Study explores SARS-CoV-2 RNA genome's functional interactions with host cell

By Colin Lightfoot, M.Sc. Infection and ImmunityReviewed by Aimee MolineuxMay 3 2022

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative virus of the current coronavirus disease 2019 (COVID-19) pandemic. Numerous vaccines in Phase III clinical trials were given emergency authorization one year after SARS-CoV-2 was discovered. Most of these vaccinations are intended to induce immune responses to the viral spike protein.

Several SARS-CoV-2 variants of concern (VOCs) have emerged, containing spike protein mutations that increase transmissibility and impair susceptibility to natural or vaccine-induced neutralizing immunity.

SARS-CoV-2, like all viruses, relies extensively on cellular proteins to complete its pathogenic life cycle. This host reliance could be leveraged to develop innovative host-centered therapies to address SARS-CoV-2 infection. Affinity purification combined with mass spectrometry (MS) enabled the detection of SARS-CoV-2 virus-host protein-protein interactions (PPIs), whereas CRISPR-Cas9 phenotypic screening revealed critical host genes and cellular pathways for the SARS-CoV-2 life cycle.

Cholesterol homeostasis, the phosphatidylinositol 3-kinase (PI3K) complex, and the sigma-1 and -2 receptors have all been identified as possible biological targets for drug repurposing.

In a recent study published on the Cell Reports server, a worldwide map of the SARS-CoV-2 genome-associated host proteins was developed by a team of researchers using the identification of RNA-binding protein by MS (ChIRP-MS) technique. The authors discovered a collection of SARS-CoV-2 host dependence variables that affect infection and could be targeted for antiviral treatments by integrating these proteomics data with gene silencing experiments.

The study

The authors used ChIRP-MS to characterize the host components that interacted with the SARS-CoV-2 genome throughout infection. A main clinical SARS-CoV-2 220/95 strain (EPI ISL 469284) was challenged to 293T cells stably expressing the human angiotensin-converting enzyme 2 (ACE2) receptor (293T-ACE2). To retain ribonucleoprotein (RNP) complexes, SARS-CoV-2-infected and -uninfected cells were treated with formaldehyde to crosslink RNA with RNA-binding proteins (RBPs).

The authors developed 129 biotinylated antisense probes corresponding to the full-length viral RNA to precisely capture SARS-CoV-2 RNA, and then utilized streptavidin beads to draw down the RNPs. SDS-PAGE was used to separate co-immunoprecipitated proteins, which were then stained with silver and evaluated by MS. The test replicates are remarkably consistent, according to correlation analyses. The SARS-CoV-2 N protein was the most prevalent viral protein. In the analyses, the replicase polyprotein pp1ab, as well as structural proteins and accessory open reading frame (ORF)9b proteins, were significantly enriched.

The authors compared the MS data from the 5 replicates using the SAINTexpress scoring method and produced a high confidence interactome. They discovered 107 human proteins that were consistently linked to the SARS-CoV-2 RNA. An extended SARS-CoV-2 interactome was discovered, along with 39 new proteins. The authors next examined the high-confidence interactome to a SARS-CoV-2 interactome, using SARS-CoV-2-infected cells that had not been cross-linked as a negative control. Seventy-eight common enriched factors were discovered, as well as a high-confidence core interactome. The expression levels of selected hits were tracked before and after infection to rule out biases caused by infection-induced host proteome alterations.

The Gene Ontology (GO) term "RNA binding" was annotated on 88% of the proteins in the extended interactome, indicating a direct interaction with the viral genome. The authors used native viral RNA immunoprecipitation (vRIP) assays on a subset of 5 components to test this hypothesis. The authors found that there was a considerable enrichment of vRNA for the 5 proteins tested as compared to the negative controls. These findings were also validated in A549-ACE2 cells that were stably expressing the various proteins. Overall, these findings show that the ChIRP method can discover proteins that interact with vRNA.

Well over 150 statistically enriched GO terms were discovered in functional assessments of the vRNA-interacting proteins, which clustered into 23 functional groupings that were shared by prior study findings. The researchers discovered host molecules involved in RNA metabolism, translation, RNA stabilization, RNP complex building, viral process, and innate immune response among them. The considerable enrichment of the RNA splicing GO term was surprising given its primarily nucleus location and involvement in mRNA maturation. According to the authors, SARS-CoV-2 RNA may sequester host RBPs involved in splicing, preventing cellular mRNA nuclear export, and favoring viral RNA translation in the cytoplasm. This concept is supported by recent research that shows SARS-CoV-2 NPS16 alters mRNA splicing to counteract innate immunity at the post-transcriptional stage.

Implications

These findings paint a picture of the SARS-CoV-2 RNA genome's functional interactions with the host cell during infection. SARS-CoV-2 engages with multiple cellular RBPs to aid viral replication, according to the authors, and some of these RBPs could be attractive targets for antiviral therapy.

This information may be useful in future research into the process of SARS-CoV-2 replication and the development of antiviral medications to combat the introduction of new human coronaviruses or variations of concern.