The effects of accessory protein mutations and other non-spike mutations on SARS-CoV-2 pathogenesis

The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to over six million deaths of approximately 500 million cases – though many tens of millions have unquestionably gone undocumented and undiagnosed. The causative agent is the 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 multiple variants, some with greater pathogenicity and/or immune evasion characteristics. A new preprint explores the contribution of these mutations to viral pathogenicity and transmission.

Introduction

The SARS-CoV-2 has a ribonucleic acid (RNA) genome composed of open reading frames (ORFs) that encode 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.

In between these genes come the accessory proteins that are unique to each virus family. These are, in one way or the other, contributory to the pathogenesis of COVID-19. For instance, the interferon signaling pathway triggers antiviral and/or inflammatory responses.

However, the SARS-CoV-2 ORF3b disrupts this pathway, while ORF7a blocks interferon-stimulated gene (ISG) BST2. Similarly, the translocation of the STAT1 protein into the nucleus, driven by interferon expression, which modulates ISG expression, is opposed by the viral ORF6.

Recommended:

---

• Biomarker in liquid biopsy for lung cancer appears more accurate in predicting immunotherapy response than tumor biopsy

---

The variants of the virus have mostly been studied in terms of the spike mutations since this protein mediates viral attachment and entry into the target host cells. The spike interacts with the host cell via the angiotensin-converting enzyme 2 (ACE2) receptor, and is the immunodominant antigen. Mutations of this gene have been associated in some cases with immune evasion.

In the current preprint, published on the bioRxiv* server, the researchers looked into the role played by the accessory proteins using synthetic genomics assembly technology. They focused on deletions in ORF3a/b, ORF6, 7a/7b, and 8 in the ancestral strain of the virus, examining the effect on replicative fitness in vitro first. Subsequently, they looked at how these affected the pathogenesis of SARS-CoV-2 infection in a mouse model.

They also built recombinant spike protein variants on a WA-1 backbone. These variants were identical to the Alpha, Beta, and Gamma spike variants and compared with the ancestral spike protein for replicative fitness and pathogenesis in mice.

What did the study show?

The scientists found that the accessory gene deletions of ORF3a and ORF3b (WA-1ΔORF3a/b) reduced the replicative fitness of the virus in mice. The deletion-virus-infected mice did not lose as much weight as the controls infected with the ancestral (WA-1) strain, and the viral load in the lung tissue on days 2 and 4 was much lower. This corroborates earlier research on this deletion.

However, unlike prior reports of attenuated clinical effects with deletions of ORF7a, ORF7b, and ORF8 in mice, no effect on viral load occurred in the current experiments. This could be due to differences in infectious dose or in different mouse strains.

The WA-1ΔORF3a/b also led to lower expression of inflammatory cytokines and chemokines than with WA-1 infection, probably due to the lower replication levels. Similar downstream effects were observed, as expected, on neutrophil recruitment. The upregulation of I14 and I15 genes drives a Type 2 T helper cell (Th2) response, seen in the severe forms of COVID-19.

This is not concordant 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 is happening earlier in these mice due to more rapid virus clearance.

Adipoq is another upregulated gene in these mice and is associated with adiponectin, a hormone that sensitizes tissues to insulin activity. Lower adiponectin levels are associated with severe respiratory failure in COVID-19.

The spike variants introduced into a WA-1 backbone were found to have no significant change in their replication, but the viral load, as measured by the viral titer in the supernatant at 72 hours, was less with the Gamma spike variant compared to the Gamma variant.

No differences were found in weight loss, virus load, or brain titer for the other two spike variants on WA-1, compared to the actual 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, the lung viral titer increased on day 2 without any difference in weight loss.

In both mouse strains, the greatest difference was seen with the Gamma spike-WA1 vs. Gamma variant, with the former showing a more severe phenotype in terms of weight loss and lung titers than the Gamma variant. In the K18-hACE2 mice, there was a trend towards higher viral titers in the brain, and higher RNA concentrations, falling short of significance.

Lung cytokines and chemokines also showed differences on days 2 and 4, again most 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 the cause of inflammation.

Genes like thpok and I15 were downregulated. Since these lead to CD4+ T cell differentiation, this finding may indicate that WA-1 non-spike genes inhibited this pathway and that this inhibition is lost with the full-fledged Gamma variant.

The latter also shows loss of interferon antagonism, mediated by non-spike genes, since the Gamma-infected mice had higher gamma-interferon levels in the lungs than Gamma-spike-WA1 mice. This runs counter to the observed higher lung titers in the latter. This difference settled by day 4, however.

What are the implications?

“This work demonstrates that ORF3a/b have substantial roles in pathogenesis and host responses to SARS-CoV-2.” Thus, mutations in the accessory proteins of the viral variants, and especially ORF3a, seem to contribute to the pathogenesis of COVID-19 with the variants. Both Beta and Gamma variants had ORF3a mutations, and these continue to be found in newer variants, indicating their importance to replicative fitness and transmissibility.

“We interpret this data to suggest that mutations outside of spike may be driving critical phenotypes of SARS-CoV-2 infection and disease.” In other words, the spike mutations often increase ACE2 binding affinity, enhancing viral entry into cells and immune evasion. Conversely, the accessory mutations reduce the clinical severity, allowing for prolonged replication and increased viral transmission.

Together, this may ensure that the virus continues to adapt with better replicative fitness, transmissibility, and pathogenicity.

*Important notice

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.

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.