Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was responsible for the COVID-19 pandemic, efficiently spreads cell-to-cell through mechanisms facilitated by its membrane glycoprotein spike. We established a dual split protein (DSP) assay based on the complementation of GFP and luciferase to quantify the fusogenic activity of the SARS-CoV-2 spike protein. We provide several lines of evidence that the spike protein of SARS-CoV-2, but not SARS-CoV-1, induced cell–cell fusion even in the absence of its receptor, angiotensin-converting enzyme 2 (ACE2). This poorly described ACE2-independent cell fusion activity of the spike protein was strictly dependent on the proteasomal cleavage of the spike by furin while TMPRSS2 was dispensable. Previous and current variants of concern (VOCs) differed significantly in their fusogenicity. The Delta spike was extremely potent compared to Alpha, Beta, Gamma and Kappa, while the Omicron spike was almost devoid of receptor-independent fusion activity. Nonetheless, for all analyzed variants, cell fusion was dependent on furin cleavage and could be pharmacologically inhibited with CMK. Mapping studies revealed that amino acids 652-1273 conferred the ACE2-independent fusion activity of the spike. Unexpectedly, residues proximal to the furin cleavage site were not of major relevance, whereas residue 655 critically regulated fusion. Finally, we found that the spike’s fusion activity in the absence of ACE2 could be inhibited by antibodies directed against its N-terminal domain (NTD) but not by antibodies targeting its receptor-binding domain (RBD). In conclusion, our BSL-1-compatible DSP assay allowed us to screen for inhibitors or antibodies that interfere with the spike’s fusogenic activity and may therefore contribute to both rational vaccine design and development of novel treatment options against SARS-CoV-2.

Human cytomegalovirus (HCMV) can cause severe clinical disease in immunocompromised individuals, such as allograft recipients and infants infected in utero. Neutralizing activity of antibodies, measured as the ability to prevent the entry of cell-free virus, has been correlated with the reduction in HCMV transmission and the severity of HCMV-associated disease. However, in vivo HCMV amplification may occur mainly via cell-to-cell spread. Thus, quantifying the inhibition of cell-to-cell transmission could be important in the evaluation of therapeutic antibodies and/or humoral responses to infection or immunization. Here, we established a quantitative plaque reduction assay, which allowed for the measurement of the capacity of antibodies to limit HCMV spread in vitro. Using an automated fluorescence spot reader, infection progression was assayed by the expansion of viral plaques during the course of infection with various GFP-expressing viruses. We found that in contrast to non-neutralizing monoclonal antibodies (mAbs), neutralizing mAbs against both glycoprotein B and H (gB and gH) could significantly inhibit viral plaque expansion of different HCMV strains and was equally efficient in fibroblasts as in epithelial cells. In contrast, an anti-pentamer mAb was active only in epithelial cells. Taken together, our data demonstrate that specific anti-HCMV mAbs can significantly limit cell-associated virus spread in vitro.

The human cytomegalovirus (HCMV) glycoprotein B (gB) is the viral fusogen required for entry into cells and for direct cell-to-cell spread of the virus. We have previously demonstrated that the exchange of the carboxy-terminal domain (CTD) of gB for the CTD of the structurally related fusion protein G of the vesicular stomatitis virus (VSV-G) resulted in an intrinsically fusion-active gB variant (gB/VSV-G). In this present study, we employed a dual split protein (DSP)-based cell fusion assay to further characterize the determinants of fusion activity in the CTD of gB. We generated a comprehensive library of gB CTD truncation mutants and identified two mutants, gB-787 and gB-807, which were fusion-competent and induced the formation of multinucleated cell syncytia in the absence of other HCMV proteins. Structural modeling coupled with site-directed mutagenesis revealed that gB fusion activity is primarily mediated by the CTD helix 2, and secondarily by the recruitment of cellular SH2/WW-domain-containing proteins. The fusion activity of gB-807 was inhibited by gB-specific monoclonal antibodies (MAbs) targeting the antigenic domains AD-1 to AD-5 within the ectodomain and not restricted to MAbs directed against AD-4 and AD-5 as observed for gB/VSV-G. This finding suggested a differential regulation of the fusion-active conformational state of both gB variants. Collectively, our findings underscore a pivotal role of the CTD in regulating the fusogenicity of HCMV gB, with important implications for understanding the conformations of gB that facilitate membrane fusion, including antigenic structures that could be targeted by antibodies to block this essential step in HCMV infection.