scholarly journals Highly Pathogenic New World Arenavirus Infection Activates the Pattern Recognition Receptor Protein Kinase R without Attenuating Virus Replication in Human Cells

2017 ◽  
Vol 91 (20) ◽  
Author(s):  
Cheng Huang ◽  
Olga A. Kolokoltsova ◽  
Elizabeth J. Mateer ◽  
Takaaki Koma ◽  
Slobodan Paessler
Keyword(s):  
Protein Kinase ◽  
Virus Replication ◽  
New World ◽  
Human Cells ◽  
Host Protein ◽  
Initiation Factor ◽  
Protein Kinase R ◽  
Highly Pathogenic ◽  
Rna Sensors ◽  
Infected Cells

ABSTRACT The arenavirus family consists of several highly pathogenic viruses, including the Old World (OW) arenavirus Lassa fever virus (LASV) and the New World (NW) Junin virus (JUNV) and Machupo virus (MACV). Host response to infection by these pathogenic arenaviruses is distinct in many aspects. JUNV and MACV infections readily induce an interferon (IFN) response in human cells, while LASV infection usually triggers an undetectable or weak IFN response. JUNV induces an IFN response through RIG-I, suggesting that the host non-self RNA sensor readily detects JUNV viral RNAs (vRNAs) during infection and activates IFN response. Double-stranded-RNA (dsRNA)-activated protein kinase R (PKR) is another host non-self RNA sensor classically known for its vRNA recognition activity. Here we report that infection with NW arenaviruses JUNV and MACV, but not OW LASV, activated PKR, concomitant with elevated phosphorylation of the translation initiation factor α subunit of eukaryotic initiation factor 2 (eIF2α). Host protein synthesis was substantially suppressed in MACV- and JUNV-infected cells but was only marginally reduced in LASV-infected cells. Despite the antiviral activity known for PKR against many other viruses, the replication of JUNV and MACV was not impaired but was slightly augmented in wild-type (wt) cells compared to that in PKR-deficient cells, suggesting that PKR or PKR activation did not negatively affect JUNV and MACV infection. Additionally, we found an enhanced IFN response in JUNV- or MACV-infected PKR-deficient cells, which was inversely correlated with virus replication. IMPORTANCE The detection of viral RNA by host non-self RNA sensors, including RIG-I and MDA5, is critical to the initiation of the innate immune response to RNA virus infection. Among pathogenic arenaviruses, the OW LASV usually does not elicit an interferon response. However, the NW arenaviruses JUNV and MACV readily trigger an IFN response in a RIG-I-dependent manner. Here, we demonstrate for the first time that pathogenic NW arenaviruses JUNV and MACV, but not the OW arenavirus LASV, activated the dsRNA-dependent PKR, another host non-self RNA sensor, during infection. Interestingly, the replication of JUNV and MACV was not restricted but was rather slightly augmented in the presence of PKR. Our data provide new evidence for a distinct interplay between host non-self RNA sensors and pathogenic arenaviruses, which also provides insights into the pathogenesis of arenaviruses and may facilitate the design of vaccines and treatments against arenavirus-caused diseases.

scholarly journals Andes Virus Nucleocapsid Protein Interrupts Protein Kinase R Dimerization To Counteract Host Interference in Viral Protein Synthesis

2014 ◽  
Vol 89 (3) ◽  
pp. 1628-1639 ◽  
Author(s):  
Zekun Wang ◽  
Mohammad A. Mir
Keyword(s):  
Protein Kinase ◽  
Viral Infection ◽  
Virus Replication ◽  
Nucleocapsid Protein ◽  
Viral Proteins ◽  
Antiviral Response ◽  
Interferon Response ◽  
Protein Kinase R ◽  
Andes Virus ◽  
Infected Cells

ABSTRACTPathogenic hantaviruses delay the type I interferon response during early stages of viral infection. However, the robust interferon response and induction of interferon-stimulated genes observed during later stages of hantavirus infection fail to combat the virus replication in infected cells. Protein kinase R (PKR), a classical interferon-stimulated gene product, phosphorylates the eukaryotic translation initiation factor eIF2α and causes translational shutdown to create roadblocks for the synthesis of viral proteins. The PKR-induced translational shutdown helps host cells to establish an antiviral state to interrupt virus replication. However, hantavirus-infected cells do not undergo translational shutdown and fail to establish an antiviral state during the course of viral infection. In this study, we showed for the first time that Andes virus infection induced PKR overexpression. However, the overexpressed PKR was not active due to a significant inhibition of autophosphorylation. Further studies revealed that Andes virus nucleocapsid protein inhibited PKR dimerization, a critical step required for PKR autophosphorylation to attain activity. The studies reported here establish a hantavirus nucleocapsid protein as a new PKR inhibitor. These studies provide mechanistic insights into hantavirus resistance to the host interferon response and solve the puzzle of the lack of translational shutdown observed in hantavirus-infected cells. The sensitivity of hantavirus replication to PKR has likely imposed a selective evolutionary pressure on hantaviruses to evade the PKR antiviral response for survival. We envision that evasion of the PKR antiviral response by NP has likely helped hantaviruses to exist during evolution and to survive in infected hosts with a multifaceted antiviral defense.IMPORTANCEProtein kinase R (PKR), a versatile antiviral host factor, shuts down the translation machinery upon activation in virus-infected cells to create hurdles for the manufacture of viral proteins. The studies reported here reveal that the hantavirus nucleocapsid protein counteracts the PKR antiviral response by inhibiting PKR dimerization, which is required for its activation. We report the discovery of a new PKR inhibitor whose expression in hantavirus-infected cells prevents the PKR-induced host translational shutdown to ensure the continuous synthesis of viral proteins required for efficient virus replication.

scholarly journals Comparison of the Innate Immune Responses to Pathogenic and Nonpathogenic Clade B New World Arenaviruses

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Hector Moreno ◽  
Rebecca Möller ◽  
Chiara Fedeli ◽  
Gisa Gerold ◽  
Stefan Kunz
Keyword(s):  
Immune Responses ◽  
New World ◽  
Transcriptome Profiling ◽  
Human Cells ◽  
Innate Immune ◽  
Type I ◽  
Innate Immune Responses ◽  
Protein Kinase R ◽  
Highly Pathogenic ◽  
Tacaribe Virus

ABSTRACT The New World (NW) arenaviruses are a diverse group of zoonotic viruses, including several causative agents of severe hemorrhagic fevers in humans. All known human-pathogenic NW arenaviruses belong to clade B, where they group into sublineages with phylogenetically closely related nonpathogenic viruses, e.g., the highly pathogenic Junin (JUNV) and Machupo viruses with the nonpathogenic Tacaribe virus (TCRV). Considering the close genetic relationship of nonpathogenic and pathogenic NW arenaviruses, the identification of molecular determinants of virulence is of great importance. The host cell’s innate antiviral defense represents a major barrier for zoonotic infection. Here, we performed a side-by-side comparison of the innate immune responses against JUNV and TCRV in human cells. Despite similar levels of viral replication, infection with TCRV consistently induced a stronger type I interferon (IFN-I) response than JUNV infection did. Transcriptome profiling revealed upregulation of a largely overlapping set of interferon-stimulated genes in cells infected with TCRV and JUNV. Both viruses were relatively insensitive to IFN-I treatment of human cells and induced similar levels of apoptosis in the presence or absence of an IFN-I response. However, in comparison to JUNV, TCRV induced stronger activation of the innate sensor double-strand RNA-dependent protein kinase R (PKR), resulting in phosphorylation of eukaryotic translation initiation factor eIF2α. Confocal microscopy studies revealed similar subcellular colocalizations of the JUNV and TCRV viral replication-transcription complexes with PKR. However, deletion of PKR by CRISPR/Cas9 hardly affected JUNV but promoted TCRV multiplication, providing the first evidence for differential innate recognition and control of pathogenic and nonpathogenic NW arenaviruses by PKR. IMPORTANCE New World (NW) arenaviruses are a diverse family of emerging zoonotic viruses that merit significant attention as important public health problems. The close genetic relationship of nonpathogenic NW arenaviruses with their highly pathogenic cousins suggests that few mutations may be sufficient to enhance virulence. The identification of molecular determinants of virulence of NW arenaviruses is therefore of great importance. Here we undertook a side-by-side comparison of the innate immune responses against the highly pathogenic Junin virus (JUNV) and the related nonpathogenic Tacaribe virus (TCRV) in human cells. We consistently found that TCRV induces a stronger type I interferon (IFN-I) response than JUNV. Transcriptome profiling revealed an overlapping pattern of IFN-induced gene expression and similar low sensitivities to IFN-I treatment. However, the double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) contributed to the control of TCRV, but not JUNV, providing the first evidence for differential innate recognition and control of JUNV and TCRV.

scholarly journals Inhibition of Protein Kinase R Activation and Upregulation of GADD34 Expression Play a Synergistic Role in Facilitating Coronavirus Replication by Maintaining De Novo Protein Synthesis in Virus-Infected Cells

2009 ◽  
Vol 83 (23) ◽  
pp. 12462-12472 ◽  
Author(s):  
Xiaoxing Wang ◽  
Ying Liao ◽  
Pei Ling Yap ◽  
Kim J. Png ◽  
James P. Tam ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Viral Replication ◽  
De Novo ◽  
Initiation Factor ◽  
Animal Cells ◽  
Protein Kinase R ◽  
Α Subunit ◽  
Infected Cells ◽  
De Novo Protein Synthesis

ABSTRACT A diversity of strategies is evolved by RNA viruses to manipulate the host translation machinery in order to create an optimal environment for viral replication and progeny production. One of the common viral targets is the α subunit of eukaryotic initiation factor 2 (eIF-2α). In this report, we show that phosphorylation of eIF-2α was severely suppressed in human and animal cells infected with the coronavirus infectious bronchitis virus (IBV). To understand whether this suppression is through inhibition of protein kinase R (PKR), the double-stranded-RNA-dependent kinase that is one of the main kinases responsible for phosphorylation of eIF-2α, cells infected with IBV were analyzed by Western blotting. The results showed that the level of phosphorylated PKR was greatly reduced in IBV-infected cells. Overexpression of IBV structural and nonstructural proteins (nsp) demonstrated that nsp2 is a weak PKR antagonist. Furthermore, GADD34, a component of the protein phosphatase 1 (PP1) complex, which dephosphorylates eIF-2α, was significantly induced in IBV-infected cells. Inhibition of the PP1 activity by okadaic acid and overexpression of GADD34, eIF-2α, and PKR, as well as their mutant constructs in virus-infected cells, showed that these viral regulatory strategies played a synergistic role in facilitating coronavirus replication. Taken together, these results confirm that IBV has developed a combination of two mechanisms, i.e., blocking PKR activation and inducing GADD34 expression, to maintain de novo protein synthesis in IBV-infected cells and, meanwhile, to enhance viral replication.

scholarly journals Immunoproteasome induction is suppressed in hepatitis C virus-infected cells in a protein kinase R-dependent manner

2016 ◽  
Vol 48 (11) ◽  
pp. e270-e270 ◽  
Author(s):  
In Soo Oh ◽  
Kathrin Textoris-Taube ◽  
Pil Soo Sung ◽  
Wonseok Kang ◽  
Xenia Gorny ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Kinase ◽  
Dependent Manner ◽  
Protein Kinase R ◽  
Infected Cells

scholarly journals The Potential Role of Protein Kinase R as a Regulator of Age-Related Neurodegeneration

2021 ◽  
Vol 13 ◽  
Author(s):  
Nicolás W. Martinez ◽  
Felipe E. Gómez ◽  
Soledad Matus
Keyword(s):  
Protein Kinase ◽  
Pathological Process ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Protein Kinase R ◽  
Eukaryotic Translation ◽  
Neurodegenerative Process ◽  
Age Related ◽  
Initiation Factor 2

There is a growing evidence describing a decline in adaptive homeostasis in aging-related diseases affecting the central nervous system (CNS), many of which are characterized by the appearance of non-native protein aggregates. One signaling pathway that allows cell adaptation is the integrated stress response (ISR), which senses stress stimuli through four kinases. ISR activation promotes translational arrest through the phosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2α) and the induction of a gene expression program to restore cellular homeostasis. However, depending on the stimulus, ISR can also induce cell death. One of the ISR sensors is the double-stranded RNA-dependent protein kinase [protein kinase R (PKR)], initially described as a viral infection sensor, and now a growing evidence supports a role for PKR on CNS physiology. PKR has been largely involved in the Alzheimer’s disease (AD) pathological process. Here, we reviewed the antecedents supporting the role of PKR on the efficiency of synaptic transmission and cognition. Then, we review PKR’s contribution to AD and discuss the possible participation of PKR as a player in the neurodegenerative process involved in aging-related pathologies affecting the CNS.

scholarly journals Severe Acute Respiratory Syndrome Coronavirus Triggers Apoptosis via Protein Kinase R but Is Resistant to Its Antiviral Activity

2008 ◽  
Vol 83 (5) ◽  
pp. 2298-2309 ◽  
Author(s):  
Verena Krähling ◽  
David A. Stein ◽  
Martin Spiegel ◽  
Friedemann Weber ◽  
Elke Mühlberger
Keyword(s):  
Protein Kinase ◽  
Severe Acute Respiratory Syndrome ◽  
Chromatin Condensation ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Protein Kinase R ◽  
Beta Interferon ◽  
Reverse Transcription Pcr ◽  
Eif2α Phosphorylation

ABSTRACT In this study, infection of 293/ACE2 cells with severe acute respiratory syndrome coronavirus (SARS-CoV) activated several apoptosis-associated events, namely, cleavage of caspase-3, caspase-8, and poly(ADP-ribose) polymerase 1 (PARP), and chromatin condensation and the phosphorylation and hence inactivation of the eukaryotic translation initiation factor 2α (eIF2α). In addition, two of the three cellular eIF2α kinases known to be virus induced, protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), were activated by SARS-CoV. The third kinase, general control nonderepressible-2 kinase (GCN2), was not activated, but late in infection the level of GCN2 protein was significantly reduced. Reverse transcription-PCR analyses revealed that the reduction of GCN2 protein was not due to decreased transcription or stability of GCN2 mRNA. The specific reduction of PKR protein expression by antisense peptide-conjugated phosphorodiamidate morpholino oligomers strongly reduced cleavage of PARP in infected cells. Surprisingly, the knockdown of PKR neither enhanced SARS-CoV replication nor abrogated SARS-CoV-induced eIF2α phosphorylation. Pretreatment of cells with beta interferon prior to SARS-CoV infection led to a significant decrease in PERK activation, eIF2α phosphorylation, and SARS-CoV replication. The various effects of beta interferon treatment were found to function independently on the expression of PKR. Our results show that SARS-CoV infection activates PKR and PERK, leading to sustained eIF2α phosphorylation. However, virus replication was not impaired by these events, suggesting that SARS-CoV possesses a mechanism to overcome the inhibitory effects of phosphorylated eIF2α on viral mRNA translation. Furthermore, our data suggest that viral activation of PKR can lead to apoptosis via a pathway that is independent of eIF2α phosphorylation.

scholarly journals Rotavirus variant replicates efficiently although encoding an aberrant NSP3 that fails to induce nuclear localization of poly(A)-binding protein

2012 ◽  
Vol 93 (7) ◽  
pp. 1483-1494 ◽  
Author(s):  
Michelle M. Arnold ◽  
Catie Small Brownback ◽  
Zenobia F. Taraporewala ◽  
John T. Patton
Keyword(s):  
Binding Protein ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Host Protein ◽  
Initiation Factor ◽  
Nuclear Accumulation ◽  
Progeny Virus ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Infected Cells

The rotavirus (RV) non-structural protein NSP3 forms a dimer that has binding domains for the translation initiation factor eIF4G and for a conserved 3′-terminal sequence of viral mRNAs. Through these activities, NSP3 has been proposed to promote viral mRNA translation by directing circularization of viral polysomes. In addition, by disrupting interactions between eIF4G and the poly(A)-binding protein (PABP), NSP3 has been suggested to inhibit translation of host polyadenylated mRNAs and to stimulate relocalization of PABP from the cytoplasm to the nucleus. Herein, we report the isolation and characterization of SA11-4Fg7re, an SA11-4F RV derivative that contains a large sequence duplication initiating within the genome segment (gene 7) encoding NSP3. Our analysis showed that mutant NSP3 (NSP3m) encoded by SA11-4Fg7re is almost twice the size of the wild-type protein and retains the capacity to dimerize. However, in comparison to wild-type NSP3, NSP3m has a decreased capacity to interact with eIF4G and to suppress the translation of polyadenylated mRNAs. In addition, NSP3m fails to induce the nuclear accumulation of PABP in infected cells. Despite the defective activities of NSP3m, the levels of viral protein and progeny virus produced in SA11-4Fg7re- and SA11-4F-infected cells were indistinguishable. Collectively, these data are consistent with a role for NSP3 in suppressing host protein synthesis through antagonism of PABP activity, but also suggest that NSP3 functions may have little or no impact on the efficiency of virus replication in widely used RV-permissive cell lines.

scholarly journals Binding and Nuclear Relocalization of Protein Kinase R by Human Cytomegalovirus TRS1

2006 ◽  
Vol 80 (23) ◽  
pp. 11817-11826 ◽  
Author(s):  
Morgan Hakki ◽  
Emily E. Marshall ◽  
Katherine L. De Niro ◽  
Adam P. Geballe
Keyword(s):  
Protein Kinase ◽  
Human Cytomegalovirus ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Underlying Mechanism ◽  
Cellular Protein ◽  
Initiation Factor ◽  
Protein Kinase R ◽  
Double Stranded Rna

ABSTRACT The human cytomegalovirus (HCMV) TRS1 and IRS1 genes block the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) and the consequent shutoff of cellular protein synthesis that occur during infection with vaccinia virus (VV) deleted of the double-stranded RNA binding protein gene E3L (VVΔE3L). To further define the underlying mechanism, we first evaluated the effect of pTRS1 on protein kinase R (PKR), the double-stranded RNA (dsRNA)-dependent eIF2α kinase. Immunoblot analyses revealed that pTRS1 expression in the context of a VVΔE3L recombinant decreased levels of PKR in the cytoplasm and increased its levels in the nucleus of infected cells, an effect not seen with wild-type VV or a VVΔE3L recombinant virus expressing E3L. This effect of pTRS1 was confirmed by visualizing the nuclear relocalization of PKR-EGFP expressed by transient transfection. PKR present in both the nuclear and cytoplasmic fractions was nonphosphorylated, indicating that it was unactivated when TRS1 was present. PKR also accumulated in the nucleus during HCMV infection as determined by indirect immunofluorescence and immunoblot analysis. Binding assays revealed that pTRS1 interacted with PKR in mammalian cells and in vitro. This interaction required the same carboxy-terminal region of pTRS1 that is necessary to rescue VVΔE3L replication in HeLa cells. The carboxy terminus of pIRS1 was also required for rescue of VVΔE3L and for mediating an interaction of pIRS1 with PKR. These results suggest that these HCMV genes directly interact with PKR and inhibit its activation by sequestering it in the nucleus, away from both its activator, cytoplasmic dsRNA, and its substrate, eIF2α.

Tumour necrosis factor α up-regulates protein kinase R (PKR)-activating protein (PACT) and increases phosphorylation of PKR and eukaryotic initiation factor 2-α in articular chondrocytes

2002 ◽  
Vol 30 (6) ◽  
pp. 886-889 ◽  
Author(s):  
S. J. Gilbert ◽  
V. C. Duance ◽  
D. J. Mason
Keyword(s):  
Protein Kinase ◽  
Tumour Necrosis ◽  
Cartilage Degradation ◽  
Initiation Factor ◽  
Tumour Necrosis Factor Α ◽  
Protein Kinase R ◽  
Initiation Factor 2 ◽  
Factor Α ◽  
Necrosis Factor

Our previous analysis of the genes regulated in cartilage at the onset of spontaneous osteoarthritis in the guinea pig knee revealed up-regulation of the gene for protein kinase R (PKR)-activating protein (PACT), which encodes the cellular activator of the protein kinase, PKR. PACT and PKR are upstream components of a number of signal transduction and gene transcription pathways used by pro-inflammatory cytokines. We have investigated the role of PACT and PKR in articular cartilage degradation using cytokine treatment of bovine primary chondrocytes and cartilage explants. Tumour necrosis factor α increased expression of PACT protein after 3 h of treatment. Furthermore, increased phosphorylation of PKR and eukaryotic initiation factor 2-α was observed. The known role of PKR in cytokine-induced signalling pathways, together with our data showing cytokine regulation of PACT and PKR in chondrocytes, reveals a novel mechanism of cartilage degradation that may be important in the pathogenesis of arthritic diseases.

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