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

Abstract

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...

Species‐specific inhibition of antiviral protein kinase R by capripoxviruses and vaccinia virus

“…VACV E3 is a multifunctional protein that inhibits other antiviral proteins with dsRNA binding capabilities, including 2 -5 -OAS in the RNaseL pathway, in addition to PKR. [37][38][39] Our findings indicate that PKR is most likely the primary target of E3 in respect to virus replication in the analyzed cells because K3 orthologs that effectively inhibited PKR completely rescued virus replication. While we have not ruled out the possibility that CaPV K3L has evolved to inhibit multiple dsRNA-mediated host restriction factors, on the basis of the known mechanism that orthopoxvirus K3 acts as a PKR pseudosubstrate, this possibility is unlikely.…”

Expression of the Vaccinia Virus Antiapoptotic F1 Protein Is Blocked by Protein Kinase R in the Absence of the Viral E3 Protein

“…This finding was somewhat surprising as F1 has been viewed as an early protein, and many VACV early proteins are expressed in the absence of E3 (17,35). However, a recent report documented that expression of the early D9 mRNA decapping enzyme also requires E3 (39). Currently, it remains unclear why F1 and D9 mRNAs display enhanced sensitivity to translational inhibition by PKR relative to that of other early mRNAs.…”

“…An alternative possibility is that E3 is required for expression of the F1 protein during infection. Although F1 is an early protein (37) and although many early proteins are expressed in the absence of E3 (38), a recent report demonstrated that expression of the early D9 mRNA decapping enzyme requires E3 (39), providing a precedent for E3-dependent early gene expression. We therefore examined the effect of deleting E3 on expression of the F1 protein.…”

Ectromelia virus accumulates less double-stranded RNA compared to vaccinia virus in BS-C-1 cells

“…Therefore, we postulate that the J2 antibody is principally detecting “free” dsRNA (i.e., not bound by E3) within poxvirus-infected cells. Interestingly, a recent report showed that the J2 antibody could be used to pull-down a E3-GFP fusion protein in an immunoprecipitation assay (Liu and Moss, 2016). The authors used a human cell line at 13 hrs.…”