The Omicron spike N679K mutation functions as a loss-of-function mutation that attenuates SARS-CoV-2 in vitro and in vivo

In a recent study published in Bio Rxiv*In a preprint server, researchers discovered that loss-of-function mutations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron variant spike protein.

study: Loss-of-function mutations in Omicron variants reduce spike protein expression and attenuate SARS-CoV-2 infection. Image Credit: JuanGaertner/Shutterstock.com

*Important Notices: Bio Rxiv We publish a non-peer-reviewed, preliminary scientific report and should not be taken as conclusive, to guide clinical practice/health-related actions, or to be treated as established information.

Background

Since the beginning of the 2019 coronavirus disease (COVID-19) pandemic, several concerned SARS-CoV-2 variants (VOCs) have emerged with different spike protein mutations.

The spike (S) protein on virions is composed of three subunits known as trimers. These subunits are called S1 and S2. Omicron’s research is primarily focused on the receptor binding domain (RBD) and its impact on infection or vaccine-induced immunity due to the numerous mutations present in the spike protein.

Mutations near the S1/S2 and furin cleavage sites (FCS) are known to contribute to the evolution of SARS-CoV-2, but their effects on omicron have not been extensively studied.

About research

In this study, researchers evaluated how the Omicron C-terminus of the SARS-CoV-2 S1 subunit (CTS1) mutation affects SARS-CoV-2 pathogenesis and transmission.

A mutant SARS-CoV-2 with N679K, P681H, and H655Y mutations was created in the WA1 backbone (YKH). We evaluated spiking in purified virions from wild-type (WT) and YKH infections. The team hypothesized that N679K could influence SARS-CoV-2 infection.

To assess this, a SARS-CoV-2 mutant with only the N679K mutation in the WA1 backbone was created. In this study, a 3- to 4-week-old golden Syrian hamster was infected with her N679K and observed for weight loss and disease progression over a 7-day period.

The team identified the cause behind the loss of function seen in the N679K mutant. Given that N679K is located next to his FCS, we assessed the effect of his N679K on proteolytic spike processing. Spiking was examined by blotting purified virions from N679K, WT, and Omicron variant BA.1.

result

The YKH mutant produced smaller plaques than the WT strain. The YKH mutant showed no reduced stock titer or replication rate in Vero E6 cells compared to the SARS-CoV-2 WT strain.

YKH-associated endpoint titers were higher at 48 hours post-infection (hpi) of Calu-3 2B4 cells compared to WT, but replication was reduced at 24 hpi. This study shows that the three mutations affect the infection dynamics of her Omicron subspecies and may provide some benefit.

The YKH spike protein, like Omicron and Delta, received more processing than the WT spike protein. At 24 hpi, the ratio of S1/S2 cleavage ratio to full-length spike ratio in YKH spikes was approximately 2.4:1, whereas WT had similar levels of S1/S2 products as full-length.

YKH mutants containing H655Y, N679K, and P681H mutations conferred higher viral endpoint yields in human respiratory cells and played a role in improving Omicron spiking.

N679K plaque sizes were smaller compared to WT at 2 and 3 days post-infection (dpi), and initial characterization indicated negligible stock titers. The observed variability in plaque size and stock titer is consistent with previous findings for 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. In this study, we found that N679K virus titers recovered by 48 hpi. This mutation appears to be a replication loss of function in both cell lines.

Hamsters infected with N679K lost less weight than hamsters infected with WT. This study found that her N679K virus titers detected in lung samples were similar to wild-type he at 2 and 4 days dpi despite significant weight loss.

At 2 dpi, the N679K mutant virus showed virus titers similar to wild-type virus in nasal washes. However, at 4 dpi, mutant virus showed reduced replication compared to wild-type virus.

This study suggests that the N679K mutation contains a loss-of-function phenotype both in vitro and in vivo. The researchers hypothesize that the effects of the P681H and H655Y mutations may mitigate this loss of function.

N679K exhibited a 66% lower signal-to-noise ratio than WT, indicating a greater reduction in spike protein compared to that in purified virions. In this study, we found that his S/N ratio in Omicron was similarly reduced, suggesting that the phenotype was consistent despite all mutations in Omicron. The N679K mutation results in lower levels of Omicron spike protein than WT.

Conclusion

The results of this study showed that the Omicron N679K mutation leads to consistent loss-of-function of subvariants. The N679K mutation reduces viral potency both in vitro and in vivo by promoting spike disassembly.

The amplifying effects of other Omicron mutations such as H655Y and P681H on spiking and infection may offset the weakening effect of the N679K mutation.

Downregulation of spike protein by N679K may affect immunity from vaccines and infections. Further studies are needed to clarify the critical impact of Omicron CTS1 mutations on SARS-CoV-2 infection.

*Important Notices: Bio Rxiv We publish a non-peer-reviewed, preliminary scientific report and should not be taken as conclusive, to guide clinical practice/health-related actions, or to be treated as established information.