Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV.

The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology1-10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct and common molecular mechanisms of these closely related coronaviruses. The TGF-ß pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org ) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2.

Identification of Myricetin as an Ebola Virus VP35-Double-Stranded RNA Interaction Inhibitor through a Novel Fluorescence-Based Assay.

Ebola virus (EBOV) is a filovirus that causes a severe and rapidly progressing hemorrhagic syndrome; a recent epidemic illustrated the urgent need for novel therapeutic agents because no drugs have been approved for treatment of Ebola virus. A key contribution to the high lethality observed during EBOV outbreaks comes from viral evasion of the host antiviral innate immune response in which viral protein VP35 plays a crucial role, blocking interferon type I production, first by masking the viral double-stranded RNA (dsRNA) and preventing its detection by the pattern recognition receptor RIG-I. Aiming to identify inhibitors of the interaction of VP35 with the viral dsRNA, counteracting the VP35 viral innate immune evasion, we established a new methodology for high-yield recombinant VP35 (rVP35) expression and purification and a novel and robust fluorescence-based rVP35-RNA interaction assay ( Z' factor of 0.69). Taking advantage of such newly established methods, we screened a small library of Sardinian natural extracts, identifying Limonium morisianum as the most potent inhibitor extract. A bioguided fractionation led to the identification of myricetin as the component that can inhibit rVP35-dsRNA interaction with an IC50 value of 2.7 µM. Molecular docking studies showed that myricetin interacts with the highly conserved region of the VP35 RNA binding domain, laying the basis for further structural optimization of potent inhibitors of VP35-dsRNA interaction.

Inhibition of HIV-1 reverse transcriptase associated activities by the hydroalcoholic extract of Casimiroa edulis seeds.

The hydroalcoholic extract obtained from the seeds of Casimiroa edulis cultivated in Sardinia (Italy) have been assayed on the two enzymatic-associated activities of the HIV-1 reverse transcriptase (RT), the RNA-dependent DNA polymerase (RDDP) and the ribonuclease H. In biochemical assays, the extract inhibited both activities in a dose-dependent manner, showing a 10-fold more potent inhibition of the HIV-1 RT RDDP activity. Furthermore, the extract was cytotoxic on K562 cell replication.

HIV-1-inhibiting activity of the essential oil of Ridolfia segetum and Oenanthe crocata.

The essential oils of Ridolfia segetum (L.) Moris and Oenanthe crocata L. (Apiaceae), collected in Sardinia (Italy), have been assayed for two enzyme-associated activities of the HIV-1 reverse transcriptase (RT): RNA-dependent DNA polymerase (RDDP) activity and ribonuclease H (RNase H) activity. In biochemical assays, the essential oils inhibited HIV-1 RT RDDP activity in a dose-dependent manner, while they were inactive towards RNase H activity. Furthermore, the oils were cytotoxic towards K (562) cell replication. GC-MS analysis of the essential oils obtained by steam distillation of the aerial parts showed that the main components of R. segetum were alpha-phellandrene, alpha-terpinolene, beta-phellandrene, and dillapiol and those of O. crocata were sabinene, TRANS-beta-ocimene, CIS-beta-ocimene, and beta-pinene.