Dual-fluorescence labeling of pseudorabies virus for live-cell tracking virus entry and replication

• DOI: 10.21203/rs.3.rs-4982958/v1
• Publication Date: 2024-10-17T14:49:42Z
• Authors: Hongxia Wu; Mingzhi Li; Li Pan; Guangtao Xiang; Lian-Feng Li; Tao Wang; Rui Luo; Yongfeng Li; Di Liu; Huanjie Zhai; Moon Assad; Xin Song; Yanjin Wang; Franck Gallardo; Yuan Sun; Huaji Qiu
• Keywords: Pseudorabies; Replication

<title>Abstract</title> Pseudorabies virus (PRV) is a neurotropic herpesvirus. It not easy to tracking the whole replication progess of PRV, especially nascent viral genome in host cells. In this study, we developed dual-fluorescence-labeled PRV (rPRV-Anchor3-mCherry) with and envelope protein gM labeled by Anchor DNA labeling system mCherry, respectively. Through single-virus rPRV-Anchor3-mCherry, observed that invaded mouse neuroblastoma Neuro2a (N2a) cells <italic>via</italic> both endocytosis plasma membrane fusion pathway. During stage, parental progeny rPRV-Anchor3-mCherry cell nuclei could be visible, nucleocapsid appeared more specifically than traditional capsid particles (rPRV-VP26-EGFP). We found numerous were produced nucleus, causing nucleus break using three-dimensional (3D) live-cell imaging electron microscopy. Moreover, Our findings confirmed simultaneously targeting <italic>UL9</italic> <italic>UL54</italic> genes CRISPR-Cas9 led complete inhibit replication. can used research multiple steps cycle.