Date of Award
Summer 8-22-2026
Document Type
Dissertation
Degree Name
Ph.D. in Biological Sciences
Organizational Unit
College of Natural Science and Mathematics, Biological Sciences
First Advisor
Schuyler van Engelenburg
Second Advisor
Erich Kushner
Third Advisor
Todd Blankenship
Fourth Advisor
Yan Qin
Copyright Statement / License for Reuse
All Rights Reserved.
Keywords
Antiviral screening, Replicon, SARS-CoV-2, Single-round viral system, Virus-like particle
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.
Copyright Date
8-2026
Publication Statement
Copyright is held by the author. User is responsible for all copyright compliance.
Rights Holder
Susiyan Jiang
Provenance
Received from ProQuest
File Format
application/pdf
Language
English (eng)
Extent
156 pgs
File Size
59 MB
Recommended Citation
Jiang, Susiyan, "A Versatile Single-Round SARS-CoV-2 System for Interrogating Antivirals and Host Determinants" (2026). Electronic Theses and Dissertations. 2775.
https://digitalcommons.du.edu/etd/2775