SARS-CoV-2 and human cold coronavirus OC43 inhibit stress granule formation through Nsp15 protein

In a recent study published in the journal PLOS Pathogens, researchers investigated the mechanisms through which the human common cold Betacoronavirus OC43 (HCoV-OC43) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibit the formation of stress granules.

Background

Stress granules are cytoplasmic aggregates containing viral ribonucleic acid (RNA) and proteins that are involved in triggering the immune system to detect and suppress viral infections. Some viruses have evolved mechanisms to prevent stress granule formation.

In the last decade, zoonotic betacoronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2 have been responsible for a staggering number of infections and deaths. Some coronavirus proteins, such as the SARS-CoV-2 nucleocapsid protein, have shown the ability to inhibit stress granule formation during ectopic overexpression.

Understanding how viruses like SARS-CoV-2 inhibit the formation of stress granules could provide therapeutic targets to improve cellular resistance to infections.

About the study

In the present study, the researchers infected human embryonic kidney (HEK) 293A cells with HCoV-OC43. They used immunofluorescence staining for T-cell internal antigen-related protein (TIAR) to detect the presence of stress granules in the infected cells at multiple time points post-infection.

To analyze the active inhibition of stress granule formation by HCoV-OC43, they also used sodium arsenite to induce stress granule formation and eukaryotic translation initiation factor 2α (eIF2α) phosphorylation in mock and virus-infected HEK 293A cells. The series of analyses were repeated in human colon adenocarcinoma (HCT-8) cells to determine if the inhibition of stress granule formation by HCoV-OC43 was specific for HEK 293A cells. The ability of HCoV-OC43 to inhibit the eIF2α phosphorylation-independent stress granule formation was also examined using Silvesterol, which initiates stress granule formation without inducing eIF2α phosphorylation.

Additionally, the analyses were repeated using immunofluorescence staining with other stress granule markers such as Ras-guanosine triphosphate (GTP)ase-activating protein SH3-domain-binding proteins 1 and 2 (G3BP1 and G3BP2), and T-cell internal antigen 1 (TIA-1), as well as eukaryotic translation initiation factors 4 subunit G and 3 subunit B (eIF4G and eIF3B), to determine whether the inhibition of stress granule formation by HCoV-OC43 was limited to stress granules containing TIAR.

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To determine the inhibition of stress granule formation by SARS-CoV-2, HEK 293A cells expressing angiotensin-converting enzyme-2 (ACE-2) were infected with SARS-CoV-2. TIAR levels in SARS-CoV-2 infected cells and mock-infected cells were compared, and reverse transcription polymerase chain reaction (RT-PCR) was used to determine the levels of G3BP1 and TIAR messenger RNA in the infected cells.

Furthermore, enhanced green fluorescent protein (EGFP)-tagged nucleocapsid protein and nonstructural protein 15 (Nsp15) of HCoV-OC43 and SARS-CoV-2 were overexpressed in HEK 293A cells to determine their role in the inhibition of stress granule formation. The role of Nsp15 in the depletion of G3BP1 mRNA and protein was also examined.

Results

The results reported no stress granule formation in cells infected with HCoV-OC43 and SARS-CoV-2. Both coronaviruses also inhibited eIF2α phosphorylation and stress granule formation from exogenous stress. Nucleocapsid protein and Nsp15 from HCoV-OC43 and SARS-CoV-2 inhibited stress granule formation when overexpressed ectopically. Additionally, the Nsp15 protein from HCoV-OC43 and SARS-CoV-2 also inhibited the eIF2α phosphorylation induced by sodium arsenite.

Furthermore, in cells infected with SARS-CoV-2, the levels of G3BP1 protein decreased sharply, and the nuclear accumulation of TIAR was observed. When cells overexpressing G3BP1 were infected with HCoV-OC43 and compared to HCoV-OC43-infected cells without G3BP1 overexpression, significantly lower levels of viral replication were observed in the cells with overexpressed G3BP1, indicating that G3BP1 played an important antiviral role.

However, although cells overexpressing G3BP1 exhibited significantly high levels of stress granule formation, a considerable number of cells infected with HCoV-OC43 also showed inhibition of stress granule formation, indicating the presence of multiple translational arrest interference mechanisms by HCoV-OC43 and SARS-CoV-2 that inhibit stress granule formation.

Conclusions

To summarize, the study investigated the inhibition of stress granule formation by two coronaviruses — SARS-CoV-2 and the human cold coronavirus OC43. The researchers also examined the role of nucleocapsid protein and Nsp15 in inhibiting stress granule formation. Additionally, the antiviral role of the G3BP1 protein was also investigated.

Overall, the results indicated that nucleocapsid protein and Nsp15 of the two viruses inhibit stress granule formation and enable viral replication through separate but complementary processes. Based on the results, the authors believe that the inactivation of stress granule formation is essential for viral replication and infection, and the mechanisms through which these viruses inhibit stress granule formation could be potential therapeutic targets.

Written by

Dr. Chinta Sidharthan

Chinta Sidharthan is a writer based in Bangalore, India. Her academic background is in evolutionary biology and genetics, and she has extensive experience in scientific research, teaching, science writing, and herpetology. Chinta holds a Ph.D. in evolutionary biology from the Indian Institute of Science and is passionate about science education, writing, animals, wildlife, and conservation. For her doctoral research, she explored the origins and diversification of blindsnakes in India, as a part of which she did extensive fieldwork in the jungles of southern India. She has received the Canadian Governor General’s bronze medal and Bangalore University gold medal for academic excellence and published her research in high-impact journals.