Autophagy-linked plasma and lysosomal membrane protein PLAC8 is a key host factor for SARS-CoV-2 entry into human cells.

Better understanding on interactions between SARS-CoV-2 and host cells should help to identify host factors that may be targetable to combat infection and COVID-19 pathology. To this end, we have conducted a genome-wide CRISPR/Cas9-based loss-of-function screen in human lung cancer cells infected with SARS-CoV-2-pseudotyped lentiviruses. Our results recapitulate many findings from previous screens that used full SARS-CoV-2 viruses, but also unveil two novel critical host factors: the lysosomal efflux transporter SPNS1 and the plasma and lysosomal membrane protein PLAC8. Functional experiments with full SARS-CoV-2 viruses confirm that loss-of-function of these genes impairs viral entry. We find that PLAC8 is a key limiting host factor, whose overexpression boosts viral infection in eight different human lung cancer cell lines. Using single-cell RNA-Seq data analyses, we demonstrate that PLAC8 is highly expressed in ciliated and secretory cells of the respiratory tract, as well as in gut enterocytes, cell types that are highly susceptible to SARS-CoV-2 infection. Proteomics and cell biology studies suggest that PLAC8 and SPNS1 regulate the autophagolysosomal compartment and affect the intracellular fate of endocytosed virions.

Luminescence-based in vivo monitoring of NF-κB activity through a gene delivery approach.

BACKGROUND: Monitoring activity of specific signaling pathways in vivo is challenging and requires highly sensitive methods to detect dynamic perturbations in whole organisms. RESULTS: In vivo gene delivery of a luciferase reporter followed by bioluminiscence imaging allows measuring NF-κB activity in mice liver and lungs. CONCLUSIONS: This protocol allows a direct measure of NF-κB activity through quantification of bioluminescence signal, demonstrating its accuracy and sensitivity in different animal models and experimental conditions. Variants could be also applied for the analysis of NF-κB activity in different tissues or for studying other signaling pathways in vivo.

Aging and chronic DNA damage response activate a regulatory pathway involving miR-29 and p53.

Aging is a multifactorial process that affects most of the biological functions of the organism and increases susceptibility to disease and death. Recent studies with animal models of accelerated aging have unveiled some mechanisms that also operate in physiological aging. However, little is known about the role of microRNAs (miRNAs) in this process. To address this question, we have analysed miRNA levels in Zmpste24-deficient mice, a model of Hutchinson-Gilford progeria syndrome. We have found that expression of the miR-29 family of miRNAs is markedly upregulated in Zmpste24(-/-) progeroid mice as well as during normal aging in mouse. Functional analysis revealed that this transcriptional activation of miR-29 is triggered in response to DNA damage and occurs in a p53-dependent manner since p53(-/-) murine fibroblasts do not increase miR-29 expression upon doxorubicin treatment. We have also found that miR-29 represses Ppm1d phosphatase, which in turn enhances p53 activity. Based on these results, we propose the existence of a novel regulatory circuitry involving miR-29, Ppm1d and p53, which is activated in aging and in response to DNA damage.