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

Myskiw, Chad; Arsenio, Janilyn; Hammett, Craig; Bruggen, Rebekah van; Deschambault, Yvon; Beausoleil, Nicole; Babiuk, Shawn; Cao, Jingxin

doi:10.1128/jvi.05505-11

Cited by 39 publications

(51 citation statements)

References 42 publications

(61 reference statements)

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“…The dsRNA binding activity of vaccinia E3 protein was examined mostly using the synthetic dsRNA analog poly(I·C) (24,25). In our laboratory, the specificity of the E3 protein binding to poly(I·C)-coated agarose beads has been demonstrated previously (18). In this study, the poly(I·C) bead pulldown assay was used to examine the dsRNA binding of vaccinia E3 mutant proteins.…”

Section: Resultsmentioning

confidence: 86%

“…Previously, it has been reported that PKR is an important regulator of apoptosis induced by the vaccinia E3L deletion mutant (18,27,28). Here, induction of the apoptotic markers cleaved poly(ADP-ribose) polymerase (PARP) and cleaved caspase-7 was examined by Western blotting.…”

Section: Suppression Of Pkr Activation By E3 Mutants Pkr Plays a Majmentioning

confidence: 97%

“…To date, however, no direct experimental data have shown that the E3 protein binds to dsRNAs in the virusinfected cells. Moreover, we have previously demonstrated that a poxvirus E3L ortholog, such as sheeppox virus 034, does not supplement the biological functions of the E3 protein, although it binds dsRNA (18). It also has been reported that the ZBD plays a role in inhibition of interferon induction (19) and full pathogenesis in vivo in mice (14,20,21).…”

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confidence: 99%

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Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA

Dueck

Chen

et al. 2015

J Virol

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Poxviridae is a diverse family of large dsDNA viruses (130 to 300 kb) which replicate exclusively in the cytoplasm of infected cells and exhibit a varied host range (1). Some members are specific to a single host, such as variola virus, the causative agent of smallpox, while others, such as vaccinia virus, display a broad host range. Poxviruses encode a number of immunomodulatory proteins, e.g., host range proteins (2). Orthopoxviruses encode a number of host range genes, such as SPI-1, K1L, C7L, p28/N1R, B5R, K3L, and E3L, the alteration or deletion of which restricts the virus replication in specific cell lines (3). These host range genes display varied restrictions and biological functions. For example, SPI-1 is required for vaccinia virus replication in PK15 and A549 cells and in mice (4, 5). K1L, an ankyrin repeat-containing protein which inhibits IkB-␣ degradation and interferon (IFN)-stimulated effectors, is required for replication in RK13 cells (6). A double deletion of C7L and K1L leads to restriction of the virus replication in PK1, RK13, and multiple human cell lines, while C7L deletion alone suppresses apoptosis and limits replication in hamster Dede cells (7). P28/N1R, an E3-RING finger ubiquitin ligase involved in protein degradation and apoptosis suppression, is required for replication in mouse macrophage (8, 9), while B5R, a membrane glycoprotein, activates Src, allowing growth in VERO, CEF, PK15, and quail (QT-6) cells (10).The most studied vaccinia host range gene is E3L. E3 protein is expressed early in the virus replication cycle and is known to suppress multiple innate immune pathways (3,11,12). E3 protein possesses biochemical capacities to bind Z-form DNA via the Z-DNA binding domain (ZBD) toward the N terminus and to bind to dsRNA through the C-terminal dsRNA binding domain (DRBD) (13,14). Since the best characterized biological functions of vaccinia virus E3 protein, such as host range function and inhibition of innate immune responses (e.g., cytokine expression, apoptosis and interferon-induced antiviral activity), all are mediated primarily via the DRBD region of E3 protein (15-17), generally it has been believed that E3 protein functions through seques-

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Section: Resultsmentioning

confidence: 86%

Section: Suppression Of Pkr Activation By E3 Mutants Pkr Plays a Majmentioning

confidence: 97%

mentioning

confidence: 99%

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Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA

Dueck

Chen

et al. 2015

J Virol

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“…Interestingly, this mutated sequence has reverted to the original sequence twice on independent branches of the tree and in viruses isolated from widely separated geographic regions. M029 has been shown to function similarly to VACV E3, binding double-stranded RNA, inhibiting protein kinase R (PKR) activation, and inhibiting IFN-␤, tumor necrosis factor alpha (TNF-␣), and interleukin-6 (IL-6) expression (45); it is a critical virulence factor in rabbit infections but also has a second function of binding RNA helicase A (RHA; DHX9), which promotes virus replication in some cell lines, so it has been described as a dual-function virulence and host range factor (46). The amino acid sequence from the related leporipoxvirus RFV is conserved from amino acid 1 to 8 and is identical to that from MYXV from amino acid 20 to 55 but is poorly conserved between residues 9 and 19; this region is also divergent in the Californian MSW strain of MYXV (47).…”

Section: Pathways To Attenuation and Virulence In Australian Isolatesmentioning

confidence: 99%

Genome Scale Evolution of Myxoma Virus Reveals Host-Pathogen Adaptation and Rapid Geographic Spread

Kerr

Rogers

Fitch

et al. 2013

J Virol

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The evolutionary interplay between myxoma virus (MYXV) and the European rabbit (Oryctolagus cuniculus) following release of the virus in Australia in 1950 as a biological control is a classic example of host-pathogen coevolution. We present a detailed genomic and phylogeographic analysis of 30 strains of MYXV, including the Australian progenitor strain Standard Laboratory Strain (SLS), 24 Australian viruses isolated from 1951 to 1999, and three isolates from the early radiation in Britain from 1954 and 1955. We show that in Australia MYXV has spread rapidly on a spatial scale, with multiple lineages cocirculating within individual localities, and that both highly virulent and attenuated viruses were still present in the field through the 1990s. In addition, the detection of closely related virus lineages at sites 1,000 km apart suggests that MYXV moves freely in geographic space, with mosquitoes, fleas, and rabbit migration all providing means of transport. Strikingly, despite multiple introductions, all modern viruses appear to be ultimately derived from the original introductions of SLS. The rapidity of MYXV evolution was also apparent at the genomic scale, with gene duplications documented in a number of viruses. Duplication of potential virulence genes may be important in increasing the expression of virulence proteins and provides the basis for the evolution of novel functions. Mutations leading to loss of open reading frames were surprisingly frequent and in some cases may explain attenuation, but no common mutations that correlated with virulence or attenuation were identified.

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“…As previous studies revealed that PKR has evolved under selection by viral inhibitors (Elde et al 2009;Rothenburg et al 2009) and that various poxvirus E3L proteins differ in their ability to inhibit human PKR (Myskiw et al 2011), we focused our studies on variola virus E3L and its natural target human PKR. Our selection of variola E3L was also spurred by the critical biodefense importance of variola virus.…”

Section: Introductionmentioning

confidence: 99%

Variola virus E3L Zα domain, but not its Z-DNA binding activity, is required for PKR inhibition

Thakur

Seo

Dever

2013

RNA

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Responding to viral infection, the interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase PKR phosphorylates translation initiation factor eIF2α to inhibit cellular and viral protein synthesis. To overcome this host defense mechanism, many poxviruses express the protein E3L, containing an N-terminal Z-DNA binding (Zα) domain and a C-terminal dsRNA-binding domain (dsRBD). While E3L is thought to inhibit PKR activation by sequestering dsRNA activators and by directly binding the kinase, the role of the Zα domain in PKR inhibition remains unclear. Here, we show that the E3L Zα domain is required to suppress the growth-inhibitory properties associated with expression of human PKR in yeast, to inhibit PKR kinase activity in vitro, and to reverse the inhibitory effects of PKR on reporter gene expression in mammalian cells treated with dsRNA. Whereas previous studies revealed that the Z-DNA binding activity of E3L is critical for viral pathogenesis, we identified point mutations in E3L that functionally uncouple Z-DNA binding and PKR inhibition. Thus, our studies reveal a molecular distinction between the nucleic acid binding and PKR inhibitory functions of the E3L Zα domain, and they support the notion that E3L contributes to viral pathogenesis by targeting PKR and other components of the cellular anti-viral defense pathway.

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

Cited by 39 publications

References 42 publications

Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA

Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA

Genome Scale Evolution of Myxoma Virus Reveals Host-Pathogen Adaptation and Rapid Geographic Spread

Variola virus E3L Zα domain, but not its Z-DNA binding activity, is required for PKR inhibition

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