We sequenced complete SARS-CoV-2 genomes from case-patients A and B (Appendix) and confirmed that these genomes were VOC Omicron (Pango lineage B.1.1.529) (Figure, panel A). Viral sequences from these 2 case-patients differed by only 1 nt. Viral sequence from case-patient A was highly similar to those of the first few reported Omicron cases identified in South Africa and Botswana
(Appendix Table 1). Because many countries have just reported detection of this VOC(https://www.gisai/hcov19-variantsExternal Link), the actual genetic diversity of this virus lineage requires further investigations.
The long branch of Omicron clade in the phylogenetic tree is attributed to the large number of mutations (Figure, panel A).
Nonsynonymous mutations were identified in the spike (S)‒encoding (n = 35) and other viral protein‒encoding (n = 22) regions (Figure, panel B). Among the nonsynonymous mutations in the S protein, 43% (n = 15) were also identified in other VOCs/variants of interest, and 31% (n = 11) were found only in VOCs (Alpha, n = 6; Beta, n = 4; Gamma, n = 5; Delta, n = 4). Some of the point mutations and deletions found in other regions are not novel and can also be found in other variants at different frequencies (Appendix Table 2). Among these non-S mutations, NSP4-T492I, NSP6-S106del, NSP6-G107del, NSP12-P323L, N-P13L, N-R203K, and N-G204R are commonly found in SARS-CoV-2 variants.
The World Health Organization classified the Omicron variant as a VOC only 2 days after being notified by the South African scientists, mainly based on the unusually large number of amino acid mutations in the spike protein and the rapid increase in South Africa. It is worrisome that the Omicron variant may transmit more easily and may replace the Delta variant which has dominated the World up to November 2021. Here, we found that the replication and fusion activity of the Omicron variant is much less dependent on TMPRSS2, while the replication and fusion of the Delta variant is greatly enhanced in VeroE6/TMPRSS2 cells.
Conformational change of the spike protein is essential in mediating the viral and cellular membrane fusion [19], which in turn allows the viral genome to reach the cytoplasm via a fusion pore. After cleavage of the S1/S2 junction by furin, the S2’ site can be cleaved by either the cell surface TMPRSS2 or the endosomal cathepsin B/L. A single-cell sequencing study showed that TMPRSS2 is highly expressed in alveolar type I and type II cells of the lung [20]. The enhancement of viral replication of the Delta variant by TMPRSS2 corroborates with previous animal studies showing more extensive infection of alveolar pneumocytes for the Delta variant [21]. In the current study, we showed that in contrast to the Delta variant, Omicron variant was not effective in using TMPRSS2 for viral replication. Our results suggest that the Omicron variant may have poorer replication in the lungs when compared with the Delta variant. Indeed, there are preliminary epidemiological studies showing that the Omicron variant may have the milder disease [22].
Although there is a marked difference in the dependence of TMPRSS2 on viral replication, there is no difference in the S2’ cleavage site between the Omicron and Delta variants. The difference in TMPRSS2 dependence may be related to the furin cleavage site, in which the Omicron variant is P681H and the Delta variant is P681R. Using a pseudovirus system, Peacock et al. have demonstrated that the TMPRSS2-mediated entry is much greater in pseudovirus carrying the polybasic furin cleavage site at the S1/S2 junction than those with the polybasic cleavage site deletion [23]. We showed that the Omicron variant is much less fusogenic when compared with the Delta variant. In addition to the replication difference in TMPRSS2 dependence, the poorer fusion activity of the Omicron variant can be attributed to the difference in the S1/S2 junction furin cleavage site. Previous studies have demonstrated that the Delta variant exhibited higher fusion activity than the wild type virus or the Alpha variant that contains P681H mutation [21,24,25]. Since syncytia formation is found in postmortem lung specimens of deceased COVID-19 patients, the fusion activity may be associated with disease severity [26].
In addition to being a protease that cleaves the spike protein for activation, TMPRSS2 also plays a role as an interferon antagonist. Overexpression of TMPRSS2 can reverse the restriction of SARS-CoV-2 replication by NCOA7, an interferon-stimulated gene [27]. It would be important to further assess how this innate immune role of TMPRSS2 will affect viral replication.
Although both VeroE6/TMPRSS2 and Calu3 exhibit high levels of TMRPSS2 expression, there was a difference in the replication of the Omicron variant between these two cell lines. While the Omicron variant could replicate to a level similar to Delta variant at 48 and 72 hpi in VeroE6/TMPRSS2 cells, the viral load of the Omicron variant remained significantly lower than the Delta variant at 48 and 72 hpi in Calu3 cells. This may be because the endocytosis is not an entry pathway for SARS-CoV-2 in Calu3 cells. Hoffmann et al. have shown that chloroquine, an endosomal acidification inhibitor, did not inhibit SARS-CoV-2 replication in Calu3 cells [28]. In Figure 3, we demonstrated that chloroquine could inhibit the replication of SARS-CoV-2, suggesting that the virus can enter VeroE6/TMPRSS2 cells by endosomal pathway.
At the time of writing, the Omicron variant has already affected over 90 countries worldwide. Our in vitro study provides a rapid phenotypic characterization of the Omicron variant which is important for the urgent public health risk assessment. It remains to be confirmed in animal models whether the difference in TMPRSS2 dependence will lead to the difference in disease severity or tissue tropism.
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