Distinct motifs in the E protein are required for SARS-CoV-2 virus particle formation and lysosomal deacidification in host cells
Koya Miura, Youichi Suzuki, Kotaro Ishida, Masashi Arakawa, Hong Wu, Yoshihiko Fujioka, Akino Emi, Koki Maeda, Ryusei Hamajima, Takashi Nakano, Takeshi Tenno, Hidekazu Hiroaki, Eiji Morita
Abstract
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.