Significance: COVID-19 is a deadly rampaging infectious disease with over 480 million cases worldwide. Unfortunately, effective therapies remain very limited. Novel antiviral agents are urgently needed to combat this global healthcare crisis. Here, we elucidate the structural basis for replicase polyprotein cleavage and substrate specificity of SARS-CoV-2 main protease (M ^pro ). Through analyzing a series of high-resolution structures of SARS-CoV-2 M ^pro throughout the proteolytic process, we demonstrate the molecular mechanism of M ^pro in proteolytic processing that confers substrate specificity. Substrate selectivity is revealed using structures of the H41A mutant in complex with six individual native cleavage substrates. Our study underscores the mechanistic function of M ^pro in the viral life cycle, which provides structural insights to develop effective inhibitors against this essential target of SARS-CoV-2.

The dNTPase SAMHD1 inhibits infection by HIV-1 and other retroviruses. In the presence of dGTP, the enzyme forms tetramers and becomes active, a process that is now elucidated by structural, biochemical and cellular analyses of human SAMHD1. Binding of dGTP to four allosteric sites promotes tetramerization and induces a conformational change in the substrate-binding pocket to activate the enzyme. SAMHD1, a dNTP triphosphohydrolase (dNTPase), has a key role in human innate immunity. It inhibits infection of blood cells by retroviruses, including HIV, and prevents the development of the autoinflammatory Aicardi–Goutières syndrome (AGS). The inactive apo-SAMHD1 interconverts between monomers and dimers, and in the presence of dGTP the protein assembles into catalytically active tetramers. Here, we present the crystal structure of the human tetrameric SAMHD1–dGTP complex. The structure reveals an elegant allosteric mechanism of activation through dGTP-induced tetramerization of two inactive dimers. Binding of dGTP to four allosteric sites promotes tetramerization and induces a conformational change in the substrate-binding pocket to yield the catalytically active enzyme. Structure-based biochemical and cell-based biological assays confirmed the proposed mechanism. The SAMHD1 tetramer structure provides the basis for a mechanistic understanding of its function in HIV restriction and the pathogenesis of AGS.

Significance: SAMHD1 is a dNTPase that depletes the cellular dNTP pool to inhibit the replication of retroviruses, including HIV-1. The dNTPase activity of SAMHD1 also enables the enzyme to be a major regulator of cellular dNTP levels in mammalian cells, in addition to be implicated in the pathogenesis of chronic lymphocytic leukemia (CLL) and Aicardi Goutières syndrome (AGS). Here we present extensive structural and enzymatic data to reveal how SAMHD1 is activated and regulated via the combined actions of GTP and all cellular dNTPs. Our work establishes a complete spectrum of nucleotide binding and the exquisite regulatory mechanism of SAMHD1 in cellular dNTP metabolism, retrovirus restriction, and the pathogenesis of CLL and AGS.