In a recent study posted to the bioRxiv* preprint server, a team of researchers analyzed the phenotypes of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), the molecular responses to infections from these VOCs, and the evolving virus-host interactions using a comparative genomic and proteomic approach.
Since the onset of the coronavirus disease 2019 (COVID-19) pandemic, multiple SARS-CoV-2 VOCs have emerged, starting with the Alpha (B.1.1.7) lineage, progressing through Beta, Delta, and Gamma lineages, to the most recent Omicron variant. While the Beta and Gamma variants were not very widespread, the Alpha, Delta, and Omicron lineages have shown high transmissibility. Phylogenetic analyses have shown that the SARS-CoV-2 VOCs have evolved independently from the wave one (W1) early-lineage viruses.
While the SARS-CoV-2 spike protein is the most mutated region in the VOCs, mutations in other regions, such as the nucleocapsid, envelope, and membrane proteins, have been known to influence immune responses. However, the molecular mechanisms of the host responses to non-synonymous mutations in regions outside the spike protein sequence have not been comprehensively examined.
About the study
In the present study, the team examined the changes in viral replication and immune responses due to mutations in the VOCs by comparing SARS-CoV-2 VOC-infected air-liquid interface cultured primary human airway epithelial (HAE) cells. The study included the Alpha, Beta, Delta, Gamma, and Omicron VOCs and two W1 isolates. Infected lung epithelial Calu-3 cells were used for messenger ribonucleic acid (mRNA) sequencing (RNA-seq), phosphoproteomics, and abundance proteomics using mass spectrometry.
Viral replication was determined from quantified single-stranded RNA levels and cumulative intensities of non-structural proteins. Additionally, the effect of structural protein alterations on viral production and infectivity was tested using a SARS-CoV-2 virus-like particle containing a luciferase encoding reporter genome and nucleocapsid, membrane, spike, and envelope structural proteins. The study also compared the phosphorylation of viral proteins across VOCs to examine the regulation of viral protein activity.
The team performed an integrative structural and sequence analysis of the mutations cataloged in the Global Initiative on Sharing Avian Influenza Data (GISAID) database to understand the adaptive VOC mutations. All the proteins in the VOCs that were mutated in comparison to the original Wuhan strain were examined using affinity purification mass spectrometry to assess the effect of mutations in protein-coding genes on the protein-protein interactions between the virus and the host.
Furthermore, an integrative computational approach was used to explore the impact of SARS-CoV-2 VOC infections on the host’s cellular biology to understand diverging host responses to viral evolution and identify targets for pan-variant antiviral therapies. Average host responses (AHR) were used to compare VOCs.
The RNA-seq data was used to plot pro-inflammatory gene induction signatures against interferon-stimulated genes to compare immunomodulation across VOCs. Levels of viral protein and RNA were analyzed to explore the association between the expression of viral proteins and the regulation of inflammatory pathways by the VOCs.
The results indicated that mutations in regions other than the SARS-CoV-2 spike protein sequences could significantly impact the interactions between the virus and the host. The study found VOC-specific differences in the expression levels of viral proteins and RNA, such as nucleocapsids and some Orf regions.
Investigation of host-virus protein-protein interactions revealed that the regulation of biological pathways in the hosts was conserved and divergent for infections with VOCs. Regulation of translational pathways in the host was highly conserved. Still, host inflammatory response modulation varied greatly for different VOCs, with the Alpha and Beta variants antagonizing the interferon-stimulated genes but not the Omicron variant.
The regulation of cellular pathways associated with cytokine and innate immune responses divergent considerably across VOCs, and the authors believe that this ability to modify the host’s innate immunity could explain the sequential replacement of dominant SARS-CoV-2 variants.
The spike protein mutations in the Omicron variants reduced the dependence of viral entry on the serine proteases, but the Omicron variants were not as effective as the Alpha and Delta variants at suppressing the host’s innate immunity. However, the more recent Omicron variants, such as BA.5 showed increased expression of antagonist genes such as Orf6, indicating that after having achieved immune escape, the emergent Omicron variants are evolving under the next strongest selective pressure of suppressing the host’s innate immune responses.
Overall, the results suggested that mutations outside of the spike protein regions significantly influence viral production and transmission. Furthermore, the SARS-CoV-2 variants have divergent protein-protein interactions with the host and different levels of protein expression, which allow them to evade the host’s innate immune responses. Additionally, the study revealed similarly regulated mRNA processing and translational pathways across VOCs that can be targeted for developing pan-variant anti-viral therapies.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.