A genome-wide arrayed CRISPR screen identifies PLSCR1 as an intrinsic barrier to SARS-CoV-2 entry that recent virus variants have evolved to resist.

Interferons (IFNs) play a crucial role in the regulation and evolution of host-virus interactions. Here, we conducted a genome-wide arrayed CRISPR knockout screen in the presence and absence of IFN to identify human genes that influence Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. We then performed an integrated analysis of genes interacting with SARS-CoV-2, drawing from a selection of 67 large-scale studies, including our own. We identified 28 genes of high relevance in both human genetic studies of Coronavirus Disease 2019 (COVID-19) patients and functional genetic screens in cell culture, with many related to the IFN pathway. Among these was the IFN-stimulated gene PLSCR1. PLSCR1 did not require IFN induction to restrict SARS-CoV-2 and did not contribute to IFN signaling. Instead, PLSCR1 specifically restricted spike-mediated SARS-CoV-2 entry. The PLSCR1-mediated restriction was alleviated by TMPRSS2 overexpression, suggesting that PLSCR1 primarily restricts the endocytic entry route. In addition, recent SARS-CoV-2 variants have adapted to circumvent the PLSCR1 barrier via currently undetermined mechanisms. Finally, we investigate the functional effects of PLSCR1 variants present in humans and discuss an association between PLSCR1 and severe COVID-19 reported recently.

Reconstituting Spore Cortex Peptidoglycan Biosynthesis Reveals a Deacetylase That Catalyzes Transamidation.

Some bacteria survive in nutrient-poor environments and resist killing by antimicrobials by forming spores. The cortex layer of the peptidoglycan cell wall that surrounds mature spores contains a unique modification, muramic-δ-lactam, that is essential for spore germination and outgrowth. Two proteins, the amidase CwlD and the deacetylase PdaA, are required for muramic-δ-lactam synthesis in cells, but their combined ability to generate muramic-δ-lactam has not been directly demonstrated. Here we report an in vitro reconstitution of cortex peptidoglycan biosynthesis, and we show that CwlD and PdaA together are sufficient for muramic-δ-lactam formation. Our method enables characterization of the individual reaction steps, and we show for the first time that PdaA has transamidase activity, catalyzing both the deacetylation of N-acetylmuramic acid and cyclization of the product to form muramic-δ-lactam. This activity is unique among peptidoglycan deacetylases and is notable because it may involve the direct ligation of a carboxylic acid with a primary amine. Our reconstitution products are nearly identical to the cortex peptidoglycan found in spores, and we expect that they will be useful substrates for future studies of enzymes that act on the spore cortex.

Characterization of Live-Attenuated Powassan Virus Vaccine Candidates Identifies an Efficacious Prime-Boost Strategy for Mitigating Powassan Virus Disease in a Murine Model.

Powassan virus (POWV) is an emerging tick-borne virus and cause of lethal encephalitis in humans. The lack of treatment or prevention strategies for POWV disease underscores the need for an effective POWV vaccine. Here, we took two independent approaches to develop vaccine candidates. First, we recoded the POWV genome to increase the dinucleotide frequencies of CpG and UpA to potentially attenuate the virus by raising its susceptibility to host innate immune factors, such as the zinc-finger antiviral protein (ZAP). Secondly, we took advantage of the live-attenuated yellow fever virus vaccine 17D strain (YFV-17D) as a vector to express the structural genes pre-membrane (prM) and envelope (E) of POWV. The chimeric YFV-17D-POWV vaccine candidate was further attenuated for in vivo application by removing an N-linked glycosylation site within the nonstructural protein (NS)1 of YFV-17D. This live-attenuated chimeric vaccine candidate significantly protected mice from POWV disease, conferring a 70% survival rate after lethal challenge when administered in a homologous two-dose regimen. Importantly, when given in a heterologous prime-boost vaccination scheme, in which vaccination with the initial chimeric virus was followed by a protein boost with the envelope protein domain III (EDIII), 100% of the mice were protected without showing any signs of morbidity. Combinations of this live-attenuated chimeric YFV-17D-POWV vaccine candidate with an EDIII protein boost warrant further studies for the development of an effective vaccine strategy for the prevention of POWV disease.