Biochemical Characterization of Emerging SARS-CoV-2 Nsp15 Endoribonuclease Variants.

Global sequencing efforts from the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, continue to provide insight into the evolution of the viral genome. Coronaviruses encode 16 nonstructural proteins, within the first two-thirds of their genome, that facilitate viral replication and transcription as well as evasion of the host immune response. However, many of these viral proteins remain understudied. Nsp15 is a uridine-specific endoribonuclease conserved across all coronaviruses. The nuclease activity of Nsp15 helps the virus evade triggering an innate immune response. Understanding how Nsp15 has changed over the course of the pandemic, and how mutations affect its RNA processing function, will provide insight into the evolution of an oligomerization-dependent endoribonuclease and inform drug design. In combination with previous structural data, bioinformatics analyses of 1.9 + million SARS-CoV-2 sequences revealed mutations across Nsp15's three structured domains (N-terminal, Middle, EndoU). Selected Nsp15 variants were characterized biochemically and compared to wild type Nsp15. We found that mutations to important catalytic residues decreased cleavage activity but increased the hexamer/monomer ratio of the recombinant protein. Many of the highly prevalent variants we analyzed led to decreased nuclease activity as well as an increase in the inactive, monomeric form. Overall, our work establishes how Nsp15 variants seen in patient samples affect nuclease activity and oligomerization, providing insight into the effect of these variants in vivo.

MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells.

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be, in part, because MERS-CoV is adept at antagonizing early innate immune pathways­interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L)­activated in response to viral double-stranded RNA (dsRNA) generated during genome replication. This is in contrast to severe acute respiratory syndrome CoV-2 (SARS-CoV-2), which we recently reported to activate PKR and RNase L and, to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of dsRNA-induced innate immune pathways. This resulted in at least tenfold attenuation of replication in human lung­derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of wild-type MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication.

Epigallocatechin Gallate Inhibits the Uridylate-Specific Endoribonuclease Nsp15 and Efficiently Neutralizes the SARS-CoV-2 Strain.

SARS-CoV-2, the coronavirus strain that initiated the COVID-19 pandemic, and its subsequent variants present challenges to vaccine development and treatment. As the coronavirus evades the host innate immune response at the initial stage of infection, the disease can have a long nonsymptomatic period. The uridylate-specific endoribonuclease Nsp15 processes the viral genome for replication and cleaves the polyU sequence in the viral RNA to interfere with the host immune system. This study screened natural compounds in vitro to identify inhibitors against Nsp15 from SARS-CoV-2. Three natural compounds, epigallocatechin gallate (EGCG), baicalin, and quercetin, were identified as potential inhibitors. Potent antiviral activity of EGCG was confirmed in plaque reduction neutralization tests with a SARS-CoV-2 strain (PRNT50 = 0.20 µM). Because the compound has been used as a functional food ingredient due to its beneficial health effects, we theorize that this natural compound may help inhibit viral replication while minimizing safety issues.

Connecting the dots on vertical transmission of SARS-CoV-2 using protein-protein interaction network analysis - Potential roles of placental ACE2 and ENDOU.

We conducted a protein-protein interaction (PPI) network study searching for proteins relevant to pregnancy-associated COVID-19 in pregnancy complicated with severe preeclampsia (sPE) and intra-amniotic infection and/or inflammation (Triple-I). PPI networks from sPE and Triple-I were intersected with the PPI network from coronavirus infection. Common proteins included the SARS-CoV-2 entry receptor ACE2 and ENDOU, a placental endoribonuclease homologous to Nsp15, a protein produced by the virus to escape host immunity. Remarkably, placental ENDOU mRNA expression far exceeded that of ACE2. Immunohistochemistry confirmed ENDOU localization at the hemochorial maternal-fetal interface. Investigation of ENDOU's relevance to vertical transmission of SARS-CoV-2 is further warranted.

Physiologic RNA targets and refined sequence specificity of coronavirus EndoU.

Coronavirus EndoU inhibits dsRNA-activated antiviral responses; however, the physiologic RNA substrates of EndoU are unknown. In this study, we used mouse hepatitis virus (MHV)-infected bone marrow-derived macrophage (BMM) and cyclic phosphate cDNA sequencing to identify the RNA targets of EndoU. EndoU targeted viral RNA, cleaving the 3' side of pyrimidines with a strong preference for U ↓ A and C ↓ A sequences (endoY ↓ A). EndoU-dependent cleavage was detected in every region of MHV RNA, from the 5' NTR to the 3' NTR, including transcriptional regulatory sequences (TRS). Cleavage at two CA dinucleotides immediately adjacent to the MHV poly(A) tail suggests a mechanism to suppress negative-strand RNA synthesis and the accumulation of viral dsRNA. MHV with EndoU (EndoUmut) or 2'-5' phosphodiesterase (PDEmut) mutations provoked the activation of RNase L in BMM, with corresponding cleavage of RNAs by RNase L. The physiologic targets of EndoU are viral RNA templates required for negative-strand RNA synthesis and dsRNA accumulation. Coronavirus EndoU cleaves U ↓ A and C ↓ A sequences (endoY ↓ A) within viral (+) strand RNA to evade dsRNA-activated host responses.