Complementation of vaccinia virus lacking the double-stranded RNA-binding protein gene E3L by human cytomegalovirus

The cellular response to viral infection often includes activation of pathways that shut off protein synthesis and thereby inhibit viral replication. In order to enable efficient replication, many viruses carry genes such as the E3L gene of vaccinia virus that counteract these host antiviral pathways. Vaccinia virus from which the E3L gene has been deleted (VVDeltaE3L) is highly sensitive to interferon and exhibits a restricted host range, replicating very inefficiently in many cell types, including human fibroblast and U373MG cells. To determine whether human cytomegalovirus (CMV) has a mechanism for preventing translational shutoff, we evaluated the ability of CMV to complement the deficiencies in replication and protein synthesis associated with VVDeltaE3L. CMV, but not UV-inactivated CMV, rescued VVDeltaE3L late gene expression and replication. Thus, complementation of the VVDeltaE3L defect appears to depend on de novo CMV gene expression and is not likely a result of CMV binding to the cell receptor or of a virion structural protein. CMV rescued VVDeltaE3L late gene expression even in the presence of ganciclovir, indicating that CMV late gene expression is not required for complementation of VVDeltaE3L. The striking decrease in overall translation after infection with VVDeltaE3L was prevented by prior infection with CMV. Finally, CMV blocked both the induction of eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation and activation of RNase L by VVDeltaE3L. These results suggest that CMV has one or more immediate-early or early genes that ensure maintenance of a high protein synthetic capacity during infection by preventing activation of the PKR/eIF2alpha phosphorylation and 2-5A oligoadenylate synthetase/RNase L pathways.     

The E3L and K3L vaccinia virus gene products stimulate translation through inhibition of the double-stranded RNA-dependent protein kinase by different mechanisms.

Vaccinia virus has evolved multiple mechanisms to counteract the interferon-induced antiviral host cell response. Recently, two vaccinia virus gene products were shown to interfere with the activity of the double-stranded RNA-dependent protein kinase (PKR): the K3L gene product and the E3L gene product. We have evaluated the efficiency by which these gene products inhibit PKR and whether they act in a synergistic manner. The effects of the two vaccinia virus gene products were compared in an in vivo system in which translation of a reporter gene (dihydrofolate reductase or eukaryotic translation initiation factor 2 alpha [eIF-2 alpha]) was inhibited because of the localized activation of PKR. In this system, the E3L gene product, and to a lesser extent the K3L gene product, potentiated translation of the reporter gene and inhibited eIF-2 alpha phosphorylation. Analysis in vitro demonstrated that the E3L gene product inhibited PKR approximately 50- to 100-fold more efficiently than the K3L gene product. However, further studies demonstrated that the mechanism of action of these two inhibitors was different. Whereas the E3L inhibitor interfered with the binding of the kinase to double-stranded RNA, the K3L inhibitor did not. We propose that the K3L inhibitor acts through its homology to eIF-2 alpha to interfere with the interaction of eIF-2 alpha with PKR. The two inhibitors did not display a synergistic effect on translation or eIF-2 alpha phosphorylation. In addition, neither K3L nor E3L expression detectably altered cellular protein synthesis.     

Ectopic Expression of Vaccinia Virus E3 and K3 Cannot Rescue Ectromelia Virus Replication in Rabbit RK13 Cells

As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV.     

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.     

Replication of modified vaccinia virus Ankara in primary chicken embryo fibroblasts requires expression of the interferon resistance gene E3L

Highly attenuated modified vaccinia virus Ankara (MVA) serves as a candidate vaccine to immunize against infectious diseases and cancer. MVA was randomly obtained by serial growth in cultures of chicken embryo fibroblasts (CEF), resulting in the loss of substantial genomic information including many genes regulating virus-host interactions. The vaccinia virus interferon (IFN) resistance gene E3L is among the few conserved open reading frames encoding viral immune defense proteins. To investigate the relevance of E3L in the MVA life cycle, we generated the deletion mutant MVA-DeltaE3L. Surprisingly, we found that MVA-DeltaE3L had lost the ability to grow in CEF, which is the first finding of a vaccinia virus host range phenotype in this otherwise highly permissive cell culture. Reinsertion of E3L led to the generation of revertant virus MVA-E3rev and rescued productive replication in CEF. Nonproductive infection of CEF with MVA-DeltaE3L allowed viral DNA replication to occur but resulted in an abrupt inhibition of viral protein synthesis at late times. Under these nonpermissive conditions, CEF underwent apoptosis starting as early as 6 h after infection, as shown by DNA fragmentation, Hoechst staining, and caspase activation. Moreover, we detected high levels of active chicken alpha/beta IFN (IFN-alpha/beta) in supernatants of MVA-DeltaE3L-infected CEF, while moderate IFN quantities were found after MVA or MVA-E3rev infection and no IFN activity was present upon infection with wild-type vaccinia viruses. Interestingly, pretreatment of CEF with similar amounts of recombinant chicken IFN-alpha inhibited growth of vaccinia viruses, including MVA. We conclude that efficient propagation of MVA in CEF, the tissue culture system used for production of MVA-based vaccines, essentially requires conserved E3L gene function as an inhibitor of apoptosis and/or IFN induction.     

Roles of vaccinia virus genes E3L and K3L and host genes PKR and RNase L during intratracheal infection of C57BL/6 mice

The importance of the 2'-5' oligoadenylate synthetase (OAS)/RNase L and double-stranded RNA (dsRNA)-dependent protein kinase (PKR) pathways in host interferon induction resulting from virus infection in response to dsRNA has been well documented. In poxvirus infections, the interactions between the vaccinia virus (VV) genes E3L and K3L, which target RNase L and PKR, respectively, serve to prevent the induction of the dsRNA-dependent induced interferon response in cell culture. To determine the importance of these host genes in controlling VV infections, mouse single-gene knockouts of RNase L and PKR and double-knockout mice were studied following intratracheal infection with VV, VVΔK3L, or VVΔE3L. VV caused lethal disease in all mouse strains. The single-knockout animals were more susceptible than wild-type animals, while the RNase L(-/-) PKR(-/-) mice were the most susceptible. VVΔE3L infections of wild-type mice were asymptomatic, demonstrating that E3L plays a critical role in controlling the host immune response. RNase L(-/-) mice showed no disease, whereas 20% of the PKR(-/-) mice succumbed at a dose of 10(8) PFU. Lethal disease was routinely observed in RNase L(-/-) PKR(-/-) mice inoculated with 10(8) PFU of VVΔE3L, with a distinct pathology. VVΔK3L infections exhibited no differences in virulence among any of the mouse constructs, suggesting that PKR is not the exclusive target of K3L. Surprisingly, VVΔK3L did not disseminate to other tissues from the lung. Hence, the cause of death in this model is respiratory disease. These results also suggest that an unanticipated role of the K3L gene is to facilitate virus dissemination.     

Expression of human papillomavirus type 6 E1, E2, L1 and L2 open reading frames in Escherichia coli

Open reading frame (ORF) fragments (putative gene fragments) from human papillomavirus type 6b (HPV-6b) were inserted into the bacterial expression vector pHK413 to provide viral antigenic determinants. Approximately 86% of the entire L1 ORF, 82% of the E2 ORF, and 52% of the L2 ORF were expressed in Escherichia coli. The E1 ORF was cloned as two fragments. The constructions containing E1n (coding for the N-terminal region) and E1c (coding for the C-terminal region) expressed 27% and 16% of the E1 ORF, respectively. Protein encoded by the L1 ORF, but not that encoded by the L2 ORF, reacted with antibodies elicited by disrupted bovine papillomavirus. These reagents will be extremely useful in unravelling the HPV-6b replication cycle.     

Generation of an Adenovirus Vector Lacking E1, E2a, E3, and All of E4 except Open Reading Frame 3

Toxicity and immunity associated with adenovirus backbone gene expression is an important hurdle to overcome for successful gene therapy. Recent efforts to improve adenovirus vectors for in vivo use have focused on the sequential deletion of essential early genes. Adenovirus vectors have been constructed with the E1 gene deleted and with this deletion in combination with an E2a, E2b, or E4 deletion. We report here a novel vector (Av4orf3nBg) lacking E1, E2a, and all of E4 except open reading frame 3 (ORF3) and expressing a beta-galactosidase reporter gene. This vector was generated by transfection of a plasmid carrying the full-length vector sequence into A30.S8 cells that express E1 and E2a but not E4. Production was subsequently performed in an E1-, E2a-, and E4-complementing cell line. We demonstrated with C57BL/6 mice that the Av4orf3nBg vector effected gene transfer with an efficiency comparable to that of the Av3nBg (wild-type E4) vector but that the former exhibited a higher level of beta-galactosidase expression. This observation suggests that E4 ORF3 alone is able to enhance RNA levels from the beta-galactosidase gene when the Rous sarcoma virus promoter is used to drive transgene expression in the mouse liver. In addition, we observed less liver toxicity in mice injected with the Av4orf3nBg vector than those injected with the Av3nBg vector at a comparable DNA copy number per cell. This study suggests that the additional deletion of E4 in an E1 and E2a deletion background may be beneficial in decreasing immunogenicity and improving safety and toxicity profiles, as well as increasing transgene capacity and expression for liver-directed gene therapy.     

Expression of the E3L gene of vaccinia virus in transgenic mice decreases host resistance to vaccinia virus and Leishmania major infections

The E3L gene of vaccinia virus (VACV) encodes the E3 protein that in cultured cells inhibits the activation of interferon (IFN)-induced proteins, double-stranded RNA-dependent protein kinase (PKR), 2′-5′-oligoadenylate synthetase/RNase L (2-5A system) and adenosine deaminase (ADAR-1), thus helping the virus to evade host responses. Here, we have characterized the in vivo E3 functions in a murine inducible cell culture system (E3L-TetOFF) and in transgenic mice (TgE3L). Inducible E3 expression in cultured cells conferred on cells resistance to the antiviral action of IFN against different viruses, while expression of the E3L gene in TgE3L mice triggered enhanced sensitivity of the animals to pathogens. Virus infection monitored in TgE3L mice by different inoculation routes (intraperitoneal and tail scarification) showed that transgenic mice became more susceptible to VACV infection than control mice. TgE3L mice were also more susceptible to Leishmania major infection, leading to an increase in parasitemia compared to control mice. The enhanced sensitivity of TgE3L mice to VACV and L. major infections occurred together with alterations in the host immune system, as revealed by decreased T-cell responses to viral antigens in the spleen and lymph nodes and by differences in the levels of specific innate cell populations. These results demonstrate that expression of the E3L gene in transgenic mice partly reverses the resistance of the host to viral and parasitic infections and that these effects are associated with immune alterations.     

Both carboxy- and amino-terminal domains of the vaccinia virus interferon resistance gene, E3L, are required for pathogenesis in a mouse model

The vaccinia virus (VV) E3L gene is responsible for providing interferon (IFN) resistance and a broad host range to VV in cell culture. The E3L gene product contains two distinct domains. A conserved carboxy-terminal domain, which is required for the IFN resistance and broad host range of the virus, has been shown to bind double-stranded RNA (dsRNA) and inhibit the antiviral dsRNA-dependent protein kinase, PKR. The amino-terminal domain, while conserved among orthopoxviruses, is dispensable in cell culture. To study the role of E3L in whole-animal infections, WR strain VV recombinants either lacking E3L (VVDeltaE3L) or expressing an amino-terminal (VVE3LDelta83N) or carboxy-terminal (VVE3LDelta26C) truncation of E3L were constructed. Whereas wild-type VV had a 50% lethal dose of approximately 10(4) PFU after intranasal infection, and elicited severe weight loss and morbidity, VVDeltaE3L was apathogenic, leading to no death, weight loss, or morbidity. VVDeltaE3L was also apathogenic after intracranial injection. Although the amino-terminal domain of E3L is dispensable for infection of cells in culture, both the amino- and carboxy-terminal domains of E3L were required for full pathogenesis in intranasal infections. These results demonstrate that the entire E3L gene is required for pathogenesis in the mouse model.     

Vaccinia virus E3 protein prevents the antiviral action of ISG15

The ubiquitin-like modifier ISG15 is one of the most predominant proteins induced by type I interferons (IFN). In this study, murine embryo fibroblast (MEFs) and mice lacking the gene were used to demonstrate a novel role of ISG15 as a host defense molecule against vaccinia virus (VACV) infection. In MEFs, the growth of replication competent Western Reserve (WR) VACV strain was affected by the absence of ISG15, but in addition, virus lacking E3 protein (VVDeltaE3L) that is unable to grow in ISG15+/+ cells replicated in ISG15-deficient cells. Inhibiting ISG15 with siRNA or promoting its expression in ISG15-/- cells with a lentivirus vector showed that VACV replication was controlled by ISG15. Immunoprecipitation analysis revealed that E3 binds ISG15 through its C-terminal domain. The VACV antiviral action of ISG15 and its interaction with E3 are events independent of PKR (double-stranded RNA-dependent protein kinase). In mice lacking ISG15, infection with VVDeltaE3L caused significant disease and mortality, an effect not observed in VVDeltaE3L-infected ISG15+/+ mice. Pathogenesis in ISG15-deficient mice infected with VVDeltaE3L or with an E3L deletion mutant virus lacking the C-terminal domain triggered an enhanced inflammatory response in the lungs compared with ISG15+/+-infected mice. These findings showed an anti-VACV function of ISG15, with the virus E3 protein suppressing the action of the ISG15 antiviral factor.     

Transforming Activity of a Novel Mutant of HPV16 E6E7 Fusion Gene

An optimized recombinant HPV16 E6E7 fusion gene (HPV16 ofE6E7) was constructed according to codon usage for mammalian cell expression, and a mutant of HPV16 ofE6E7 fusion gene (HPV16 omfE6E7) was generated by site-directed mutagenesis at L57G, C113R for the E6 protein and C24G, E26G for the E7 protein for HPV16 ofE6E7 [patent pending (CN 101100672)]. The HPV16 omfE6E7 gene constructed in this work not only lost the transformation capability to NIH 3T3 cells and tumorigenicity in SCID mice, but also maintain...     

Structural insights into E2–E3 interaction for LC3 lipidation

The members of the LC3/Atg8 family of proteins are covalently attached to phagophore and autophagosomal membranes. At the last step of the LC3 lipidation cascade, LC3 is transferred from the E2 enzyme ATG3 to phosphatidylethanolamine (PE). This transfer is stimulated by the ATG12–ATG5-ATG16L1 E3 complex, but the mechanism is not fully understood. We recently found that ATG12 of the E3 binds to a short sequence in the flexible region (FR) of ATG3 with high affinity, and that this interaction is critical for E2–E3 complex formation. These findings, together with detailed structural analyses of this interaction, define the properties of ATG12 and provide new insights of how LC3 transfer begins with ATG3 recruitment by ATG12.     

Structural insights into E2–E3 interaction for LC3 lipidation

The members of the LC3/Atg8 family of proteins are covalently attached to phagophore and autophagosomal membranes. At the last step of the LC3 lipidation cascade, LC3 is transferred from the E2 enzyme ATG3 to phosphatidylethanolamine (PE). This transfer is stimulated by the ATG12–ATG5-ATG16L1 E3 complex, but the mechanism is not fully understood. We recently found that ATG12 of the E3 binds to a short sequence in the flexible region (FR) of ATG3 with high affinity, and that this interaction is critical for E2–E3 complex formation. These findings, together with detailed structural analyses of this interaction, define the properties of ATG12 and provide new insights of how LC3 transfer begins with ATG3 recruitment by ATG12.     

Loss of protein kinase PKR expression in human HeLa cells complements the vaccinia virus E3L deletion mutant phenotype by restoration of viral protein synthesis

The E3L proteins encoded by vaccinia virus bind double-stranded RNA and mediate interferon resistance, promote virus growth, and impair virus-mediated apoptosis. Among the cellular proteins implicated as targets of E3L is the protein kinase regulated by RNA (PKR). To test in human cells the role of PKR in conferring the E3L mutant phenotype, HeLa cells stably deficient in PKR generated by an RNA interference-silencing strategy were compared to parental and control knockdown cells following infection with either an E3L deletion mutant (ΔE3L) or wild-type (WT) virus. The growth yields of WT virus were comparable in PKR-sufficient and -deficient cells. By contrast, the single-cycle yield of ΔE3L virus was increased by nearly 2 log₁₀ in PKR-deficient cells over the impaired growth in PKR-sufficient cells. Furthermore, virus-induced apoptosis characteristic of the ΔE3L mutant in PKR-sufficient cells was effectively abolished in PKR-deficient HeLa cells. The viral protein synthesis pattern was altered in ΔE3L-infected PKR-sufficient cells, characterized by an inhibition of late viral protein expression, whereas in PKR-deficient cells, late protein accumulation was restored. Phosphorylation of both PKR and the α subunit of protein synthesis initiation factor 2 (eIF-2α) was elevated severalfold in ΔE3L-infected PKR-sufficient, but not PKR-deficient, cells. WT virus did not significantly increase PKR or eIF-2α phosphorylation in either PKR-sufficient or -deficient cells, both of which supported efficient WT viral protein production. Finally, apoptosis induced by infection of PKR-sufficient HeLa cells with ΔE3L virus was blocked by a caspase antagonist, but mutant virus growth was not rescued, suggesting that translation inhibition rather than apoptosis activation is a principal factor limiting virus growth.     

DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. Two DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns. Dnmt3L, a member of the Dnmt3 family, has been reported to be necessary for maternal methylation imprinting, possibly by interacting with Dnmt3a and/or Dnmt3b (Hata, K., Okano, M., Lei, H., and Li, E. (2002) Development 129, 1983-1993). In the present study, the effect of DNMT3L, a human homologue of Dnmt3L, on the DNA methylation activity of mouse Dnmt3a and Dnmt3b was examined in vitro. DNMT3L enhanced the DNA methylation activity of Dnmt3a and Dnmt3b about 1.5-3-fold in a dose-dependent manner but did not enhance the DNA methylation activity of Dnmt1. Although the extents of stimulation were different, a stimulatory effect on the DNA methylation activity was observed for all of the substrate DNA sequences examined, such as those of the maternally methylated SNRPN and Lit-1 imprinting genes, the paternally methylated H19 imprinting gene, the CpG island of the myoD gene, the 5 S ribosomal RNA gene, an artificial 28-bp DNA, poly(dG-dC)-poly(dG-dC), and poly(dI-dC)-poly(dI-dC). DNMT3L could not bind to DNA but could bind to Dnmt3a and Dnmt3b, indicating that the stimulatory effect of DNMT3L on the DNA methylation activity may not be due to the guiding of Dnmt3a and Dnmt3b to the targeting DNA sequence but may comprise a direct effect on their catalytic activity. The carboxyl-terminal half of DNMT3L was found to be responsible for the enhancement of the enzyme activity.