Spindler, K. R. | Search bioRxiv

bioRxiv2026

Salmonella Typhi asparaginase-dependent activation of GCN2 promotes bacterial killing in murine macrophages

Many intracellular pathogens stimulate host cell stress by directly or indirectly causing an imbalance in host nutrients; depletion of amino acid pools in particular can act as a danger signal to infected cells. Using a restrictive host model of Salmonella enterica serovar Typhi (S. Typhi) infection, we identify early induction the integrated stress response (ISR) by viable bacteria, but not heat-killed bacteria. Genetic deletion of the amino acid sensing ISR kinase GCN2 (also known as EIF2AK4) prevented early ISR activation during S. Typhi infection, and murine macrophages lacking GCN2 show impaired bacterial clearance and decreased cytokine output. Supplementation of wildtype C57BL/6 murine macrophages with only the non-essential amino acid asparagine was sufficient to suppress S. Typhi-induced ISR activation and deletion of S. Typhi ansB, encoding an asparaginase, prevented ISR activation during infection. Pharmacological inhibition of mammalian target of rapamycin (mTOR), the other major amino acid sensing pathway in eukaryotic cells, prevented GCN2 activation and ISR induction in murine macrophages, indicating an upstream role for mTOR in signaling to GCN2. These findings suggest a role for the ISR in macrophage innate immune responses to S. Typhi infection and highlight a potential difference in nutrient-dependent signaling between the S. Typhi-susceptible human host and the restrictive murine host centered around asparagine, mTOR, and GCN2.

immunology↗

bioRxiv2019

Enhanced replication of mouse adenovirus type 1 following virus-induced degradation of protein kinase R (PKR)

Protein kinase R (PKR) plays a major role in activating host immunity during infection by sensing dsRNA produced by viruses. Once activated by dsRNA, PKR phosphorylates the translation factor eIF2, halting cellular translation. Many viruses have methods of inhibiting PKR activation or its downstream effects, circumventing protein synthesis shutdown. These include sequestering dsRNA or producing proteins that bind to and inhibit PKR activation. Here we describe our finding that in multiple cell types, PKR was depleted during mouse adenovirus type 1 (MAV-1) infection. MAV-1 did not appear to be targeting PKR at a transcriptional or translational level because total PKR mRNA levels and levels of PKR mRNA bound to polysomes were unchanged or increased during MAV-1 infection. However, inhibiting the proteasome reduced the PKR depletion seen in MAV-1-infected cells, whereas inhibiting the lysosome had no effect. This suggests that proteasomal degradation alone is responsible for PKR degradation during MAV-1 infection. Time course experiments indicate that the degradation occurs early after infection. Infecting cells with UV-inactivated virus prevented PKR degradation, whereas inhibiting viral DNA replication did not. Together these results suggest that an early viral gene is responsible. Degradation of PKR is a rare mechanism to oppose PKR activity, and it has only been described in six RNA viruses. To our knowledge, this is the first example of a DNA virus counteracting PKR by degrading it.\n\nImportanceThe first line of defense in cells during viral infection is the innate immune system, which is activated by different viral products. PKR is a part of this innate immune system and is induced by interferon and activated by dsRNA produced by DNA and RNA viruses. PKR is such an important part of the antiviral response that many viral families have gene products to counteract its activation or the resulting effects of its activity. Although a few RNA viruses degrade PKR, this method of counteracting PKR has not been reported for any DNA viruses. MAV-1 does not encode virus-associated RNAs, a human adenoviral defense against PKR activation. Instead, MAV-1 degrades PKR, and it is the first DNA virus reported to do so. The innate immune evasion by PKR degradation is a previously unidentified way for a DNA virus to circumvent the host antiviral response.

microbiology↗