A Versatile Single-Round SARS-CoV-2 System for Interrogating Antivirals and Host Determinants

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) highlighted the need for experimental systems that enable investigation of viral replication, assembly, and host-virus interactions while minimizing the biosafety constraints associated with infectious virus. Although authentic SARS-CoV-2 remains the gold standard for coronavirus research, its use requires biosafety level 3 (BSL-3) containment, limiting experimental accessibility and throughput. Existing alternatives, including pseudovirus and virus-like particle (VLP) systems, reproduce only selected stages of the viral life cycle. The overall objective of this dissertation was to develop complementary BSL-2-compatible experimental platforms for mechanistic studies of SARS-CoV-2 assembly, replication organelle organization, host determinants, and antiviral activity. Three complementary SARS-CoV-2 platforms were developed and evaluated. An internal ribosome entry site (IRES)-based plasmid system enabled coordinated expression of the four structural proteins and efficient production of NanoLuc reporter-containing VLPs for quantitative analysis of structural protein function and VLP assembly. A Replicase–Structural (RepStruct) system incorporating components of the viral replication machinery generated VLP-like particles but failed to support the intended replication-transcription program, illustrating the challenges of reconstructing coronavirus replication using synthetic viral genomes. Building on these findings, a hybrid platform combining transient structural protein expression with a stable BHK-21 SARS-CoV-2 replicon successfully integrated reporter-containing VLP production with sustained intracellular viral RNA replication. The hybrid system demonstrated robust sensitivity to inhibitors targeting viral replication, structural protein assembly, and host secretory trafficking, providing a versatile platform for antiviral evaluation under BSL-2 conditions. In addition to platform development, this work provides new biological insights into SARS-CoV-2 replication and assembly. Fluorescence microscopy demonstrated heterogeneous structural protein incorporation into VLPs generated by different production strategies and identified ORF3a as dispensable for reporter-containing VLP production. Studies of host membrane remodeling revealed preferential association of TMEM41B, but not Sec23A, with coronavirus-induced membrane structures. Correlative cryogenic structured illumination microscopy and focused ion beam-scanning electron microscopy further demonstrated that structural protein complementation of the stable replicon recapitulates key ultrastructural hallmarks of SARS-CoV-2 infection, including double-membrane vesicle replication organelles, replication-associated electron-dense complexes, and membrane budding intermediates. Collectively, these complementary BSL-2 platforms provide accessible tools for investigating coronavirus biology and evaluating antiviral strategies.