Heliothis virescens ascovirus 3h blocks the cell cycle of Spodoptera exigua fat body cells at G2 /M phase by downregulating cyclin B1 and cyclin-dependent kinase 1

Abstract

Ascoviruses are double-stranded DNA viruses that are pathogenic to noctuid larvae. In vitro infection causes the cells to fail to replicate and proliferate normally. However, the molecular mechanisms are unclear. In this study, the transmission electron microscopy data of infected-Spodoptera exigua (Hübner) fat body cells (SeFB, IOZCAS-SpexII-A cells) showed that virions were internalized in phagocytic vesicles, but not in the nucleus. FACS of cell-cycle progression was performed in SeFB cells infected with Heliothis virescens ascovirus 3h (HvAV-3h). The cell cycle phase distributions of the SeFB cells were G₁ = 29.52 ± 1.10%, S = 30.33 ± 1.19%, and G₂ /M = 40.06 ± 0.75%. The cell culture doubling time was approximately 24 h. The G₁ , S, and G₂ /M phases were each approximately 8 h. The unsynchronized or synchronized cells were arrested at G₂ /M phase after infection with HvAV-3h. Our data also showed that cells with more than 4N DNA content appeared in the HvAV-3h-treated group. While the mRNA levels of cyclin B₁ , cyclin H, and cyclin-dependent kinase 1 (CDK1) were downregulated after HvAV-3h infection, the mRNA expression levels of cyclin A, cyclin D, and cyclin B₂ were not significantly changed. Western blotting results showed that the expression of cyclin B₁ and CDK1 in infected SeFB cells within 24 h postinfection (hpi), and HvAV-3h infection inhibited the expression of cyclin B₁ and CDK1 at 12-24 hpi. Overall, these data implied that HvAV-3h infection leads to an accumulation of cells in the G₂ /M phases by downregulating the expression of cyclin B₁ and CDK1.

Keywords: CDK1; Heliothis virescens ascovirus 3h; SeFB cell line; cell cycle; cyclin B.