic translation initiation factor 2 (eIF2) occurring at early time points after infection. Permissive PKR(o/o) cells were converted to MVMp-restrictive cells after reintroduction of the PKR gene by transfection. Conversely, regulated expression of the vaccinia virus E3 protein, a PKR inhibitor, in MEFs prevented eIF2alpha phosphorylation and increased MVMp protein synthesis. In vitro-synthesized genome-length R1 mRNA of MVMp was a potent activator of PKR. Virus-resistant primary MEFs and NIH 3T3 cells responded to MVMp infection with significant increases in eIF2alpha phosphorylation. In contrast, virus-permissive mouse (PKR(o/o), BHK21, and A9) and human transformed (NB324K fibroblast, U373 glioma, and HepG2 hepatoma) cells consistently showed no significant increase in the level of eIF2alpha phosphorylation following MVMp infection. The synthesis of the viral NS1 protein was inversely correlated with the steady-state PKR levels. Our results show that the PKR-mediated antiviral response is an important mechanism for control of productive MVMp infection, and its impairment in human transformed cells allowed efficient MVMp gene expression. PKR translational control may therefore contribute to the oncolysis of MVMp and other autonomous parvoviruses.

The identification and characterization of a monoclonal antibody to the vaccinia virus E3 protein

Monoclonal antibodies with reactivity to vaccinia virus specific proteins are useful reagents to study the proteins as well as to help understand aspects of the poxvirus life cycle. Using a vaccinia virus proteomics microarray, we found a hybridoma (MAb 3015B2) from a vaccinia virus vaccinated mouse that reacted with the product of the E3L gene. The specificity to the E3 protein was confirmed by Western blotting and immunofluorescence of cells infected with either wild-type vaccinia virus or a mutant virus with the E3L gene deleted. Antibody reactivity with E3 was also seen in cells transfected with a plasmid expressing the E3 protein. A panel of mutated vaccinia viruses with truncations in the E3L gene revealed that while MAb 3015B2 reacted with E3 lacking the C-terminal 7 amino acids, it lost reactivity with a mutant E3 lacking the C-terminal 26 amino acids. This indicates that the antigenic site recognized by 3015B2 is on the C-terminus, somewhere between amino acids 164 through 183. The antibody also recognizes the E3 protein encoded by other orthopoxviruses. This antibody will be useful for further investigations of the E3 protein as well as a useful reagent to indicate vaccinia virus early protein expression.

Vaccinia virus double-stranded RNA-binding protein E3 does not interfere with siRNA-mediated gene silencing in mammalian cells

Vaccinia virus (VACV) evolved several strategies to evade antiviral cellular defence. The vaccinia virus E3 protein for example binds and sequesters double stranded RNA (dsRNA) and counteracts interferon action. We were interested to find out whether and to what extend E3 interferes with RNA silencing mediated by short interfering RNA (siRNA) in mammalian cells. We could show that the expression of a VACV-encoded marker gene can be efficiently inhibited by siRNA independently of the presence of the E3 protein. In addition, expression of E3 had no impact on RNA polymerase III promoter-derived shRNA-induced silencing of a cellular gene in human cells. Both VACV early and late gene expression could be inhibited by siRNA. Furthermore, downregulation of the expression of the E3L gene itself by siRNA in VACV infected cells produced the previously described phenotype of a knock-out virus, which illustrates the power of siRNA for vaccinia virus gene function analysis.

Mutational analysis of the vaccinia virus E3 protein defines amino acid residues involved in E3 binding to double-stranded RNA.

Alanine-substitution mutations were targeted to 14 amino acid residues within the double-stranded (ds) RNA binding motif (dsRBM) of the vaccinia virus E3 protein. Substitutions at six positions--Glu-124, Phe-135, Phe-148, Lys-167, Arg-168, and Lys-171--caused significant reductions in dsRNA binding. These six residues are conserved in the two dsRBMs for which structural information is available (Escherichia coli RNase III and Drosophila melanogaster staufen) and in many other members of the dsRBM protein family. Residues we show to be important for dsRNA binding by vaccinia virus E3 map to the same face of the dsRBM structure and are thus likely to compose part of the RNA binding site.