Vaccinia Virus K1L and C7L Inhibit Antiviral Activities Induced by Type I Interferons

ABSTRACT Cellular tropism of vaccinia virus (VACV) is regulated by host range genes, including K1L, C7L, and E3L. While E3L is known to support viral replication by antagonizing interferon (IFN) effectors, including PKR, the exact functions of K1L and C7L are unclear. Here, we show that K1L and C7L can also inhibit antiviral effectors induced by type I IFN. In human Huh7 and MCF-7 cells, a VACV mutant lacking both K1L and C7L (vK1L−C7L−) replicated as efficiently as wild-type (WT) VACV, even in the presence of IFN. However, pretreating the cells with type I IFN, while having very little effect on WT VACV, blocked the replication of vK1L−C7L− at the step of intermediate viral gene translation. Restoring either K1L or C7L to vK1L−C7L− fully restored the IFN resistance phenotype. The deletion of K1L and C7L from VACV did not affect the ability of the virus to inhibit IFN signaling or its ability to inhibit the phosphorylation of PKR and the α subunit of eukaryotic initiation factor 2, indicating that K1L and C7L function by antagonizing an IFN effector(s) but with a mechanism that is different from those of IFN antagonists previously identified for VACV. Mutations of K1L that inactivate the host range function also rendered K1L unable to antagonize IFN, suggesting that K1L supports VACV replication in mammalian cells by antagonizing the same antiviral factor(s) that is induced by IFN in Huh7 cells.

Download Full-text

Orthopoxvirus K3 orthologs show virus- and host-specific inhibition of the antiviral protein kinase PKR

PLoS Pathogens ◽

10.1371/journal.ppat.1009183 ◽

2021 ◽

Vol 17 (1) ◽

pp. e1009183

Author(s):

Chorong Park ◽

Chen Peng ◽

M. Julhasur Rahman ◽

Sherry L. Haller ◽

Loubna Tazi ◽

...

Keyword(s):

Protein Kinase ◽

Vaccinia Virus ◽

Host Range ◽

Translation Initiation Factor ◽

Host Cells ◽

Initiation Factor ◽

Specific Inhibition ◽

Antiviral Protein ◽

Range Function ◽

Species Specific

The antiviral protein kinase R (PKR) is an important host restriction factor, which poxviruses must overcome to productively infect host cells. To inhibit PKR, many poxviruses encode a pseudosubstrate mimic of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), designated K3 in vaccinia virus. Although the interaction between PKR and eIF2α is highly conserved, some K3 orthologs from host-restricted poxviruses were previously shown to inhibit PKR in a species-specific manner. To better define this host range function, we compared the sensitivity of PKR from 17 mammals to inhibition by K3 orthologs from closely related orthopoxviruses, a genus with a generally broader host range. The K3 orthologs showed species-specific inhibition of PKR and exhibited three distinct inhibition profiles. In some cases, PKR from closely related species showed dramatic differences in their sensitivity to K3 orthologs. Vaccinia virus expressing the camelpox virus K3 ortholog replicated more than three orders of magnitude better in human and sheep cells than a virus expressing vaccinia virus K3, but both viruses replicated comparably well in cow cells. Strikingly, in site-directed mutagenesis experiments between the variola virus and camelpox virus K3 orthologs, we found that different amino acid combinations were necessary to mediate improved or diminished inhibition of PKR derived from different host species. Because there is likely a limited number of possible variations in PKR that affect K3-interactions but still maintain PKR/eIF2α interactions, it is possible that by chance PKR from some potential new hosts may be susceptible to K3-mediated inhibition from a virus it has never previously encountered. We conclude that neither the sensitivity of host proteins to virus inhibition nor the effectiveness of viral immune antagonists can be inferred from their phylogenetic relatedness but must be experimentally determined.

Download Full-text

Varicella-Zoster Virus IE63, a Major Viral Latency Protein, Is Required To Inhibit the Alpha Interferon-Induced Antiviral Response

Journal of Virology ◽

10.1128/jvi.00325-07 ◽

2007 ◽

Vol 81 (15) ◽

pp. 7844-7851 ◽

Cited By ~ 56

Author(s):

Aruna P. N. Ambagala ◽

Jeffrey I. Cohen

Keyword(s):

Varicella Zoster Virus ◽

Melanoma Cells ◽

Alpha Interferon ◽

Parental Virus ◽

Initiation Factor ◽

Viral Gene ◽

Α Subunit ◽

Varicella Zoster ◽

In Cells ◽

Hsv 1

ABSTRACT Varicella-zoster virus (VZV) open reading frame 63 (ORF63) is the most abundant transcript expressed during latency in human sensory ganglia. VZV with ORF63 deleted is impaired for replication in melanoma cells and fibroblasts and for latency in rodents. We found that replication of the ORF63 deletion mutant is fully complemented in U2OS cells, which have been shown to complement the growth of herpes simplex virus type 1 (HSV-1) ICP0 mutants. Since HSV-1 ICP0 mutants are hypersensitive to alpha interferon (IFN-α), we examined the effect of IFN-α on VZV replication. Replication of the ORF63 mutant in melanoma cells was severely inhibited in the presence of IFN-α, in contrast to other VZV mutants that were similarly impaired for replication or to parental virus. The VZV ORF63 mutant was not hypersensitive to IFN-γ. IFN-α inhibited viral-gene expression in cells infected with the ORF63 mutant at a posttranscriptional level. Since IFN-α stimulates gene products that can phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF-2α) and inhibit translation, we determined whether cells infected with the ORF63 mutant had increased phosphorylation of eIF-2α compared with cells infected with parental virus. While phosphorylated eIF-2α was undetectable in uninfected cells or cells infected with parental virus, it was present in cells infected with the ORF63 mutant. Conversely, expression of IE63 (encoded by ORF63) in the absence of other viral proteins inhibited phosphorylation of eIF-2α. Since IFN-α has been shown to limit VZV replication in human skin xenografts, the ability of VZV IE63 to block the effects of the cytokine may play a critical role in VZV pathogenesis.

Download Full-text

GCN2 phosphorylation of eIF2α activates NF-κB in response to UV irradiation

Biochemical Journal ◽

10.1042/bj20041164 ◽

2005 ◽

Vol 385 (2) ◽

pp. 371-380 ◽

Cited By ~ 102

Author(s):

Hao-Yuan JIANG ◽

Ronald C. WEK

Keyword(s):

Uv Irradiation ◽

Mammalian Cells ◽

Transcriptional Control ◽

Control Cell ◽

Nuclear Factor Κb ◽

Initiation Factor ◽

Α Subunit ◽

Eif2α Phosphorylation ◽

Eif2α Kinase ◽

Secondary Function

In response to UV irradiation, mammalian cells elicit a gene expression programme designed to repair damage and control cell proliferation and apoptosis. Important members of this stress response include the NF-κB (nuclear factor-κB) family. However, the mechanisms by which UV irradiation activates NF-κB are not well understood. In eukaryotes, a variety of environmental stresses are recognized and remediated by a family of protein kinases that phosphorylate the α subunit of eIF2 (eukaryotic initiation factor-2). In the present study we show that NF-κB in MEF (murine embryo fibroblast) cells is activated by UV-C and UV-B irradiation through a mechanism requiring eIF2α phosphorylation. The primary eIF2α kinase in response to UV is GCN2 (general control non-derepressible-2), with PEK/PERK (pancreatic eIF2α kinase/RNA-dependent-protein-kinase-like endoplasmic-reticulum kinase) carrying out a secondary function. Our studies indicate that lowered protein synthesis accompanying eIF2α phosphorylation, combined with eIF2α kinase-independent turnover of IκBα (inhibitor of κBα), reduces the levels of IκBα in response to UV irradiation. Release of NF-κB from the inhibitory IκBα would facilitate NF-κB entry into the nucleus and targeted transcriptional control. We also find that loss of GCN2 in MEF cells significantly enhances apoptosis in response to UV exposure similar to that measured in cells deleted for the RelA/p65 subunit of NF-κB. These results demonstrate that GCN2 is central to recognition of UV stress, and that eIF2α phosphorylation provides resistance to apoptosis in response to this environmental insult.

Download Full-text

A Poxvirus Host Range Protein, CP77, Binds to a Cellular Protein, HMG20A, and Regulates Its Dissociation from the Vaccinia Virus Genome in CHO-K1 Cells

Journal of Virology ◽

10.1128/jvi.00207-06 ◽

2006 ◽

Vol 80 (15) ◽

pp. 7714-7728 ◽

Cited By ~ 28

Author(s):

Jye-Chian Hsiao ◽

Chien-Chiang Chao ◽

Ming-Jer Young ◽

Yu-Tai Chang ◽

Er-Chieh Cho ◽

...

Keyword(s):

Amino Acids ◽

Vaccinia Virus ◽

Host Range ◽

Viral Genome ◽

Cellular Protein ◽

Deletion Mapping ◽

Viral Dna ◽

Virus Growth ◽

Range Function ◽

Viral Host Range

ABSTRACT Vaccinia virus does not grow in Chinese hamster ovary (CHO-K1) cells in the absence of a viral host range factor, cowpox protein CP77. In this study, CP77 was fused to the C terminus of green fluorescence protein (GFP-CP77) and a series of nested deletion mutants of GFP-CP77 was constructed for insertion into a vaccinia virus host range mutant, VV-hr, and expressed from a viral early promoter. Deletion mapping analyses demonstrated that the N-terminal 352 amino acids of CP77 were sufficient to support vaccinia virus growth in CHO-K1 cells, whereas the C-terminal residues 353 to 668 were dispensable. In yeast two-hybrid analyses, CP77 bound to a cellular protein, HMG20A, and GST pulldown analyses showed that residues 1 to 234 of CP77 were sufficient for this interaction. After VV-hr virus infection of CHO-K1 cells, HMG20A was translocated from the nucleus to viral factories and bound to the viral genome via the HMG box region. In control VV-hr-infected CHO-K1 cells, binding of HMG20A to the viral genome persisted from 2 to 8 h postinfection (h p.i.); in contrast, when CP77 was expressed, the association of HMG20A with viral genome was transient, with little HMG20A remaining bound at 8 h p.i. This indicates that dissociation of HMG20A from viral factories correlates well with CP77 host range activity in CHO-K1 cells. Finally, in cells expressing a CP77 deletion protein (amino acids 277 to 668) or a ΔANK5 mutant that did not support vaccinia virus growth and did not contain the HMG20A binding site, HMG20A remained bound to viral DNA, demonstrating that the binding of CP77 to HMG20A is essential for its host range function. In summary, our data revealed that a novel cellular protein, HMG20A, the dissociation of which from viral DNA is regulated by CP77, providing the first cellular target regulated by viral host range CP77 protein.

Download Full-text

Longitudinal system-based analysis of transcriptional responses to type I interferons

Physiological Genomics ◽

10.1152/physiolgenomics.00058.2009 ◽

2009 ◽

Vol 38 (3) ◽

pp. 362-371 ◽

Cited By ~ 20

Author(s):

D. J. Pappas ◽

G. Coppola ◽

P. A. Gabatto ◽

F. Gao ◽

D. H. Geschwind ◽

...

Keyword(s):

T Cells ◽

Translation Initiation Factor ◽

Type I Interferons ◽

Initiation Factor ◽

Type I ◽

Type I Ifn ◽

Transcriptional Responses ◽

Statistical Measures ◽

Differential Responses ◽

Ifn Treatment

Type I interferons (IFNs) are pleiotropic cytokines that modulate both innate and adaptive immune responses. They have been used to treat autoimmune disorders, cancers, and viral infection and have been demonstrated to elicit differential responses within cells, despite sharing a single receptor. The molecular basis for such differential responses has remained elusive. To identify the mechanisms underlying differential type I IFN signaling, we used whole genome microarrays to measure longitudinal transcriptional events within human CD4+ T cells treated with IFN-α2b or IFN-β1a. We identified differentially regulated genes, analyzed them for the enrichment of known promoter elements and pathways, and constructed a network module based on weighted gene coexpression network analysis (WGCNA). WGCNA uses advanced statistical measures to find interconnected modules of correlated genes. Overall, differential responses to IFN in CD4+ T cells related to three dominant themes: migration, antigen presentation, and the cytotoxic response. For migration, WGCNA identified subtype-specific regulation of pre-mRNA processing factor 4 homolog B and eukaryotic translation initiation factor 4A2, which work at various levels within the cell to affect the expression of the chemokine CCL5. WGCNA also identified sterile α-motif domain-containing 9-like ( SAMD9L) as critical in subtype-independent effects of IFN treatment. RNA interference of SAMD9L expression enhanced the migratory phenotype of activated T cells treated with IFN-β compared with controls. Through the analysis of the dynamic transcriptional events after differential IFN treatment, we were able to identify specific signatures and to uncover novel genes that may underpin the type I IFN response.

Download Full-text

Modified Vaccinia Virus Ankara Triggers Type I IFN Production in Murine Conventional Dendritic Cells via a cGAS/STING-Mediated Cytosolic DNA-Sensing Pathway

PLoS Pathogens ◽

10.1371/journal.ppat.1003989 ◽

2014 ◽

Vol 10 (4) ◽

pp. e1003989 ◽

Cited By ~ 87

Author(s):

Peihong Dai ◽

Weiyi Wang ◽

Hua Cao ◽

Francesca Avogadri ◽

Lianpan Dai ◽

...

Keyword(s):

Dendritic Cells ◽

Vaccinia Virus ◽

Type I ◽

Type I Ifn ◽

Dna Sensing ◽

Modified Vaccinia Virus Ankara

Download Full-text

Skeletal muscle eEF2 and 4EBP1 phosphorylation during endurance exercise is dependent on intensity and muscle fiber type

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.90806.2008 ◽

2009 ◽

Vol 296 (2) ◽

pp. R326-R333 ◽

Cited By ~ 38

Author(s):

Adam J. Rose ◽

Bruno Bisiani ◽

Bodil Vistisen ◽

Bente Kiens ◽

Erik A. Richter

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Exercise Intensity ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Fiber Type ◽

Mrna Translation ◽

Initiation Factor ◽

Type I ◽

Α Subunit

Protein synthesis in skeletal muscle is known to decrease during exercise, and it has been suggested that this may depend on the magnitude of the relative metabolic stress within the contracting muscle. To examine the mechanisms behind this, the effect of exercise intensity on skeletal muscle eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) phosphorylation, key components in the mRNA translation machinery, were examined together with AMP-activated protein kinase (AMPK) in healthy young men. Skeletal muscle eEF2 phosphorylation at Thr56 increased during exercise but was not influenced by exercise intensity, and was lower than rest 30 min after exercise. On the other hand, 4EBP1 phosphorylation at Thr37/46 decreased during exercise, and this decrease was greater at higher exercise intensities and was similar to rest 30 min after exercise. AMPK activity, as indexed by AMPK α-subunit phosphorylation at Thr172 and phosphorylation of the AMPK substrate ACCβ at Ser221, was higher with higher exercise intensities, and these indices were higher than rest after high-intensity exercise only. Using immunohistochemistry, it was shown that the increase in skeletal muscle eEF2 Thr56 phosphorylation was restricted to type I myofibers. Taken together, these data suggest that the depression of skeletal muscle protein synthesis with endurance-type exercise may be regulated at both initiation (i.e., 4EBP1) and elongation (i.e., eEF2) steps, with eEF2 phosphorylation contributing at all exercise intensities but 4EBP1 dephosphorylation contributing to a greater extent at high vs. low exercise intensities.

Download Full-text

Pancreatic eukaryotic initiation factor-2α kinase (PEK) homologues in humans, Drosophila melanogaster and Caenorhabditis elegans that mediate translational control in response to endoplasmic reticulum stress

Biochemical Journal ◽

10.1042/bj3460281 ◽

2000 ◽

Vol 346 (2) ◽

pp. 281-293 ◽

Cited By ~ 59

Author(s):

Ruchira SOOD ◽

Amy C. PORTER ◽

Kun MA ◽

Lawrence A. QUILLIAM ◽

Ronald C. WEK

Keyword(s):

Endoplasmic Reticulum ◽

Drosophila Melanogaster ◽

Caenorhabditis Elegans ◽

Er Stress ◽

Translational Control ◽

Mammalian Cells ◽

Transmembrane Protein ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Α Subunit

In response to different cellular stresses, a family of protein kinases regulates translation by phosphorylation of the α subunit of eukaryotic initiation factor-2 (eIF-2α). Recently, we identified a new family member, pancreatic eIF-2α kinase (PEK) from rat pancreas. PEK, also referred to as RNA-dependent protein kinase (PKR)-like endoplasmic reticulum (ER) kinase (PERK) is a transmembrane protein implicated in translational control in response to stresses that impair protein folding in the ER. In this study, we identified and characterized PEK homologues from humans, Drosophila melanogaster and Caenorhabditis elegans. Expression of human PEK mRNA was found in over 50 different tissues examined, with highest levels in secretory tissues. In mammalian cells subjected to ER stress, we found that elevated eIF-2α phosphorylation was coincident with increased PEK autophosphorylation and eIF-2α kinase activity. Activation of PEK was abolished by deletion of PEK N-terminal sequences located in the ER lumen. To address the role of C. elegans PEK in translational control, we expressed this kinase in yeast and found that it inhibits growth by hyperphosphorylation of eIF-2α and inhibition of eIF-2B. Furthermore, we found that vaccinia virus K3L protein, an inhibitor of the eIF-2α kinase PKR involved in an anti-viral defence pathway, also reduced PEK activity. These results suggest that decreased translation initiation by PEK during ER stress may provide the cell with an opportunity to remedy the folding problem prior to introducing newly synthesized proteins into the secretory pathway.

Download Full-text

PS3-38 Vaccinia virus encoded Type I IFN antagonist can suppress IFN signaling independently of IFN binding

Cytokine ◽

10.1016/j.cyto.2010.07.377 ◽

2010 ◽

Vol 52 (1-2) ◽

pp. 89

Author(s):

Murugabaskar Balan ◽

Ahmed Lasfar ◽

Sergey V. Smirnov ◽

Walid Abushahba ◽

Sergei V. Kotenko

Keyword(s):

Vaccinia Virus ◽

Type I ◽

Type I Ifn

Download Full-text

Response of Three Different Viruses to Interferon Priming and Dithiothreitol Treatment of Avian Cells

Journal of Virology ◽

10.1128/jvi.01175-16 ◽

2016 ◽

Vol 90 (18) ◽

pp. 8328-8340 ◽

Cited By ~ 1

Author(s):

Irene Lostalé-Seijo ◽

José Martínez-Costas ◽

Javier Benavente

Keyword(s):

Protein Kinase ◽

Vaccinia Virus ◽

Vesicular Stomatitis Virus ◽

Rna Binding ◽

Initiation Factor ◽

Type I ◽

Avian Reovirus ◽

Double Stranded Rna ◽

Vesicular Stomatitis ◽

Eif2 Phosphorylation

ABSTRACTWe have previously shown that the replication of avian reovirus (ARV) in chicken cells is much more resistant to interferon (IFN) than the replication of vesicular stomatitis virus (VSV) or vaccinia virus (VV). In this study, we have investigated the role that the double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays in the sensitivity of these three viruses toward the antiviral action of chicken interferon. Our data suggest that while interferon priming of avian cells blocks vaccinia virus replication by promoting PKR activation, the replication of vesicular stomatitis virus appears to be blocked at a pretranslational step. Our data further suggest that the replication of avian reovirus in chicken cells is quite resistant to interferon priming because this virus uses strategies to downregulate PKR activation and also because translation of avian reovirus mRNAs is more resistant to phosphorylation of the alpha subunit of initiation factor eIF2 than translation of their cellular counterparts. Our results further reveal that the avian reovirus protein sigmaA is able to prevent PKR activation and that this function is dependent on its double-stranded RNA-binding activity. Finally, this study demonstrates that vaccinia virus and avian reovirus, but not vesicular stomatitis virus, express/induce factors that counteract the ability of dithiothreitol to promote eIF2 phosphorylation. Our data demonstrate that each of the three different viruses used in this study elicits distinct responses to interferon and to dithiothreitol-induced eIF2 phosphorylation when infecting avian cells.IMPORTANCEType I interferons constitute the first barrier of defense against viral infections, and one of the best characterized antiviral strategies is mediated by the double-stranded RNA-activated protein kinase R (PKR). The results of this study revealed that IFN priming of avian cells has little effect on avian reovirus (ARV) replication but drastically diminishes the replication of vaccinia virus (VV) and vesicular stomatitis virus (VSV) by PKR-dependent and -independent mechanisms, respectively. Our data also demonstrate that the dsRNA-binding ability of ARV protein sigmaA plays a key role in the resistance of ARV toward IFN by preventing PKR activation. Our findings will contribute to improve the current understanding of the interaction of viruses with the host's innate immune system. Finally, it would be of interest to uncover the mechanisms that allow avian reovirus transcripts to be efficiently translated under conditions (moderate eIF2 phosphorylation) that block the synthesis of cellular proteins.

Download Full-text