MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells

Middle East respiratory syndrome coronavirus (MERS CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be in part because MERS CoV is adept at antagonizing early innate immune pathways; these include interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase ribonuclease L (OAS/RNase L), all activated in response to viral double stranded (ds)RNA generated during genome replication. This is in contrast to SARS CoV 2, which we recently reported activates PKR and RNase L and to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of the dsRNA-induced innate immune pathways. This resulted in ten-fold attenuation of replication in human lung derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of WT MERS CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA induced innate immunity during MERS CoV infection in order to optimize viral replication.

GADD34-mediated dephosphorylation of eIF2α facilitates pseudorabies virus replication by maintaining de novo protein synthesis

Viruses have evolved multiple strategies to manipulate their host’s translational machinery for the synthesis of viral proteins. A common viral target is the alpha subunit of eukaryotic initiation factor 2 (eIF2α). In this study, we show that global protein synthesis was increased but the eIF2α phosphorylation level was markedly decreased in porcine kidney 15 (PK15) cells infected with pseudorabies virus (PRV), a swine herpesvirus. An increase in the eIF2α phosphorylation level by salubrinal treatment or transfection of constructs expressing wild-type eIF2α or an eIF2α phosphomimetic [eIF2α(S51D)] attenuated global protein synthesis and suppressed PRV replication. To explore the mechanism involved in the inhibition of eIF2α phosphorylation during PRV infection, we examined the phosphorylation status of protein kinase R-like endoplasmic reticulum kinase (PERK) and double-stranded RNA-dependent protein kinase R (PKR), two kinases that regulate eIF2α phosphorylation during infection with numerous viruses. We found that the level of neither phosphorylated (p)-PERK nor p-PKR was altered in PRV-infected cells or the lungs of infected mice. However, the expression of growth arrest and DNA damage-inducible protein 34 (GADD34), which promotes eIF2α dephosphorylation by recruiting protein phosphatase 1 (PP1), was significantly induced both in vivo and in vitro. Knockdown of GADD34 and inhibition of PP1 activity by okadaic acid treatment led to increased eIF2α phosphorylation but significantly suppressed global protein synthesis and inhibited PRV replication. Collectively, these results demonstrated that PRV induces GADD34 expression to promote eIF2α dephosphorylation, thereby maintaining de novo protein synthesis and facilitating viral replication.

Mechanism of Chronic Stress-Induced Glutamatergic Neuronal Damage in the Basolateral Amygdaloid Nucleus

Stress is a ubiquitous part of our life, while appropriate stress levels can help improve the body’s adaptability to the environment. However, sustained and excessive levels of stress can lead to the occurrence of multiple devastating diseases. As an emotional center, the amygdala plays a key role in the regulation of stress-induced psycho-behavioral disorders. The structural changes in the amygdala have been shown to affect its functional characteristics. The amygdala-related neurotransmitter imbalance is closely related to psychobehavioral abnormalities. However, the mechanism of structural and functional changes of glutamatergic neurons in the amygdala induced by stress has not been fully elucidated. Here, we identified that chronic stress could lead to the degeneration and death of glutamatergic neurons in the lateral amygdaloid nucleus, resulting in neuroendocrine and psychobehavioral disorders. Therefore, our studies further suggest that the Protein Kinase R-like ER Kinase (PERK) pathway may be therapeutically targeted as one of the key mechanisms of stress-induced glutamatergic neuronal degeneration and death in the amygdala.

Increased Post-Hypoxic Oxidative Stress and Activation of the PERK Branch of the UPR in Trap1-Deficient Drosophila melanogaster Is Abrogated by Metformin

Hypoxia is known to impair mitochondrial and endoplasmic reticulum (ER) homeostasis. Post-hypoxic perturbations of the ER proteostasis result in the accumulation of misfolded/unfolded proteins leading to the activation of the Unfolded Protein Response (UPR). Mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) is reported to preserve mitochondrial membrane potential and to impede reactive oxygen species (ROS) production thereby protecting cells from ER stress as well as oxidative stress. The first-line antidiabetic drug Metformin has been attributed a neuroprotective role after hypoxia. Interestingly, Metformin has been reported to rescue mitochondrial deficits in fibroblasts derived from a patient carrying a homozygous TRAP1 loss-of-function mutation. We sought to investigate a putative link between Metformin, TRAP1, and the UPR after hypoxia. We assessed post-hypoxic/reperfusion longevity, mortality, negative geotaxis, ROS production, metabolic activity, gene expression of antioxidant proteins, and activation of the UPR in Trap1-deficient flies. Following hypoxia, Trap1 deficiency caused higher mortality and greater impairments in negative geotaxis compared to controls. Similarly, post-hypoxic production of ROS and UPR activation was significantly higher in Trap1-deficient compared to control flies. Metformin counteracted the deleterious effects of hypoxia in Trap1-deficient flies but had no protective effect in wild-type flies. We provide evidence that TRAP1 is crucially involved in the post-hypoxic regulation of mitochondrial/ER stress and the activation of the UPR. Metformin appears to rescue Trap1-deficiency after hypoxia mitigating ROS production and downregulating the pro-apoptotic PERK (protein kinase R-like ER kinase) arm of the UPR.

Porphyromonas gingivalis-Induced Neuroinflammation in Alzheimer’s Disease

“Chronic” periodontitis and its keystone pathogen Porphyromonas gingivalis have repeatedly been associated with Alzheimer’s disease (AD). Pathological hallmarks in AD are brain accumulations of amyloid-beta and neurofibrillary tangles consisting of aggregated and hyperphosphorylated tau. In addition, neuroinflammation induced by P. gingivalis has increasingly been recognized as a factor in the pathogenesis of AD. The present mini-review discusses possible mechanisms for the induction of neuroinflammation by P. gingivalis in AD, involving factors such as pro-inflammatory mediators, amyloid-beta, tau, microglia, cathepsin B, and protein kinase R. Inflammagens of P. gingivalis such as lipopolysaccharide and gingipains are also discussed.

Induction of UPR Promotes Interferon Response to Inhibit PRRSV Replication via PKR and NF-κB Pathway

Porcine reproductive and respiratory syndrome virus (PRRSV) was previously shown to induce a certain level of cellular stress during viral replication. Unfolded protein response (UPR) is a cellular stress response responsible for coping with stress and cellular survival. However, the pathway leading to the induction of UPR that may influence PRRSV replication is still unknown. Here, we found that PRRSV infection induced UPR prior to interferon response. Induction of UPR significantly enhanced the expression of interferon and interferon-related genes, thus leading to the suppression of PRRSV infection. Next, we explored the underlying mechanisms of UPR-induced antiviral response. We found that induction of UPR promoted the expression of protein kinase R (PKR), and PKR was highly correlated with the reduction of PRRSV replication. Furthermore, tunicamycin stimulation and PKR overexpression activated NF-κB and interferon response at the early stage of PRRSV infection, thus reinforcing the expression of type I interferons and proinflammatory cytokines and leading to inhibition of PRRSV. In addition, PRRSV nsp4 was shown to reduce the expression of PKR. These findings might have implications for our understandings of the host’s immune mechanism against PRRSV and a new strategy of PRRSV to evade the host antiviral immunity.

Regulation of innate immune responses in macrophages differentiated in the presence of vitamin D and infected with dengue virus 2

A dysregulated or exacerbated inflammatory response is thought to be the key driver of the pathogenesis of severe disease caused by the mosquito-borne dengue virus (DENV). Compounds that restrict virus replication and modulate the inflammatory response could thus serve as promising therapeutics mitigating the disease pathogenesis. We and others have previously shown that macrophages, which are important cellular targets for DENV replication, differentiated in the presence of bioactive vitamin D (VitD3) are less permissive to viral replication, and produce lower levels of pro-inflammatory cytokines. Therefore, we here evaluated the extent and kinetics of innate immune responses of DENV-2 infected monocytes differentiated into macrophages in the presence (D3-MDMs) or absence of VitD3 (MDMs). We found that D3-MDMs expressed lower levels of RIG I, Toll-like receptor (TLR)3, and TLR7, as well as higher levels of SOCS-1 in response to DENV-2 infection. D3-MDMs produced lower levels of reactive oxygen species, related to a lower expression of TLR9. Moreover, although VitD3 treatment did not modulate either the expression of IFN-α or IFN-β, higher expression of protein kinase R (PKR) and 2′-5′-oligoadenylate synthetase 1 (OAS1) mRNA were found in D3-MDMs. Importantly, the observed effects were independent of reduced infection, highlighting the intrinsic differences between D3-MDMs and MDMs. Taken together, our results suggest that differentiation of MDMs in the presence of VitD3 modulates innate immunity in responses to DENV-2 infection.

Protein Kinase R in Bacterial Infections: Friend or Foe?

The global antimicrobial resistance crisis poses a significant threat to humankind in the coming decades. Challenges associated with the development of novel antibiotics underscore the urgent need to develop alternative treatment strategies to combat bacterial infections. Host-directed therapy is a promising new therapeutic strategy that aims to boost the host immune response to bacteria rather than target the pathogen itself, thereby circumventing the development of antibiotic resistance. However, host-directed therapy depends on the identification of druggable host targets or proteins with key functions in antibacterial defense. Protein Kinase R (PKR) is a well-characterized human kinase with established roles in cancer, metabolic disorders, neurodegeneration, and antiviral defense. However, its role in antibacterial defense has been surprisingly underappreciated. Although the canonical role of PKR is to inhibit protein translation during viral infection, this kinase senses and responds to multiple types of cellular stress by regulating cell-signaling pathways involved in inflammation, cell death, and autophagy – mechanisms that are all critical for a protective host response against bacterial pathogens. Indeed, there is accumulating evidence to demonstrate that PKR contributes significantly to the immune response to a variety of bacterial pathogens. Importantly, there are existing pharmacological modulators of PKR that are well-tolerated in animals, indicating that PKR is a feasible target for host-directed therapy. In this review, we provide an overview of immune cell functions regulated by PKR and summarize the current knowledge on the role and functions of PKR in bacterial infections. We also review the non-canonical activators of PKR and speculate on the potential mechanisms that trigger activation of PKR during bacterial infection. Finally, we provide an overview of existing pharmacological modulators of PKR that could be explored as novel treatment strategies for bacterial infections.