Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: spike (S), envelope (E), membrane (M), and nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of β-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected versus transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is relocalized at endoplasmic reticulum (ER)–Golgi intermediate compartment (ERGIC) or Golgi compartments upon coexpression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M, and N are required for optimal production of virus-like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.
Highlights
At the end of 2019, SARS-Cov-2 emerged in China through zoonotic transmission and led to the COVID-19 pandemic, cumulating by end of September 2020 to over 31 million cases and more than 950,000 deaths worldwide [1]
We detected in SARSCoV-2-infected cells and their supernatant both a predominant noncleaved S form, denoted as S0, and a cleaved form of S, denoted as S2, which is likely induced from S0 processing by furin [13], an ubiquitous protein convertase localized within the cell secretory pathway [15]
We detected the S2* form that was predominantly expressed in 293T cells transfected with S (Fig. 1B). While both S0 and S2 were the prominent forms detected in purified SARS-CoV-2 viral particles, we did not observe secretion of particles containing S in the supernatant of cells expressing S alone (Fig. 1A), suggesting that some other viral proteins are required for secretion of S-containing particles
Summary
At the end of 2019, SARS-Cov-2 emerged in China through zoonotic transmission and led to the COVID-19 pandemic, cumulating by end of September 2020 to over 31 million cases and more than 950,000 deaths worldwide [1]. While both S0 and S2 were the prominent forms detected in purified SARS-CoV-2 viral particles, we did not observe secretion of particles containing S in the supernatant of cells expressing S alone (Fig. 1A), suggesting that some other viral proteins are required for secretion of S-containing particles.
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