Sindbis Macrodomain Poly-ADP-Ribose Hydrolase Activity Is Important for Viral RNA Synthesis.

ADP-ribosylation is a highly dynamic posttranslational modification frequently studied in stress response pathways with recent attention given to its role in response to viral infection. Notably, the alphaviruses encode catalytically active macrodomains capable of ADP-ribosylhydrolase (ARH) activities, implying a role in remodeling the cellular ADP-ribosylome. This report decouples mono- and poly-ARH contributions to macrodomain function using a newly engineered Sindbis virus (SINV) mutant with attenuated poly-ARH activity. Our findings indicate that viral poly-ARH activity is uniquely required for high titer replication in mammalian systems. Despite translating incoming genomic RNA as efficiently as WT virus, mutant viruses have a reduced capacity to establish productive infection, offering a more complete understanding of the kinetics and role of the alphavirus macrodomain with important implications for broader ADP-ribosyltransferase biology. IMPORTANCE Viral macrodomains have drawn attention in recent years due to their high degree of conservation in several virus families (e.g., coronaviruses and alphaviruses) and their potential druggability. These domains erase mono- or poly-ADP-ribose, posttranslational modifications written by host poly-ADP-ribose polymerase (PARP) proteins, from undetermined host or viral proteins to enhance replication. Prior work determined that efficient alphavirus replication requires catalytically active macrodomains; however, which form of the modification requires removal and from which protein(s) had not been determined. Here, we present evidence for the specific requirement of poly-ARH activity to ensure efficient productive infection and virus replication.

Replication and single-cycle delivery of SARS-CoV-2 replicons.

Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. Experimental systems that do not rely on viruses capable of spread are needed for potential use in lower-containment settings. In this work, we use a yeast-based reverse genetics system to develop spike-deleted SARS-CoV-2 self-replicating RNAs. These noninfectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate replicon delivery particles for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and viral variant characterization.

Inborn errors of type I IFN immunity in patients with life-threatening COVID-19.

Clinical outcome upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ranges from silent infection to lethal coronavirus disease 2019 (COVID-19). We have found an enrichment in rare variants predicted to be loss-of-function (LOF) at the 13 human loci known to govern Toll-like receptor 3 (TLR3)- and interferon regulatory factor 7 (IRF7)-dependent type I interferon (IFN) immunity to influenza virus in 659 patients with life-threatening COVID-19 pneumonia relative to 534 subjects with asymptomatic or benign infection. By testing these and other rare variants at these 13 loci, we experimentally defined LOF variants underlying autosomal-recessive or autosomal-dominant deficiencies in 23 patients (3.5%) 17 to 77 years of age. We show that human fibroblasts with mutations affecting this circuit are vulnerable to SARS-CoV-2. Inborn errors of TLR3- and IRF7-dependent type I IFN immunity can underlie life-threatening COVID-19 pneumonia in patients with no prior severe infection.