Enhanced Synthesis of Foreign Nuclear Protein Stimulates Viral Reproduction via the Induction of γ-Thionin Expression

Abstract

Plants are a promising platform for recombinant protein production. Here we propose a novel approach to increase the level of viral vector-mediated recombinant protein synthesis. This approach is based on the hypothesis that antiviral protection is weakened during the antibacterial cellular response. We suggested that introduced to the cell foreign nuclear localized proteins, including effectors such as bacterial nucleomodulins, can interfere with the import of cellular nuclear proteins and launch antibacterial defense reactions, creating favorable conditions for cytoplasmic virus reproduction. Here, we performed synthesis of an artificial nuclear protein—red fluorescent protein (mRFP) fused with a nuclear localization sequence (NLS)—in plant cells as a mimetic of a bacterial effector. Superproduction of mRFP:NLS induced Nicotiana benthamiana γ-thionin (NbγThio) mRNA accumulation. Both NLS-containing protein synthesis and increased NbγThio expression stimulated reproduction of the viral vector based on the genome of crucifer-infecting tobacco mosaic virus (crTMV) in N. benthamiana leaves. We isolated the NbγThio gene promoter (PrγThio) and showed that PrγThio activity sharply increased in response to massive synthesis of GFP fused with NLS. We conclude that NLS-induced PrγThio activation and increased accumulation of Nbγthio mRNA led to the stimulation of GFP expression from crTMV: GFP vector in the transient expression system.

Nicotiana benthamiana Methanol-Inducible Gene (MIG) 21 Encodes a Nucleolus-Localized Protein That Stimulates Viral Intercellular Transport and Downregulates Nuclear Import

“…To test this assumption, we obtained recombinant 6xHis-NbMIG21 in Escherichia coli cells and performed in vitro binding of this protein with different promoter regions, analyzing the results using a gel retardation assay ( Figure 11 ). We used sequences of several available promoters of various N. benthamiana genes, including PrKPILP [ 38 ], PrThio [ 39 ], PrNtPME, and PrAELP [ 21 ], cauliflower mosaic virus 35S promoter and identified in this work PrMIG21, expecting that promoters contain common regulatory elements, which may be important for NbMIG21p binding.…”

“…But the side effect of this cellular reaction is sensitivity to the virus: viral vector reproduction is stimulated both upon a γ-thionin elevated expression and massive synthesis of foreign NLS-containing proteins. The exact mechanism of these effects is unknown, but it likely involves interference with nucleocytoplasmic transport [ 39 ].…”

Nicotiana benthamiana Methanol‐Inducible Gene (MIG) 21 Encodes a Nucleolus‐Localized Protein that Stimulates Viral Intercellular Transport and Downregulates Nuclear Import

“…And it is not the only example of balancing between resistance and susceptibility: recently it was shown that increased production of NLS-containing proteins (as a mimetic of nucleomodulins) in plant cell leads to the induction of γthionin, a factor of antibacterial defense response, but the side effect of this cellular reaction is sensitivity to the virus -viral vector reproduction is stimulated both upon γ-thionin elevated expression or massive synthesis of foreign NLS-containing proteins. The exact mechanism of these effects is unknown but likely it involves interference with nucleocytoplasmic transport [40].…”

“…To test this assumption, we obtained recombinant 6xHis-MIG21 in Escherichia coli cells and performed in vitro binding of this protein with different promoter regions analyzing results by gel retardation assay (Figure 10). We used sequences of several available promoters of various N. benthamiana genes including PrKPILP [39], PrThio [40], PrNtPME and PrAELP [21], and identified here PrMIG21 as well as cauliflower mosaic virus 35S promoter, expecting that promoters contain common regulatory elements, which may be important for NbMIG21 binding.…”