Generation and characterization of UL41 null pseudorabies virus variant in vitro and in vivo

Chao Ye,Xuefei Cheng,Jingjing Xu,Guoxin Li,Jing Chen,Shasha Zhou,Jiqiang Wu,Zhiqing Yu,Tao Wang,Guangzhi Tong,Wu Tong,Hao Zheng

doi:10.1186/s12985-018-1025-4

Chao Ye, Xuefei Cheng + Show 10 more

Open Access

https://doi.org/10.1186/s12985-018-1025-4

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Abstract

BackgroundThe alphaherpesvirus virion host shutoff (vhs) gene, UL41, can induce degradation of host mRNAs and shut off host protein synthesis. The roles of vhs in HSV-1 and HSV-2 have been studied extensively in previous studies, however, relatively little is known about the vhs protein of PRV.MethodsA novel method combining CRISPR/Cas9 and Gibson assembly was developed to generate UL41 null PRV variant. The properties of UL41 null PRV in vitro and in vivo were further characterized. And the vhs activity of UL41 protein of PRV variant was evaluated by luciferase assay, Western-blot and RT-qPCR.ResultsGibson assembly based on homologous recombination can accomplish one-step insertion of viral DNA fragments into donor plasmids efficiently (> 80%). Cas9/gRNA further largely enhanced the efficiency of homologous recombination. Using this method we were able to rapidly generate the UL41 null and revertant viruses of PRV variant. Compared to wild type (JS-2012), the UL41 null virus showed significantly smaller plaques and lower titers in Vero cells and impaired lethality and neuroinvasion in mice. Further the UL41 protein from different PRV strains exhibited unequal vhs activity in vitro, which of JS-2012 showed significantly weaker vhs activity than that of European-American strains. In addition UL41 null virus can also significantly decrease the expression of host genes during the early period of infection, which suggests other viral factors may be also involved in host shutoff.ConclusionsCRISPR/Cas9 combined with Gibson assembly efficiently generated UL41 null PRV. Compared to wild type, UL41 null PRV showed impaired both replication capability in vitro and neuroinvasion in vivo. Further UL41 protein of PRV variant showed significantly weaker vhs activity than that of PRV SC (European-American-like strain), suggesting the deficiency of vhs activity by the PRV variant UL41 protein.

Highlights

The alphaherpesvirus virion host shutoff gene, UL41, can induce degradation of host mRNAs and shut off host protein synthesis
Large DNA viruses engineered by Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 have been reported, including adenovirus, herpes simplex virus 1 (HSV-1) [9], and even Pseudorabies virus (PRV) [10]
In addition UL41 null virus can significantly decrease the expression of host genes during the early period of infection, which suggests other viral factors may be involved in host shutoff

Summary

Introduction

The alphaherpesvirus virion host shutoff (vhs) gene, UL41, can induce degradation of host mRNAs and shut off host protein synthesis. Pseudorabies virus (PRV) is a member of the alphaherpesviridae subfamily [1]. Large DNA viruses engineered by CRISPR/Cas have been reported, including adenovirus, herpes simplex virus 1 (HSV-1) [9], and even PRV [10]. Gibson assembly, developed by Dr Daniel Gibson and colleagues, is a novel cloning method that eliminates the need for restriction enzyme digestion when cloning DNA fragments into a linearized vector [12]. It allows to assemble and clone oligos in a single step under isothermal conditions, and requires only a linearized vector and linear inserts with short sequences at each end that are homologous to one another

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