The current coronavirus disease 2019 (COVID-19) pandemic has resulted in more than six million deaths out of an estimated 500 million cases – although many tens of millions have undoubtedly remained undocumented and undiagnosed. The causative agent is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged in December 2019 in Wuhan before spreading to all parts of the world.
The virus has mutated into several variants, some with greater pathogenicity and/or immune evasion characteristics. A new preprint explores the contribution of these mutations to viral pathogenicity and transmission.
SARS-CoV-2 has a ribonucleic acid (RNA) genome composed of open reading frames (ORFs) that code for replicase proteins, structural proteins, and accessory proteins. The replicase gene is located at the 5′ end by ORF1a/b, followed by the four structural genes for the spike, envelope, membrane and nucleocapsid components of the virus.
Between these genes are the accessory proteins that are unique to each family of viruses. These contribute, in one way or another, to the pathogenesis of COVID-19. For example, the interferon signaling pathway triggers antiviral and/or inflammatory responses.
However, SARS-CoV-2 ORF3b disrupts this pathway, while ORF7a blocks the interferon-stimulated gene (ISG) BST2. Similarly, the translocation of the STAT1 protein in the nucleus, driven by the expression of interferon, which modulates the expression of ISG, is countered by the viral ORF6.
Virus variants have mainly been studied in terms of spike mutations since this protein mediates viral attachment and entry into target host cells. The spike interacts with the host cell via the angiotensin converting enzyme 2 (ACE2) receptor and is the immunodominant antigen. Mutations in this gene have been associated in some cases with immune evasion.
In the current preprint, published on the bioRxiv* server, researchers investigated the role played by accessory proteins using synthetic genomic assembly technology. They focused on deletions in ORF3a/b, ORF6, 7a/7b and 8 in the ancestral strain of the virus, first examining the effect on replicative fitness in vitro. Subsequently, they examined how these affected the pathogenesis of SARS-CoV-2 infection in a mouse model.
They also constructed recombinants spike protein variants on a WA-1 backbone. These variants were identical to spike variants Alpha, Beta, and Gamma and compared to the ancestral spike protein for replicative fitness and pathogenesis in mice.
What did the study show?
Scientists found that deletions of ORF3a and ORF3b accessory genes (WA-1ΔORF3a/b) reduced the replicative ability of the virus in mice. Mice infected with the deletion virus did not lose as much weight as controls infected with the ancestral strain (WA-1), and the viral load in lung tissue on days 2 and 4 was much lower. This supports previous research on this deletion.
However, contrary to previous reports of attenuated clinical effects with ORF7a, ORF7b, and ORF8 deletions in mice, no effect on viral load occurred in current experiments. This could be due to differences in infectious dose or different strains of mice.
WA-1ΔORF3a/ba also leads to lower expression of inflammatory cytokines and chemokines than with WA-1 infection, likely due to lower replication levels. Similar downstream effects were observed, as expected, on neutrophil recruitment. Upregulation of the I14 and I15 genes drives a type 2 (Th2) T helper cell response, seen in severe forms of COVID-19.
This is not consistent with the less severe clinical phenotype of WA-1ΔORF3a/b. A possible explanation for the upregulation, rather than the expected downregulation, of these cytokines could be their association with tissue repair, which occurs earlier in these mice due to faster virus clearance.
Adipoq is another upregulated gene in these mice and is associated with adiponectin, a hormone that sensitizes tissues to insulin activity. Lower levels of adiponectin are associated with severe respiratory failure in COVID-19.
Spike variants introduced into a WA-1 backbone were found to have no significant change in their replication, but viral load, as measured by viral titer in the supernatant at 72 hours, was lower with the variant. advanced Gamma compared to the Gamma A variant.
No differences were found in weight loss, viral load, or brain titer for the other two spike variants on WA-1, compared to true variants, in K18-hACE2 mice. However, with BALB/c mice, the Beta spike-WA1 strain produced an attenuated phenotype compared to the Beta variant, while with the Alpha spike-WA1 strain, lung viral titer increased at day 2 without any difference. of weight loss.
In both mouse strains, the greatest difference was observed with the Gamma spike-WA1 variant compared to the Gamma variant, with the former exhibiting a more severe phenotype in terms of weight loss and lung titers than the Gamma variant. In K18-hACE2 mice, there was a trend for higher brain viral titers and higher RNA concentrations, below significance.
Lung cytokines and chemokines also showed differences on days 2 and 4, again more significantly in the Gamma spike-WA1 strain compared to the Gamma strain. Neutrophil recruitment genes were the most affected, with CXCL5 showing the greatest increase in expression. Interestingly, this gene is a major neutrophil attractant in COVID-19 and is implicated as a cause of inflammation.
Genes like thpok and I15 were down-regulated. Since these lead to CD4+ T cell differentiation, this finding may indicate that the WA-1 spikeless genes inhibited this pathway and that this inhibition is lost with the full-fledged Gamma variant.
The latter also shows a loss of interferon antagonism, mediated by spikeless genes, since Gamma-infected mice had higher levels of interferon-gamma in the lungs than Gamma-spike-WA1 mice. This goes against the higher lung titers observed in the latter. However, this difference resolved by day 4.
What are the implications?
“This work demonstrates that ORF3a/b play an important role in the pathogenesis and host responses to SARS-CoV-2.” Thus, mutations in the accessory proteins of the viral variants, and in particular ORF3a, seem to contribute to the pathogenesis of COVID-19 with the variants. Both the beta and gamma variants had ORF3a mutations, and these continue to be found in newer variants, indicating their importance for replicative fitness and transmissibility.
“We interpret these data to suggest that mutations outside the peak may underlie critical phenotypes of SARS-CoV-2 infection and disease..” In other words, spike mutations often increase ACE2 binding affinity, increasing viral entry into cells and immune evasion. Conversely, accessory mutations reduce clinical severity, allowing for prolonged replication and increased viral transmission.
Together, this can ensure that the virus continues to adapt with better replicative fitness, transmissibility and pathogenicity.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.
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