Distinct motifs in the E protein are required for SARS-CoV-2 virus particle formation and lysosomal deacidification in host cells

ABSTRACT Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is a major public health concern, but the mechanisms underlying its viral particle formation are not well understood. In this study, we established a system for producing virus-like particles (VLPs) by expressing four structural proteins that make up SARS-CoV-2 virus particles in cells and used a spike (S) protein fused with the HiBiT peptide as a marker for evaluating VLP production. Using this system, we confirmed that the E protein plays an important role in VLP release. Both the co-expression of VPS4A K173Q and ORF3A and treatment with bafilomycin A1 enhanced VLP release. These results suggest that VLPs are released in an endosomal sorting complex required for transport-independent manner and that lysosomal dysfunction is required for the efficient release of VLPs. Screening various E protein mutants revealed that the F56/Y57/Y59 amyloidization motif and the D72/L73/L74/V75 PDZ-binding motif (PBM) are critical for E protein function in VLP release. We also found that E protein expression led to an increase in the pH of lysosomes and that the N15 residue required for viroporin activity, the C40/C43 consensus sequence, or the K63 dibasic motif are required for its function. However, amyloidization or PBM mutations did not affect lysosomal deacidification, suggesting that the mechanisms of E protein activity during VLP formation and lysosomal deacidification are distinct. Overall, this study highlights the importance of the E protein in SARS-CoV-2 viral particle formation, and the results may be useful in the development of drugs that inhibit this process. IMPORTANCE Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), has caused a global public health crisis. The E protein, a structural protein found in this virus particle, is also known to be a viroporin. As such, it forms oligomeric ion channels or pores in the host cell membrane. However, the relationship between these two functions is poorly understood. In this study, we showed that the roles of E protein in virus particle and viroporin formation are distinct. This study contributes to the development of drugs that inhibit SARS-CoV-2 virus particle formation. Additionally, we designed a highly sensitive and high-throughput virus-like particle detection system using the HiBiT tag, which is a useful tool for studying the release of SARS-CoV-2.