Molecular characterization of the medaka (Oryzias latipes) Hsp90ab1

Nervous necrosis virus (NNV) can infect many species of fish and has caused severe economic losses to the aquaculture industry worldwide. Recently, there is a wealth of research indicating that heat shock protein 90ab1 (Hsp90ab1) is a receptor for red-spotted grouper nervous necrosis virus (RGNNV), however the potential function has not been examined so far. In this study, medaka (Oryzias latipes) hsp90ab1 gene (OlHsp90ab1) was identified, which contained 13 exons with a genome length of 5191 bp and coding sequences of 2175 bp. OlHsp90ab1 had 724 amino acids, containing three regions including low complexity regions, histidine kinase-like ATPases region and coiled coil regions. Phylogenetic tree analysis revealed that OlHsp90ab1 clustered closely with homolog in Fundulus heteroclitus. Moreover, the transcript of hsp90ab1 was constitutively expressed in all adult tissues. Following RGNNV infection in medaka, the relative expression of hsp90ab1 increased in the eye and heart, while decreased in the spleen, brain, gill, liver, and kidney. Overexpression of OlHsp90ab1 in GE cells significantly increased viral gene transcription. Meanwhile, medaka mutants named OlHsp90ab1△5 and OlHsp90ab1△17a were established using CRISPR-Cas9 system. Unexpectedly, homozygous mutants (Hsp90ab1^-/-) died 2 weeks post-hatching. Moreover, heterozygote mutants (Hsp90ab1^+/-) and wild type (WT) medakas were infected with RGNNV. However, the expression of RGNNV RNA2 gene in different tissues of Hsp90ab1^+/- showed no significant difference compared to WT medaka after RGNNV infection within one week. Taken together, medaka Hsp90ab1 plays a crucial role in the organism's growth and development, and involves in the immune response process while it is not a suitable candidate to generate the anti-RGNNV strain.