Tracking of single virus: Dual fluorescent labeling of pseudorabies virus for observing entry and replication in the
Mingzhi Li1, Li Pan1, Caoyuan Ma1
1State Key Laboratory for Animal Disease Control and Prevention Harbin Veterinary Research Institute, CAAS 678 Haping Road, Harbin, Heilongjiang 150069, China.
Abstract:
Pseudorabies virus (PRV) is a neurotropic herpesvirus. It is not easy to be track the whole replication progress of PRV, especially the nascent viral genome in the host cells. In this study, we developed a dual-fluorescence-labeled PRV (rPRV-Anchor3-mCherry) with the viral genome and the envelope protein gM labeled by ANCHOR DNA labeling system and mCherry, respectively. Through single-virus tracking of rPRV-Anchor3-mCherry, we observed that PRV invaded mouse neuroblastoma Neuro-2a cells via both endocytosis and plasma membrane fusion pathway. During the replication stage, parental and progeny viral genome of rPRV-Anchor3-mCherry in the cell nuclei could be visible, and viral nucleocapsid appeared more specifically than traditional capsid protein labeled PRV particles (rPRV-VP26-EGFP). We found that numerous progeny viral particles were produced in the nuclear, causing the nucleus membrane to break using three-dimensional (3D) live-cell imaging and electron microscopy. Moreover, our findings confirmed that simultaneously targeting of the UL9 and UL54 genes using a CRISPR-Cas9 system led to the complete inhibition PRV replication. rPRV-Anchor3-mCherry can be used to research multiple steps of the viral cycle.
Insights
This study introduces a dual-fluorescence Pseudorabies virus (PRV) for tracking viral replication. The new tool reveals PRV entry pathways and genome replication, aiding in understanding herpesvirus infection.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Pseudorabies virus (PRV), a neurotropic herpesvirus, presents challenges in tracking its replication, particularly the nascent viral genome within host cells.
- Understanding PRV's life cycle is crucial for developing effective antiviral strategies.
Purpose of the Study:
- To develop a novel dual-fluorescence-labeled PRV (rPRV-Anchor3-mCherry) for visualizing viral genome and envelope protein dynamics.
- To investigate PRV entry mechanisms and replication processes in host cells.
Main Methods:
- Development of a dual-fluorescence-labeled PRV (rPRV-Anchor3-mCherry) using ANCHOR DNA labeling and mCherry.
- Single-virus tracking, 3D live-cell imaging, and electron microscopy.
- CRISPR-Cas9 gene editing to target UL9 and UL54 genes.
Main Results:
- PRV invasion of Neuro-2a cells occurs through both endocytosis and plasma membrane fusion.
- Parental and progeny viral genomes are visible in the nucleus during replication, with nucleocapsids appearing more specifically than with traditional labeling.
- Extensive progeny viral particle production in the nucleus leads to nuclear membrane breakdown.
- Simultaneous targeting of UL9 and UL54 genes completely inhibits PRV replication.
Conclusions:
- The rPRV-Anchor3-mCherry system provides a powerful tool for studying multiple stages of the PRV life cycle.
- Visualizing PRV genome replication and particle production offers new insights into herpesvirus pathogenesis.
- CRISPR-Cas9 targeting of specific genes presents a potential therapeutic approach for PRV infections.
