Display of epitopes on the surface of tobacco mosaic virus: Impact of charge and isoelectric point of the epitope on virus-host interactions

The biophysical properties of the tobacco mosaic tobamovirus (TMV) coat protein (CP) make it possible to display foreign peptides on the surface of TMV. The immunogenic epitopes G5-24 from the rabies virus (RV) glycoprotein, and 5B19 from murine hepatitis virus (MHV) S-glycoprotein were successfully displayed on the surface of TMV, and viruses accumulated to high levels in infected leaves of Nicotiana tabacum Xanthinn. The peptide RB19, which contains an arginine residue plus the 5B19 epitope fused to the CP (TMV-RB19), resulted in the induction of necrotic local lesions on inoculated leaves of N. tabacum Xanthi-nn and cell death of infected BY2 protoplasts. RNA dot blot assays confirmed that expression of the acidic and basic pathogenesis-related PR2 genes were induced in infected Xanthi-nn leaf tissue. TMV that carried epitope 31D from the RV nucleoprotein did not accumulate in inoculated tobacco leaves. Analysis of hybrid CPs predicted that the isoelectric points (pI):charge value was 5.31:-2 for wild-type CP, 5.64:-1 for CP-RB19, and 9.14:+2 for CP-31D. When acidic amino acids were inserted in CP-RB19 and CP-31D to bring their pI:charge to near that of wild-type CP, the resulting viruses TMV-RB19E and TMV-4D:31D infected N. tabacum Xanthi-nn plants and BY2 protoplasts without causing cell death. These data show the importance of the pI of the epitope and its effects on the hybrid CP pI:charge value for successful epitope display as well as the lack of tolerance to positively charged epitopes on the surface of TMV.