SAMD9L acts as an antiviral factor against HIV-1 and primate lentiviruses by restricting viral and cellular translation.

Sterile alpha motif domain-containing proteins 9 and 9-like (SAMD9/9L) are associated with life-threatening genetic diseases in humans and are restriction factors of poxviruses. Yet, their cellular function and the extent of their antiviral role are poorly known. Here, we found that interferon-stimulated human SAMD9L restricts HIV-1 in the late phases of replication, at the posttranscriptional and prematuration steps, impacting viral translation and, possibly, endosomal trafficking. Surprisingly, the paralog SAMD9 exerted an opposite effect, enhancing HIV-1. More broadly, we showed that SAMD9L restricts primate lentiviruses, but not a gammaretrovirus (MLV), nor 2 RNA viruses (arenavirus MOPV and rhabdovirus VSV). Using structural modeling and mutagenesis of SAMD9L, we identified a conserved Schlafen-like active site necessary for HIV-1 restriction by human and a rodent SAMD9L. By testing a gain-of-function constitutively active variant from patients with SAMD9L-associated autoinflammatory disease, we determined that SAMD9L pathogenic functions also depend on the Schlafen-like active site. Finally, we found that the constitutively active SAMD9L strongly inhibited HIV, MLV, and, to a lesser extent, MOPV. This suggests that the virus-specific effect of SAMD9L may involve its differential activation/sensing and the virus ability to evade from SAMD9L restriction. Overall, our study identifies SAMD9L as an HIV-1 antiviral factor from the cell autonomous immunity and deciphers host determinants underlying the translational repression. This provides novel links and therapeutic avenues against viral infections and genetic diseases.

Metabolism-dependent secondary effect of anti-MAPK cancer therapy on DNA repair.

Amino acid bioavailability impacts mRNA translation in a codon-dependent manner. Here, we report that the anti-cancer MAPK inhibitors (MAPKi) decrease the intracellular concentration of aspartate and glutamate in melanoma cells. This coincides with the accumulation of ribosomes on codons corresponding to these amino acids and triggers the translation-dependent degradation of mRNAs encoding aspartate- and glutamate-rich proteins, involved in DNA metabolism such as DNA replication and repair. Consequently, cells that survive MAPKi degrade aspartate and glutamate likely to generate energy, which simultaneously decreases their requirement for amino acids due to the downregulation of aspartate- and glutamate-rich proteins involved in cell proliferation. Concomitantly, the downregulation of aspartate- and glutamate-rich proteins involved in DNA repair increases DNA damage loads. Thus, DNA repair defects, and therefore mutations, are at least in part a secondary effect of the metabolic adaptation of cells exposed to MAPKi.

Translation-dependent and -independent mRNA decay occur through mutually exclusive pathways defined by ribosome density during T cell activation.

mRNA translation and decay are tightly interconnected processes both in the context of mRNA quality-control pathways and for the degradation of functional mRNAs. Cotranslational mRNA degradation through codon usage, ribosome collisions, and the recruitment of specific proteins to ribosomes is an important determinant of mRNA turnover. However, the extent to which translation-dependent mRNA decay (TDD) and translation-independent mRNA decay (TID) pathways participate in the degradation of mRNAs has not been studied yet. Here we describe a comprehensive analysis of basal and signal-induced TDD and TID in mouse primary CD4+ T cells. Our results indicate that most cellular transcripts are decayed to some extent in a translation-dependent manner. Our analysis further identifies the length of untranslated regions, the density of ribosomes, and GC3 content as important determinants of TDD magnitude. Consistently, all transcripts that undergo changes in ribosome density within their coding sequence upon T cell activation display a corresponding change in their TDD level. Moreover, we reveal a dynamic modulation in the relationship between GC3 content and TDD upon T cell activation, with a reversal in the impact of GC3- and AU3-rich codons. Altogether, our data show a strong and dynamic interconnection between mRNA translation and decay in mammalian primary cells.

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades").

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the development of numerous innovative applications. However, delivery of the Cas9 protein and single-guide RNA (sgRNA) into target cells can be technically challenge. Classical viral vectors, such as those derived from lentiviruses (LVs) or adeno-associated viruses (AAVs), allow for efficient delivery of transgenes coding for the Cas9 protein and its associated sgRNA in many primary cells and in vivo. Nevertheless, these vectors can suffer from drawbacks such as integration of the transgene in the target cell genome, a limited cargo capacity, and long-term expression of the Cas9 protein and guide RNA in target cells. To overcome some of these problems, a delivery vector based on the murine Leukemia virus (MLV) was developed to package the Cas9 protein and its associated guide RNA in the absence of any coding transgene. By fusing the Cas9 protein to the C-terminus of the structural protein Gag from MLV, virus-like particles (VLPs) loaded with the Cas9 protein and sgRNA (named "Nanoblades") were formed. Nanoblades can be collected from the culture medium of producer cells, purified, quantified, and used to transduce target cells and deliver the active Cas9/sgRNA complex. Nanoblades deliver their ribonucleoprotein (RNP) cargo transiently and rapidly in a wide range of primary and immortalized cells and can be programmed for other applications, such as transient transcriptional activation of targeted genes, using modified Cas9 proteins. Nanoblades are capable of in vivo genome-editing in the liver of injected adult mice and in oocytes to generate transgenic animals. Finally, they can be complexed with donor DNA for "transfection-free" homology-directed repair. Nanoblade preparation is simple, relatively low-cost, and can be easily carried out in any cell biology laboratory.