In a recent study posted in the bioRxiv* preprint server, researchers explored the impact of a loss-of-function mutation in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants on the expression of the spike protein.
Study: Loss-of-function mutation in Omicron variants reduces spike protein expression and attenuates SARS-CoV-2 infection. Image Credit: JuanGaertner/Shutterstock.com
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Background
Several SARS-CoV-2 variants of concern (VOCs) with diverse spike protein mutations have emerged since the coronavirus disease 2019 (COVID-19) pandemic began.
The spike (S) protein on virions comprises three subunits, known as a trimer. These subunits are called S1 and S2. Omicron studies have primarily concentrated on the receptor-binding domain (RBD) and its effect on infection- or vaccine-induced immunity due to the numerous mutations present in the spike protein.
Mutations near the S1/S2 cleavage site and furin cleavage site (FCS) are known to contribute to the evolution of SARS-CoV-2, but their impact on Omicron has not been extensively researched.
About the study
In the present study, researchers assessed how the SARS-CoV-2 Omicron C-terminus of the S1 subunit (CTS1) mutations impact SARS-CoV-2 pathogenesis and infection.
A mutant SARS-CoV-2 with N679K, P681H, and H655Y mutations was created in the WA1 backbone (YKH). Spike processing in purified virions from wild-type (WT) and YKH infection was evaluated. The team hypothesized that N679K could affect SARS-CoV-2 infection.
A SARS-CoV-2 mutant with only an N679K mutation in the WA1 backbone was creat to assess this. The study involved infecting three- to four-week-old golden Syrian hamsters with N679K and noting weight loss and disease progression for seven days.
The team determined the cause behind the loss of function seen in the N679K mutant. The effects of N679K on proteolytic spike processing were evaluated, considering its location next to the FCS. Spike processing was examined by blotting purified virions from N679K, WT, and Omicron variant BA.1.
Results
The YKH mutant produced smaller plaques than the WT strain. The YKH mutant displayed no reduction in stock titers or replication kinetics within Vero E6 cells compared to the SARS-CoV-2 WT strain.
The endpoint titers associated with YKH were higher at 48 hours post-infection (hpi) in Calu-3 2B4 cells in comparison to WT, although replication was decreased at 24 hpi. The study indicated that the three mutations might influence the Omicron variant’s infection dynamics, potentially providing some benefits.
The YKH spike protein underwent more processing than the WT spike protein, similar to Omicron and Delta. At 24 hpi, the YKH spike had an S1/S2 cleavage ratio to a full-length spike ratio of approximately 2.4:1, while the WT had similar levels of S1/S2 product as full length.
The YKH mutant, which contains the H655Y, N679K, and P681H mutations, led to higher viral endpoint yields within human respiratory cells and played a role in the improved spike processing of Omicron.
The N679K plaque sizes were smaller at two and three days post-infection (dpi) compared to WT, and stock titers were negligibly lower according to the initial characterization. The observed variations in plaque size and stock titers align with previous findings on most Omicron strains.
The N679K mutant showed reduced replication in Calu-3 2B4 and Vero E6 cells at 24 hpi, in contrast to the minimal differences observed in YKH replication kinetics. The study found that N679K viral titer recuperated by 48 hpi; the mutation appears to be a loss-of-function for replication in both cell lines.
N679K-infected hamsters showed less body weight loss in comparison to WT-infected hamsters. The study found that despite significant weight loss, the N679K viral titers detected in the lung samples were similar to the wild type at two and four days dpi.
At two dpi, the N679K mutant virus showed similar viral titers to the wild-type virus in nasal washes. However, at four dpi, the mutant virus exhibited decreased replication compared to the wild-type virus.
The study suggests that the N679K mutation includes a loss-of-function phenotype both in vitro and in vivo. The researchers hypothesize that the effect of the P681H and H655Y mutations may mitigate this loss of function.
N679K exhibited a 66% lower S/N ratio than WT, indicating a greater decline in spike protein relative to the decrease in purified virions. The study found that Omicron’s S/N ratio decreased similarly, suggesting that the phenotype remains consistent despite all Omicron mutations. The N679K mutation leads to lower levels of the Omicron spike protein than the WT.
Conclusion
The study findings showed that the Omicron N679K mutation leads to consistent loss of function in subvariants. The N679K mutation reduces the virus’s strength both in vitro and in vivo by enhancing spike degradation.
The amplifying effects of other Omicron mutations, such as H655Y and P681H on spike processing and infection may offset the N679K mutation’s weakening effect.
The reduced spike protein expression caused by N679K could impact immunity resulting from vaccines and infection. Further research is needed to clarify the significant impact of the Omicron CTS1 mutations on SARS-CoV-2 infection.
*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.
