Structural insights into SARS-CoV-2 nonstructural protein 4 (nsp4) biogenesis

Area of Science:

• Virology
• Structural Biology
• Cell Biology

Background:

• SARS-CoV-2 requires a replication-transcription complex (RTC) for propagation.
• Nonstructural protein 4 (nsp4) is a key component of the RTC, involved in endoplasmic reticulum (ER) membrane rearrangements and double-membrane vesicle (DMV) formation.
• The precise membrane topology and function of nsp4 in SARS-CoV-2 remain incompletely understood.

Purpose of the Study:

• To elucidate the membrane topology of SARS-CoV-2 nsp4.
• To understand the structural basis of nsp4's role in viral replication.
• To investigate the formation of DMVs and associated membrane structures.

Main Methods:

• Biochemical analysis to determine protein features.
• Molecular dynamics simulations to predict folding and membrane disposition.
• Analysis of glycosylation patterns.

Main Results:

• SARS-CoV-2 nsp4 possesses a partially cleaved signal peptide and three transmembrane segments.
• The N-terminus is oriented towards the ER lumen, while the C-terminus faces the cytoplasm.
• A non-canonical glycosylation sequon (N¹³¹IC) is not glycosylated in mammalian cells.
• Molecular dynamics simulations confirmed nsp4's membrane disposition and folding.

Conclusions:

• The determined membrane topology of nsp4 provides structural insights into its function.
• nsp4 is essential for ER membrane rearrangements and the formation of DMVs and double-membrane pores.
• These structures are critical for SARS-CoV-2 RNA replication and transport, advancing understanding of viral propagation mechanisms.