NOD-Like Receptors: Guards of Cellular Homeostasis Perturbation during Infection

The innate immune system relies on families of pattern recognition receptors (PRRs) that detect distinct conserved molecular motifs from microbes to initiate antimicrobial responses. Activation of PRRs triggers a series of signaling cascades, leading to the release of pro-inflammatory cytokines, chemokines and antimicrobials, thereby contributing to the early host defense against microbes and regulating adaptive immunity. Additionally, PRRs can detect perturbation of cellular homeostasis caused by pathogens and fine-tune the immune responses. Among PRRs, nucleotide binding oligomerization domain (NOD)-like receptors (NLRs) have attracted particular interest in the context of cellular stress-induced inflammation during infection. Recently, mechanistic insights into the monitoring of cellular homeostasis perturbation by NLRs have been provided. We summarize the current knowledge about the disruption of cellular homeostasis by pathogens and focus on NLRs as innate immune sensors for its detection. We highlight the mechanisms employed by various pathogens to elicit cytoskeleton disruption, organelle stress as well as protein translation block, point out exemplary NLRs that guard cellular homeostasis during infection and introduce the concept of stress-associated molecular patterns (SAMPs). We postulate that integration of information about microbial patterns, danger signals, and SAMPs enables the innate immune system with adequate plasticity and precision in elaborating responses to microbes of variable virulence.

Germline SAMD9L truncation variants trigger global translational repression

SAMD9L is an interferon-induced tumor suppressor implicated in a spectrum of multisystem disorders, including risk for myeloid malignancies and immune deficiency. We identified a heterozygous de novo frameshift variant in SAMD9L in an infant with B cell aplasia and clinical autoinflammatory features who died from respiratory failure with chronic rhinovirus infection. Autopsy demonstrated absent bone marrow and peripheral B cells as well as selective loss of Langerhans and Purkinje cells. The frameshift variant led to expression of a truncated protein with interferon treatment. This protein exhibited a gain-of-function phenotype, resulting in interference in global protein synthesis via inhibition of translational elongation. Using a mutational scan, we identified a region within SAMD9L where stop-gain variants trigger a similar translational arrest. SAMD9L variants that globally suppress translation had no effect or increased mRNA transcription. The complex-reported phenotype likely reflects lineage-dominant sensitivities to this translation block. Taken together, our findings indicate that interferon-triggered SAMD9L gain-of-function variants globally suppress translation.

The flavivirus polymerase NS5 regulates translation of viral genomic RNA

Flaviviruses, including dengue virus and Zika virus, contain a single-stranded positive sense RNA genome that encodes viral proteins essential for replication and also serves as the template for new genome synthesis. As these processes move in opposite directions along the genome, translation must be inhibited at a defined point following infection to clear the template of ribosomes to allow efficient replication. Here, we demonstrate in vitro and in cell-based assays that the viral RNA polymerase, NS5, inhibits translation of the viral genome. By reconstituting translation in vitro using highly purified components, we show that this translation block occurs at the initiation stage and that translation inhibition depends on NS5-RNA interaction, primarily through association with the 5′ replication promoter region. This work supports a model whereby expression of a viral protein signals successful translation of the infecting genome, prompting a switch to a ribosome depleted replication-competent form.

Transient arrest in proteasomal degradation during inhibition of translation in the unfolded protein response

The UPR (unfolded protein response) activates transcription of genes involved in proteasomal degradation. However, we found that in its early stages the UPR leads to a transient inhibition of proteasomal disposal of cytosolic substrates (p53 and p27kip1) and of those targeted to ER (endoplasmic reticulum)-associated degradation (uncleaved precursor of asialoglycoprotein receptor H2a). Degradation resumed soon after the protein synthesis arrest that occurs in early UPR subsided. Consistent with this, protein synthesis inhibitors blocked ubiquitin/proteasomal degradation. Ubiquitination was inhibited during the translation block, suggesting short-lived E3 ubiquitin ligases as candidate depleted proteins. This was indeed the case for p53 whose E3 ligase, Mdm2 (murine double minute 2), when overexpressed, restored the degradation, whereas a mutant Mdm2 in its acidic domain restored the ubiquitination but did not completely restore the degradation. Inhibition of proteasomal degradation early in UPR may prevent depletion of essential short-lived factors during the translation arrest. Stabilization of p27 through this mechanism may explain the cell cycle arrest in G1 when translation is blocked by inhibitors or by the UPR.

Poly(A) tail shortening is the translation-dependent step in c-myc mRNA degradation.

The highly unstable c-myc mRNA has been shown to be stabilized in cells treated with protein synthesis inhibitors. We have studied this phenomenon in an effort to gain more insight into the degradation pathway of this mRNA. Our results indicate that the stabilization of c-myc mRNA in the absence of translation can be fully explained by the inhibition of translation-dependent poly(A) tail shortening. This view is based on the following observations. First, the normally rapid shortening of the c-myc poly(A) tail was slowed down by a translation block. Second, c-myc messengers which carry a short poly(A) tail, as a result of prolonged actinomycin D or 3'-deoxyadenosine treatment, were not stabilized by the inhibition of translation. We propose that c-myc mRNA degradation proceeds in at least two steps. The first step is the shortening of long poly(A) tails. This step requires ongoing translation and thus is responsible for the delay in mRNA degradation observed in the presence of protein synthesis inhibitors. The second step involves rapid degradation of the body of the mRNA, possibly preceded by the removal of the short remainder of the poly(A) tail. This last step is independent of translation.

Poly(A) tail shortening is the translation-dependent step in c-myc mRNA degradation

The highly unstable c-myc mRNA has been shown to be stabilized in cells treated with protein synthesis inhibitors. We have studied this phenomenon in an effort to gain more insight into the degradation pathway of this mRNA. Our results indicate that the stabilization of c-myc mRNA in the absence of translation can be fully explained by the inhibition of translation-dependent poly(A) tail shortening. This view is based on the following observations. First, the normally rapid shortening of the c-myc poly(A) tail was slowed down by a translation block. Second, c-myc messengers which carry a short poly(A) tail, as a result of prolonged actinomycin D or 3'-deoxyadenosine treatment, were not stabilized by the inhibition of translation. We propose that c-myc mRNA degradation proceeds in at least two steps. The first step is the shortening of long poly(A) tails. This step requires ongoing translation and thus is responsible for the delay in mRNA degradation observed in the presence of protein synthesis inhibitors. The second step involves rapid degradation of the body of the mRNA, possibly preceded by the removal of the short remainder of the poly(A) tail. This last step is independent of translation.