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. 2023 Mar 13:14:1112580.
doi: 10.3389/fmicb.2023.1112580. eCollection 2023.

Regulated control of virus replication by 4-hydroxytamoxifen-induced splicing

Zhenghao Zhao  1 , Busen Wang  1 , Shipo Wu  1 , Zhe Zhang  1 , Yi Chen  1 , Jinlong Zhang  1 , Yudong Wang  1 , Danni Zhu  1   2 , Yao Li  1 , Jinghan Xu  1 , Lihua Hou  1 , Wei Chen  1
Affiliations

Regulated control of virus replication by 4-hydroxytamoxifen-induced splicing

Zhenghao Zhao et al. Front Microbiol. .

Abstract

Designing a modified virus that can be controlled to replicate will facilitate the study of pathogenic mechanisms of virus and virus-host interactions. Here, we report a universal switch element that enables precise control of virus replication after exposure to a small molecule. Inteins mediate a traceless protein splicing-ligation process, and we generate a series of modified vesicular stomatitis virus (VSV) with intein insertion into the nucleocapsid, phosphoprotein, or large RNA-dependent RNA polymerase of VSV. Two recombinant VSV, LC599 and LY1744, were screened for intein insertion in the large RNA-dependent RNA polymerase of VSV, and their replication was regulated in a dose-dependent manner with the small molecule 4-hydroxytamoxifen, which induces intein splicing to restore the VSV replication. Furthermore, in the presence of 4-hydroxytamoxifen, the intein-modified VSV LC599 replicated efficiently in an animal model like a prototype of VSV. Thus, we present a simple and highly adaptable tool for regulating virus replication.

Keywords: intein; post-translation regulation; small molecule switch; vesicular stomatitis virus; virus regulation.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Experimental design and plasmids construction. (A) Schematic representation of the generation of recombinant 4-HT-regulated VSV that are characterized by high reproduction in 4-HT+ media but replication incompetence in 4-HT− media. Moreover, the replication capacity was assayed by measuring the GFP expression of VSV (P2). (B) Virus genome schemes of VSV: wild type VSV, VSV (dG)-GFP, VSV (dG)-GFP-N-intein, VSV (dG)-GFP-P-intein, and VSV (dG)-GFP-L-intein.
Figure 2
Figure 2
Screening regulation of intein insertion sites in viral proteins of VSV. (A–C) The structures and function domains of VSV nucleoprotein (A), phosphoprotein (B), the large RNA polymerase (C), and the intein insertion sites selected on the structures. N protein domains (Green and Zhang, 2006): N-terminal domain (NNTD); C-terminal domain (NCTD); and RNA-binding sites are shown in red vertical lines. P protein domains (Jenni and Bloyet, 2020): N-terminal domain (PNTD); L protein-binding domain (PL); oligomerization (POD); and C-terminal domain (PCTD). L protein domains (Jenni and Bloyet, 2020): RNA-dependent RNA polymerase domain (RdRp); capping domain (Cap); connector domain (CD); methyltransferase (MT); and C-terminal domain (CTD). (D) The GFP expression level of the recombinant 4-HT-regulated VSV and the percentages of GFP-positive cells were analyzed by FACS (E). Significance of results were evaluated by Mann Whitney test between two groups. p value less than 0.05 was considered significant. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant.
Figure 3
Figure 3
Characterization of the replication of the intein-inserted VSV in cells. (A) Virus genome schemes of VSVs: wild type VSV, VSV-Luc, LC599, and LY1744. (B) Cell morphology of BHK-T7 cells infected by LC599 (P2) and LY1744 (P2). The 4-HT+ group could form syncytia mediated by the expression of VSV G protein (red arrow). (C) 4-HT regulated the replication of LC599 (P2) and LY1744 (P2) in a dose-dependent manner. (D) The replication of LC599 (P2) and LY1744 (P2) with or without 4-HT. (E) The growth curve of VSV-Luc, LC599, and LY1744. (F) BHK-T7 cells were infected with P1 generation recombinant virus (4-HT+), and the culture supernatant harvested post-infection was defined as P2 generation. The culture supernatant harvested post-infection with P2 generation (4-HT+) was defined as P3 generation and so on. The 4-HT-regulated capacity on different passages of LC599 and LY1744 was detected. Significance of results were evaluated by Mann Whitney test between two groups. p value less than 0.05 was considered significant. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant.
Figure 4
Figure 4
Characterization of the replication of the intern-modified VSV in mice. (A) Effect of intracranial (i.c.) virus infection with the indicated viruses on the survival rates and body weights of BALB/c mice (n = 10). (B) Luminescence imaging in challenged mice on day 3. Luminescence intensity (photons/s/cm2/sr) was represented in false colors. (C) Changes of the whole-brain luminescence signals. (D) Detection of the virus titers in the brain tissues of a mouse (n = 10) on day 5. (E) Viral RNA transcripts of N genes in the brain (5 dpi) were measured by quantitative real-time PCR. Data were plotted for individual mice (n = 10). (F) Representative brain sections were isolated on day 5 after the challenge of mice. Brain sections were stained with hematoxylin and eosin (HE), original magnification 10×. Lymphocyte infiltration (blue arrow), perivascular cuffing (red arrow), and lymphocyte aggregation (blue arrow). Significance of results were evaluated by Mann Whitney test between two groups. p value less than 0.05 was considered significant. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant.
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