Journal: Viruses

Article Title: Exploiting the Achilles' Heel of Viral RNA Processing to Develop Novel Antivirals.

doi: 10.3390/v17010054

Figure Lengend Snippet: Figure 3. 5342191 inhibits HCoV-229E and HCoV-OC43 replication. (A) A schematic of the exper- imental protocol. Huh7 cells were infected with indicated coronavirus strains for 1 h; cells were washed and then treated with either DMSO or 5342191. Media were harvested and levels of viral RNA release into media were determined by RTqPCR. Cells were lysed for RNA extraction to measure levels of intracellular viral RNA using RTqPCR, and viral protein production via Western blotting. (B) Effect of 5342191 concentrations on HCoV-229E RNA accumulation in media (by RTqPCR) and cell viability (assessed using Alamar blue). (C) A correlation of viral RNA release and infectious virus was produced. Infected cells were treated with EC50 (1.2 µM) or EC90 (2.5 µM) doses of 5342191 for 24 h, then media were harvested, and viral RNA accumulation in media was measured by RTqPCR, infectious virus by TCID50 assay. (D) Cells were infected with the HCoV-OC43 virus at an MOI of 2 and then treated with either DMSO or 2.5 µM 5342191 for 24 h. Culture supernatants were harvested and analyzed by RTqPCR to measure the release of HCoV OC43 into the culture media. (E) Top: a schematic of the coronavirus virus genome and subgenomic RNAs generated. Shown are the positions of primer sets used to measure genomic or total viral RNAs. Bottom: infected cells were treated with DMSO or 2.5 µM 5342191 for 24 h. Extracellular viral RNA accumulation was measured by RTqPCR. Total RNA was extracted from cells and the abundance of genomic or total viral RNA (genomic and nested RNAs) was measured by RTqPCR. (F) Representative immunoblots showing the impact of 5342191 on the production of the nucleocapsid (N) protein of the HCoV-229E virus (top) and the N protein and spike (S) protein of the HCoV-OC43 virus (bottom). ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Total RNA was extracted from cells using the BioRad Aurum Total RNA Lysis Kit (BioRad) as per the manufacturer’s instructions.

Techniques: Infection, RNA Extraction, Western Blot, Virus, Produced, TCID50 Assay, Generated

Journal: Viruses

Article Title: Exploiting the Achilles' Heel of Viral RNA Processing to Develop Novel Antivirals.

doi: 10.3390/v17010054

Figure Lengend Snippet: Figure 8. 5342191 inhibits PR8 influenza A virus replication. (A) A549 cells were infected at an MOI of 2 with PR8 for 1 h, and media were removed and replaced with media containing DMSO or indicated doses of 5342191. At 24 hpi, media were harvested, and levels of viral RNA released were determined by RTqPCR. (B) To correlate the level of viral RNA released to the infectious virus, infected cells were treated with doses of 5342191 required to reduce PR8 RNA accumulation in media by 90% (EC90, 0.104 µM). Media were assessed in parallel to determine the effect of the compound addition on the level of infectious virus present as determined by TCID50 assay. (C–E) A549 cells were infected with PR8 at an MOI of 2 for 1 h, and media were removed and replaced with media containing DMSO or 0.104 µM 5342191. At 24 hpi, cells were harvested, (C) expression of viral NP or NS1 was assessed by the western blot or (D) total RNA extracted, and intracellular levels of indicated viral RNAs were determined by RTqPCR. (E) Ratio of M2 versus M1 or NS2 versus NS1 to assess the impact of 5342191 on viral RNA splicing. Shown are the results from n > 3 independent assays. **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

Article Snippet: Total RNA was extracted from cells using the BioRad Aurum Total RNA Lysis Kit (BioRad) as per the manufacturer’s instructions.

Techniques: Virus, Infection, TCID50 Assay, Expressing, Western Blot

Journal: Viruses

Article Title: Exploiting the Achilles' Heel of Viral RNA Processing to Develop Novel Antivirals.

doi: 10.3390/v17010054

Figure Lengend Snippet: Figure 9. 5342191 acts post-entry to inhibit influenza replication. A549 cells were infected at an MOI of 2 with PR8 for 1 h, and then media were replaced. Media were harvested at various times post-infection and (A) the level of viral RNA in media was determined by RTqPCR; (B,C) cells were harvested and (B) protein lysates were used for western blot to detect PR8 NP or NS1 proteins, or (C) total RNA extracted and the intracellular abundance of viral RNAs (M1, NP, and NS1) was determined by RTqPCR. (D) To assess whether 5342191 could inhibit virus replication at times after infection, cells were infected with PR8, MOI 2, for 1 h, then 0.104 µM of 5342191 was added 1, 6, 9, or 12 hpi. At 24 hpi, media was harvested, and levels of viral RNA were determined by RTqPCR. ****, p < 0.0001.

Article Snippet: Total RNA was extracted from cells using the BioRad Aurum Total RNA Lysis Kit (BioRad) as per the manufacturer’s instructions.

Techniques: Infection, Western Blot, Virus