Nervous necrosis virus induced vacuolization is a Rab5- and actin-dependent process

ABSTRACT

Cytoplasmic vacuolization is commonly induced by bacteria and viruses, reflecting the complex interactions between pathogens and the host. However, their characteristics and formation remain unclear. Nervous necrosis virus (NNV) infects more than 100 global fish species, causing enormous economic losses. Vacuolization is a hallmark of NNV infection in host cells, but remains a mystery. In this study, we developed a simple aptamer labelling technique to identify red-spotted grouper NNV (RGNNV) particles in fixed and live cells to explore RGNNV-induced vacuolization. We observed that RGNNV-induced vacuolization was positively associated with the infection time and virus uptake. During infection, most RGNNV particles, as well as viral genes, colocalized with vacuoles, but not giant vacuoles > 3 μm in diameter. Although the capsid protein (CP) is the only structural protein of RGNNV, its overexpression did not induce vacuolization, suggesting that vacuole formation probably requires virus entry and replication. Given that small Rab proteins and the cytoskeleton are key factors in regulating cellular vesicles, we further investigated their roles in RGNNV-induced vacuolization. Using live cell imaging, Rab5, a marker of early endosomes, was continuously located in vacuoles bearing RGNNV during giant vacuole formation. Rab5 is required for vacuole formation and interacts with CP according to siRNA interference and Co-IP analysis. Furthermore, actin formed distinct rings around small vacuoles, while vacuoles were located near microtubules. Actin, but not microtubules, plays an important role in vacuole formation using chemical inhibitors. These results provide valuable insights into the pathogenesis and control of RGNNV infections.

KEYWORDS:

• RGNNV
• vacuolization
• Rab5
• aptamer
• imaging

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data Availability statement

The raw data supporting the conclusions of this article will be made available by the authors without undue reservation to any qualified researcher.

Ethics statement

All relevant ethical safeguards were met for animal experimentation, and all animal experiments in this study were approved by the Animal Care and Use Committee of the College of Marine Sciences, South China Agricultural University.

Additional information

Funding

This work was supported by grants from the National Natural Science Foundation of China (U20A20102; 41976104), Innovation Group Project of Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai) (311021006), China Agriculture Research System of MOF and MARA (CARS-47-G16), and Doctoral Workstation Foundation of Guangdong Second Provincial General Hospital (No. 2021BSGZ023).