Dimming the corona: studying SARS-coronavirus-2 at reduced biocontainment level using replicons and virus-like particles

Abstract

The coronavirus-induced disease 19 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections, has had a devastating impact on millions of lives globally, with severe mortality rates and catastrophic social implications. Developing tools for effective vaccine strategies and platforms is essential for controlling and preventing the recurrence of such pandemics. Moreover, molecular virology tools that facilitate the study of viral pathogens, impact of viral mutations, and interactions with various host proteins are essential. Viral replicon- and virus-like particle (VLP)-based systems are excellent examples of such tools. This review outlines the importance, advantages, and disadvantages of both the replicon- and VLP-based systems that have been developed for SARS-CoV-2 and have helped the scientific community in dimming the intensity of the COVID-19 pandemic.

Keywords: COVID-19; SARS-CoV-2; coronavirus; drug discovery; replicon; vaccines; virus-like particles.

Fig 1

SARS-CoV-2 genome and early life cycle. (A) SARS-CoV-2 genome organization includes ORF 1a and 1b, which result in the polyproteins 1a and 1ab (pp1a and pp1ab) comprising 16 nsps; nsp1, disrupting host messenger RNA translation; nsp2, disrupting signal transduction; nsp3, multidomain protein involved in polyprotein processing [papain-like protease (PLpro)], double-membrane vesicle (DMV) formation, and immune evasion; nsp4, DMV formation; nsp5, main protease for polyprotein processing [3C-like protease (3CLpro)]; nsp6, DMV formation; nsp7/8, scaffold proteins for replication-transcription complex; nsp9, RNA-binding protein; nsp10, binder/scaffold of nsp14; nsp12, RNA-dependent RNA polymerase and nucleotidyltransferase (NiRAN) activities; nsp13, helicase; nsp14, exoribonuclease activity (proofreading); nsp15, endonuclease (EndoU) degrades dsRNA; nsp16, 5′ cap formation. Other ORFs code for the structural proteins (S, E, M, and N) and accessory proteins (shown as gray boxes, ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, and ORF10). (B) SARS-CoV-2 life cycle. After a SARS-CoV-2 virion enters the cell, host ribosomes translate the released positive-sense RNA genome. Some viral proteins are embedded in the endoplasmic reticulum (ER) where they contort the ER membrane into DMVs and other convoluted membrane (CM) structures to hide the replication-transcription complex, and replication intermediates, from the innate immune system as it synthesizes viral RNA.

Fig 2

Schematic of SARS-CoV-2 replicon systems and VLP reporter strategies. (A) Replicon systems generate a translated reporter signal encoded by electroporated RNA transcribed in vitro (1) or are encoded by mRNA transcribed by cellular RNA polymerases from a DNA template, e.g., transfected or electroporated DNA plasmids (2) or stable cell lines that either persistently harbor SARS-CoV-2 replicon by containing replicon-coding DNA integrated into their genome or are based on self-replicating RNA expressing a selectable reporter (3). (B) Examples of VLP (4) and replicon-derived VLP (5) systems. Expressing the structural proteins (S, E, M, and N) in producer cells results in the production of empty VLPs (4). VLPs can enter target cells through receptors, but they cannot undergo replication inside them. Co-transfection of a replicon with plasmids expressing these structural proteins results in rdVLPs (5). rdVLPs can package the replicon and thus can both enter target cells and undergo one round of infection. In similar approaches, the N protein can be omitted from the replicon genome or S, E, and M (but not all three) may be included in the replicon sequence. Promoters other than CMV (cytomegalovirus) may also be used. Figure created with BioRender.