Rescue of vaccinia virus lacking the E3L gene by mutants of E3L

Chang, Hwai-Wen; Uribe, Libia Herrero; Jacobs, Bertram L.

doi:10.1128/jvi.69.10.6605-6608.1995

Cited by 108 publications

(49 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Poxviruses, including VACV, encode numerous proteins that protect against a variety of innate defenses including those triggered by dsRNA (8)(9)(10). The VACV E3 dsRNA binding protein plays an important role: mutations in the C-terminal dsRNA binding domain result in increased interferon sensitivity and a severe host range defect involving activation of PKR, RNase L, and interferon regulatory factor 3 (IRF3) (11)(12)(13)(14)(15)(16)(17). Roles of PKR and RNase L pathways were suggested by partially restoring replication of a VACV E3 deletion mutant in PKR-or RNase L-deficient mouse embryo fibroblasts (16).…”

mentioning

confidence: 99%

Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense Proteins Revealed with CRISPR-Cas9 Knockout Cell Lines and Vaccinia Virus Mutants

Liu

Moss

2016

J Virol

View full text Add to dashboard Cite

Vaccinia virus (VACV) decapping enzymes and cellular exoribonuclease Xrn1 catalyze successive steps in mRNA degradation and prevent double-stranded RNA (dsRNA) accumulation, whereas the viral E3 protein can bind dsRNA. We showed that dsRNA and E3 colocalized within cytoplasmic viral factories in cells infected with a decapping enzyme mutant as well as with wild-type VACV and that they coprecipitated with antibody. An E3 deletion mutant induced protein kinase R (PKR) and eukaryotic translation initiation factor alpha (eIF2␣) phosphorylation earlier and more strongly than a decapping enzyme mutant even though less dsRNA was made, leading to more profound effects on viral gene expression. Human HAP1 and A549 cells were genetically modified by clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) to determine whether the same pathways restrict E3 and decapping mutants. The E3 mutant replicated in PKR knockout (KO) HAP1 cells in which RNase L is intrinsically inactive but only with a double knockout (DKO) of PKR and RNase L in A549 cells, indicating that both pathways decreased replication equivalently and that no additional dsRNA pathway was crucial. In contrast, replication of the decapping enzyme mutant increased significantly (though less than that of wild-type virus) in DKO A549 cells but not in DKO HAP1 cells where a smaller increase in viral protein synthesis occurred. Xrn1 KO A549 cells were viable but nonpermissive for VACV; however, wild-type and mutant viruses replicated in triple-KO cells in which RNase L and PKR were also inactivated. Since KO of PKR and RNase L was sufficient to enable VACV replication in the absence of E3 or Xrn1, the poor replication of the decapping mutant, particularly in HAP1 DKO, cells indicated additional translational defects. IMPORTANCEViruses have evolved ways of preventing or counteracting the cascade of antiviral responses that double-stranded RNA (dsRNA) triggers in host cells. We showed that the dsRNA produced in excess in cells infected with a vaccinia virus (VACV) decapping enzyme mutant and by wild-type virus colocalized with the viral E3 protein in cytoplasmic viral factories. Novel human cell lines defective in either or both protein kinase R and RNase L dsRNA effector pathways and/or the cellular 5= exonuclease Xrn1 were prepared by CRISPR-Cas9 gene editing. Inactivation of both pathways was necessary and sufficient to allow full replication of the E3 mutant and reverse the defect cause by inactivation of Xrn1, whereas the decapping enzyme mutant still exhibited defects in gene expression. The study provided new insights into functions of the VACV proteins, and the well-characterized panel of CRISPR-Cas9-modified human cell lines should have broad applicability for studying innate dsRNA pathways. D ouble-stranded RNA (dsRNA) is a principal viral pathogenassociated molecular pattern that is recognized by cellular sensors, including oligoadenylate synthetase (OAS), protein kinase R (PKR), Toll-like receptors, retinoic acid-inducible gene-I...

show abstract

mentioning

confidence: 99%

Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense Proteins Revealed with CRISPR-Cas9 Knockout Cell Lines and Vaccinia Virus Mutants

Liu

Moss

2016

J Virol

View full text Add to dashboard Cite

show abstract

“…20 Both plants and animals produce a number of these viral suppressors, adding support to the evolved antiviral nature of RNA silencing. 3,21,22 Furthermore, plants that have defective RNA silencing pathways are susceptible to certain viruses. 22 Since its discovery in animal systems, the dsRNA-activated arm of antiviral immunity that degrades a specific gene has been successfully exploited with the use of RNAi.…”

Section: Dsrna and Gene Silencingmentioning

confidence: 99%

Immune responses to dsRNA: Implications for gene silencing technologies

2005

View full text Add to dashboard Cite

Nucleic acid‐induced gene silencing, such as RNA interference (RNAi), induces a multitude of responses in addition to the knockdown of a gene. This is best understood in the context of the antiviral immune response, from which the processes of RNAi are thought to be derived. Viral challenge of a vertebrate host leads to an intricate series of responses that orchestrate antiviral immunity. The success of this multifaceted system in overcoming viral encounters hinges on complex pathogen–host interactions. One aspect of these interactions, the nucleic acid‐based immune response, is key to the successful resolution of a viral challenge. In particular, dsRNA, a nucleic acid associated with viral replication, is involved in numerous interactions contributing to induction, activation and regulation of antiviral mechanisms. Specifically, dsRNA is responsible for stimulating important protective responses, such as the activation of dicer‐related antiviral pathways, induction of type 1 IFN, and stimulation of dsRNA‐activated protein kinase and oligoadenylate synthetase. Furthermore, the modulation and shaping of this overall immune response is facilitated through nucleic acid interactions with pattern recognition receptors such as toll‐like receptor 3. These diverse dsRNA‐induced antiviral responses have implications for biotechnologies that use dsRNA to harness one arm of the host antiviral machinery for silencing a specific target gene. The interlinked nature of these response elements means that it may be difficult to completely isolate one element from the other arms of the antiviral response program of an organism. Thus, it is beneficial to understand all aspects of the immune response to dsRNA in order to manipulate these systems and minimize unwanted non‐specific effects.

show abstract

“…The vaccinia virus-encoded E3 protein mitigates the antiviral effects of interferon by blocking cellular response pathways dependent on double-stranded (ds) RNA (2,4,21). Genetic and biochemical experiments suggest that the biological activity of E3 derives primarily, if not exclusively, from its capacity to bind to dsRNA (4,5,6,21). The C-terminal half of the 190-amino-acid E3 polypeptide is sufficient to bind dsRNA in vitro (4,22).…”

mentioning

confidence: 99%

“…To better understand the basis for dsRNA recognition by the vaccinia virus E3 protein, we have examined the effects of single-amino-acid substitutions within the dsRBM on RNA binding. Our work builds on that of Jacobs and coworkers, who defined E3 as the virus-encoded inhibitor of PKR, localized its dsRNA binding function to the C-terminal half, and showed that alterations at selected residues within the conserved dsRBM could affect dsRNA binding by E3 protein translated in vitro (4)(5)(6)21). Our approach has been to study the structure and function of the E3 by using purified recombinant protein.…”

mentioning

confidence: 99%

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

Shuman

1996

J Virol

View full text Add to dashboard Cite

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.

show abstract

Rescue of vaccinia virus lacking the E3L gene by mutants of E3L

Cited by 108 publications

References 21 publications

Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense Proteins Revealed with CRISPR-Cas9 Knockout Cell Lines and Vaccinia Virus Mutants

Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense Proteins Revealed with CRISPR-Cas9 Knockout Cell Lines and Vaccinia Virus Mutants

Immune responses to dsRNA: Implications for gene silencing technologies

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

Contact Info

Product

Resources

About