Functional analysis of the UL24 protein of suid herpesvirus 1
The UL24 homologous genes are conserved in alphaherpesviruses. However, the proximity of the UL24 gene and the UL23 gene encoding for thymidine kinase (TK) in the genome of suid herpesvirus 1 (SuHV-1) makes it difficult to mutate UL24 without affecting the expression of the TK gene, and thus functional studies of the UL24 gene have lagged behind. In this study, CRISPR/Cas9 and homologous recombination were adopted to generate UL24 and TK mutant viruses. Deletion of either the UL24 or the TK gene resulted in significantly reduced SuHV-1 replication and spread capacity in Vero cells. However, UL24-deleted virus still maintained a certain degree of lethality in mice, while TK-deleted viruses completely lost their lethality in mice. Similarly, neurovirulence of UL24-deleted virus in mice was not significantly affected compared to parental virus. In comparison, infection with the TK-deleted viruses resulted in significantly reduced neurovirulence and complete loss of lethality. In addition, and for the first time, viral UL24 protein was found to be expressed late during SuHV-1 infection; enhanced green fluorescence protein (eGFP) labeled UL24 protein was shown to be localized in the nucleus via heterologous expression. In conclusion, the UL24 gene of SuHV-1 encodes a nuclear-localized viral protein and acts as a minor virulence-associated factor compared to the TK gene.
KeywordsSuid herpesvirus 1 UL24 Thymidine kinase CRISPR/Cas9 Virulence
This study was supported by the National Key Research and Development Program of China (2016YFD0500100), China Agriculture Research System (NYCYTX-009), and Shanghai Municipal Agriculture Science and Technology Key Project (2015, 1-7 and 2016, 4-2).
CY and JC conducted the most experiments and drafted the manuscript. GT critically revised the manuscript. XC, SZ, SJ, JX, HZ, WT, GL helped with the experiments. All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no confict of interest.
All applicable international, national, and institutional guidelines for the care and use of animals were followed.
Research involving human and animal participants
The animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, China. The animal ethics approved number was SHVRI-MO-2017070588.
- 1.Pellett PE, Davison AJ, Eberle R, Ehlers B, Hayward GS, Lacoste V et al (2011) Herpesvirales. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) Virus taxonomy: Ninth report of the international committee on taxonomy of viruses. Elsevier Academic Press, London, p 99–107Google Scholar
- 2.Pejsak ZK, Truszczyni MJ (2006) Truszczynsk: Aujeszky’s disease (pseudorabies). In: Straw BE, Zimmerman JJ, D’Allaire S, Taylor DJ (eds) Diseases of swine, 9th ed. Blackwell Publishing Ltd, Ames, p 419–433Google Scholar
- 11.Jacobson JG, Martin SL, Coen DM (1989) A conserved open reading frame that overlaps the herpes simplex virus thymidine kinase gene is important for viral growth in cell culture. J Virol 63:1839–1843Google Scholar