Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Visualization of Early RNA Replication Kinetics of SARS-CoV-2 by Using Single Molecule RNA-Fish Combined with Immunofluorescence

Rajiv Pathak
,
Carolina Eliscovich
ORCID logo ,
Ignacio Mena
ORCID logo ,
Anastasija Čupić
,
Kartik Chandran
,
Rohit Jangra
,
Adolfo García-Sastre
ORCID logo ,
Robert Singer
,
Ganjam V Kalpana
* ORCID logo
Version 1 : Received: 18 January 2024 / Approved: 19 January 2024 / Online: 19 January 2024 (07:07:04 CET)

A peer-reviewed article of this Preprint also exists.

Pathak, R.; Eliscovich, C.; Mena, I.; Cupic, A.; Rutkowska, M.; Chandran, K.; Jangra, R.K.; García-Sastre, A.; Singer, R.H.; Kalpana, G.V. Visualization of Early RNA Replication Kinetics of SARS-CoV-2 by Using Single Molecule RNA-FISH Combined with Immunofluorescence. Viruses 2024, 16, 262. Pathak, R.; Eliscovich, C.; Mena, I.; Cupic, A.; Rutkowska, M.; Chandran, K.; Jangra, R.K.; García-Sastre, A.; Singer, R.H.; Kalpana, G.V. Visualization of Early RNA Replication Kinetics of SARS-CoV-2 by Using Single Molecule RNA-FISH Combined with Immunofluorescence. Viruses 2024, 16, 262.

Abstract

SARS-CoV-2 infection remains a global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood including the kinetics of formation of genomic RNA (gRNA), sub-genomic RNA (sgRNA) and replication centers/organelles (ROs). We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA, immunofluorescence to detect nsp3 protein, a marker for the formation RO, and carried out a time course analysis. We found that single molecules of gRNA are visible within the cytoplasm at 30 minutes post-infection (p.i.). Starting from 2 hr. p.i., most of the viral RNA existed in clusters/speckles, some of which were surrounded by single molecules of sgRNA. These speckles associated with nsp3 protein starting at 3 hr. p.i., indicating that these were precursors to ROs. Furthermore, RNA replication was asynchronous as cells with RNA at all stages of replication were found at any given time point. Our probes detected the SARS-COV-2 variants of concern, and BA.1 strain exhibited slower rate of replication kinetics than the WA1 strain. Our results provide insights into the kinetics of SARS-CoV-2 early post-entry events, which will facilitate identification of new therapeutic targets to early stage replication to combat COVID-19.

Keywords

SARS-CoV-2; COVID-19; gRNA and sgRNA replication; Replication Organelles (RO); nsp3; Variants of Concern (VOC)

Subject

Biology and Life Sciences, Virology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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