Omicron spike N679K mutation acts as a loss-of-function mutation attenuating SARS-CoV-2 in vitro and in vivo

In a recent study published in the bioRxiv* preprint server, researchers explored the impact of a loss-of-function mutation in Omicron variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 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*Important Notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.

Background

Several concerning SARS-CoV-2 (COV) variants with various spike protein mutations have emerged since the start of the coronavirus disease 2019 (COVID-19) pandemic.

The spike (S) protein on virions comprises three subunits, called trimers. These subunits are called S1 and S2. Omicron’s studies have primarily focused on the receptor binding domain (RBD) and its effect on infection or vaccine-induced immunity due to the many mutations present in the spike protein.

Mutations near the S1/S2 cleavage site and the furin cleavage site (FCS) are known to contribute to the evolution of SARS-CoV-2, but their impact on Omicron has not been investigated. extensive research.

About the study

In the present study, researchers assessed how the SARS-CoV-2 Omicron C-terminus of the S1 subunit (CTS1) mutations impact the pathogenesis and infection of SARS-CoV-2 .

A SARS-CoV-2 mutant with N679K, P681H and H655Y mutations was created in the WA1 (YKH) backbone. 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 created to assess this. The study involved infecting three to four week old golden Syrian hamsters with N679K and recording weight loss and disease progression for seven days.

The team determined the cause of the loss of function observed in the N679K mutant. The effects of N679K on proteolytic spike processing were assessed, given its location next to FCS. Spike processing was examined by drinking virions purified 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 in Vero E6 cells compared to the SARS-CoV-2 WT strain.

YKH-associated endpoint titers were higher at 48 hours post-infection (hpi) in Calu-3 2B4 cells compared to WT, although replication decreased at 24 hpi. The study indicated that all three mutations could influence the infection dynamics of the Omicron variant, potentially offering some advantages.

The YKH spike protein has undergone more processing than the WT spike protein, similar to Omicron and Delta. At 24 hpi, the YKH peak had a full length peak S1/S2 cleavage ratio of approximately 2.4:1, while the WT had similar levels of full length S1/S2 product. .

The YKH mutant, which contains the H655Y, N679K, and P681H mutations, led to higher viral yields in human respiratory cells and played a role in improving Omicron spike processing.

N679K plaque sizes were smaller at two and three days post infection (dpi) compared to WT, and stock titers were slightly lower according to initial characterization. The observed variations in plaque size and stock titers are consistent with previous results 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 viral titer N679K recovered 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 compared to WT-infected hamsters. The study found that despite significant weight loss, N679K viral titers detected in lung samples were similar to wild-type at two and four days dpi.

At two days per inch, the N679K mutant virus showed viral titers similar to wild-type virus in nasal washes. However, at four days per inch, the mutant virus exhibited reduced 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 could mitigate this loss of function.

N679K exhibited a 66% lower S/N ratio than WT, indicating a greater decrease in spike protein compared to the decrease in purified virions. The study found that the S/N ratio of Omicron similarly decreased, suggesting that the phenotype remains consistent despite all Omicron mutations. The N679K mutation leads to lower levels of the Omicron spike protein than WT.

Conclusion

The results of the study showed that the Omicron N679K mutation causes consistent loss of function in the subvariants. The N679K mutation reduces virus strength in vitro and in vivo by enhancing spike breakdown.

The enhancing effects of other Omicron mutations, such as H655Y and P681H on spike processing and infection, may offset the weakening effect of the N679K mutation.

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 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 considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.