Vaccinia Virus E3L Research Articles

Complementation of Deletion of the Vaccinia Virus E3L Gene by theEscherichia coliRNase III Gene

This work investigated whether theEscherichia coliRNase III gene,rnc+, could complement vp1080, a mutant vaccinia virus that is deleted of its E3L gene. Like E3L,rnc+ codes for a dsRNA binding protein that contains an additional nucleolytic activity.Rncgenes were cloned into the eukaryotic expression vector pMTVa−, expressed in COS-1 cells, and shown to be functional. Transient rescue experiments in HeLa cells demonstrated that the cleavage function of thernc+ gene was necessary for full rescue of vp1080. Thernc70 gene, which encodes a product deficient in catalytic activity but still capable of binding to dsRNA, rescued vp1080 weakly. Thernc105 gene, which encodes a product that cannot bind or cleave dsRNA, was unable to rescue vp1080. Therncgenes were also inserted into the E3L locus of vp1080. While recombinants containing thernc+ gene or thernc70 gene regained the IFN resistance phenotype in RK13cells, full host range of vaccinia virus was only restored in the recombinant containing thernc+ gene. Thus, the ability of RNase III to process dsRNA appears to be necessary to restore the host range phenotype. The vp-rnc 105 recombinant behaved similarly to vp1080.

Host-range restriction of vaccinia virus E3L-specific deletion mutants.

The vaccinia virus (VV) E3L gene product functions as a dsRNA binding protein that is involved in conferring an interferon-resistant phenotype upon the virus. Studies with a vaccinia virus (VV) E3L- deletion mutant (vP1080) have also demonstrated that the E3L gene product is critical for productive replication on certain cell substrates. While E3L was found to be nonessential for replication in chick embryo fibroblasts (CEFs), virus specifically deleted of E3L was found to be replication deficient in Vero, HeLa, and murine L929 cells. Further, the temporal block in replication appears to differ in these cell systems, as evidenced by the observed timing of protein synthesis inhibition. In Vero cells infected with the VV E3L- mutant, there was no detectable protein synthesis after 2 hr post-infection, whereas in L929 cells normal protein patterns were observed even at late times post-infection. Expression of a heterologous dsRNA binding protein, the reovirus sigma 3 protein, by the E3L- mutant virus restored near wild-type growth characteristics, suggesting the critical nature for regulating dsRNA levels in VV-infected cells.

Reversal of the interferon-sensitive phenotype of a vaccinia virus lacking E3L by expression of the reovirus S4 gene.

The vaccinia virus (VV) E3L gene, which encodes a potent inhibitor of the interferon (IFN)-induced, double-stranded RNA (dsRNA)-dependent protein kinase, PKR, is thought to be involved in the IFN-resistant phenotype of VV. The E3L gene products, p25 and p20, act as inhibitors of PKR, presumably by binding and sequestering activator dsRNA from the kinase. In this study we demonstrate that VV with the E3L gene specifically deleted (vP1080) was sensitive to the antiviral effects of IFN and debilitated in its ability to rescue vesicular stomatitis virus from the antiviral effects of IFN. Infection of L929 cells with E3L-minus virus led to rRNA degradation typical of activation of the 2'-5'-oligoadenylate synthetase/RNase L system, and extracts of infected cells lacked the PKR-inhibitory activity characteristic of wild-type VV. The reovirus S4 gene, which encodes a dsRNA-binding protein (sigma 3) that can also inhibit PKR activation by binding and sequestering activator dsRNA, was inserted into vP1080. The resultant virus (vP1112) was partially resistant to the antiviral effects of IFN in comparison with vP1080. Further studies demonstrated that transient expression of the reovirus sigma 3 protein rescued E3L-minus VV replication in HeLa cells. In these studies, rescue by sigma 3 mutants correlated with their ability to bind dsRNA. Finally, vP112 was also able to rescue the replication of the IFN-sensitive virus vesicular stomatitis virus in a manner similar to that of wild-type VV. Together, these results suggest that the reovirus S4 gene can replace the VV E3L gene with respect to interference with the IFN-induced antiviral activity.

Proteins that interact with PKR

The in vitro activities of recombinant gene products of the vaccinia virus E3L and K3L genes have been compared. These proteins are both potent inhibitors of the dsRNA activated protein kinase (PKR) as assayed in cell-free translation systems or with purified PKR. The two gene products function at similar molar concentrations. Both proteins are expressed early in vaccinia virus infection suggesting that vaccinia virus maintains redundant mechanisms for the down regulation of PKR. The K3L gene product can be shown to be associated with PKR in vaccinia virus infected cells. The activities of the vaccinia virus PKR inhibitors are compared with other viral protein inhibitors of PKR. A variety of cellular proteins have also been identified by their ability to inhibit PKR activity or to prevent PKR activation. These cellular PKR interacting proteins have been uncovered from the studies of viral strategies to prevent PKR activation, as well as from studies looking at the effects of growth control, growth factors or oncogene expression on PKR activity. A picture emerges of PKR fulfilling a complex regulatory role in cell function with the regulation of its activity as part of a complex cascade interfacing with the signal transduction/cell cycle control machinery.

Nuclear Localization of a Double-Stranded RNA-Binding Protein Encoded by the Vaccinia Virus E3L Gene

We produced a B cell hybridoma (TW2.3) from vaccinia virus-infected mice that secreted a monoclonal antibody (MAb) reactive with a 25-kDA early viral protein that was localized by laser scanning confocal microscopy to the nucleus and cytoplasmic viral factory regions of infected cells. By cell-free translation of mRNA selected by hybridization to a complete library of vaccinia virus DNA fragments, the immunoreactive polypeptide was mapped to open reading frame E3L. The RNA start site of an early promoter was located 26 nucleotides upstream of the first methionine codon of E3L. Evidence was obtained that translation initiation occurs in vivo and in vitro at both the first and second methionine codons to produce major and minor polypeptides of 25 and 19 kDa, respectively. Both polypeptides bound double-stranded RNA, confirming the recent report of H.-W. Chang, J. C. Watson, and B. L. Jacobs ( Proc. Natl, Acad. Sci. USA 89, 4825-4829, 1992). Other vaccinia virus proteins were not required for the nuclear localization of the E3L protein, since MAb TW2.3 bound to the nuclei of uninfected cells that were transfected with the E3L gene under the control of the SV40 early promoter. We also demonstrated that the E3L protein can bind to nuclei of aldehyde fixed and detergent permeabilized uninfected cells. This binding was abrogated by treatment of the cells with RNase but not DNase. The nuclear and cytoplasmic locations of the double-stranded RNA binding protein are consistent with multiple functions in the vaccinia virus infectious cycle.

Identification of a Conserved Motif That Is Necessary for Binding of the Vaccinia Virus E3L Gene Products to Double-Stranded RNA

The E3L gene of vaccinia virus encodes the double-stranded (ds) RNA binding proteins p20 and p25 that exhibit inhibitory activity for the IFN-induced, P 1/eIF-2α protein kinase. A region in the E3L encoded proteins (residues 156-180) shares a high degree of similarity with several proteins that bind double-helical RNA including the P 1/eIF-2α kinase, bacterial and yeast RNase III, and a human transactivator response element/Rev response element binding protein. In this study, mutants of E3L proteins were constructed in order to determine the region of the proteins required for dsRNA binding and kinase inhibitory activity. Our data indicate that both the region necessary for dsRNA binding and for kinase inhibitory activity are located at the carboxyl terminus of the protein. The E3L proteins with 7 amino acids deleted from the carboxyl terminus (184-190) could bind to dsRNA, but with lower affinity than could the full-length protein. This protein did not detectably inhibit kinase in vitro. Deletion of 26 amino acids from the carboxyl terminus of the E3L proteins (165-190) abolished dsRNA binding activity and kinase inhibitory activity. In addition, mutations at amino acid 164, 167, or 174 severely inhibited binding to dsRNA. On the other hand, deletion of 83 amino acids from the amino terminus did not affect the proteins' ability to bind dsRNA or inhibit kinase. These results suggest that a region of sequence between amino acids 164 and 183 is necessary for E3L proteins' dsRNA binding activity. This region lies within the homologous domain that the E3L proteins share with other dsRNA binding proteins.

Mechanism of interferon action: Identification of a RNA binding domain within the N-terminal region of the human RNA-dependent P1/eIF-2α protein kinase

A molecular cDNA clone of the human RNA-dependent P1/eIF-2α protein kinase was expressed in Escherichia coli. Mutant P1 proteins were examined for RNA binding activity by Northwestern blot analysis using the reovirus s1 mRNA, an activator of the kinase; the adenovirus VAI RNA, an inhibitor of kinase activation; or human immunodeficiency virus (HIV) TAR RNA as probe. Analysis of TrpE-P1 deletion mutant fusion proteins revealed that the 11-kDa N-terminal region of the P1 protein bound reovirus s1 mRNA, adenovirus VAI RNA, and HIV TAR RNA. Neither s1 RNA, VAI RNA, nor TAR RNA was bound by truncated P1 proteins which lacked the N-terminal 98 amino acids. Computer analysis revealed that the human protein P1 sequence corresponding to amino acid residues within the N-terminal RNA binding domain displays high homology (>54% identity; 61 to 94% similarity) with two animal virus proteins which possess RNA binding activity (vaccinia virus E3L; rotavirus VP2) and two proteins of unknown function (murine TIK; rotavirus NS34), but which are likely RNA binding proteins.