Three heterologous proteins simultaneously expressed from a chimeric potyvirus: infectivity, stability and the correlation of genome and virion lengths.

Three heterologous proteins were simultaneously expressed from a chimeric potyvirus Potato virus A (PVA) in Nicotiana benthamiana. The genes for green fluorescent protein of Aequoria victoriae ("G"; 714 nucleotides, nt), luciferase of Renilla reniformis ("L", 933 nt) and beta-glucuronidase of Escherichia coli ("U", 1806 nt) were inserted into the engineered cloning sites at the N-terminus of the P1 domain, the junction of P1 and helper component protein (HC-Pro), and the junction of the viral replicase (NIb) and coat protein (CP), respectively, in an infectious PVA cDNA. The proteins were expressed as part of the viral polyprotein and subsequently released by cleavage at the flanking proteolytic cleavage sites by P1 (one site) or the NIa-Pro proteinase (other sites). The engineered viral genome (pGLU, 13311 nt) was 39.2% larger than wild-type PVA (9565 nt) and infected plants of N. benthamiana systemically. pGLU was stable and expressed all three heterologous proteins, also following the second infection cycle initiated by sap-inoculation of new plants with the progeny viruses. The gene for GUS showed some inherent instabilities, as also reported in other studies. Accumulation of pGLU in infected leaves was lower by a magnitude as compared to the vector viruses pG0U and p0LU used to express two heterologous proteins. Hence, pGLU may have reached the maximum genome size that can still function and complete the PVA infection cycle. Examination of virions by electron microscopy indicated that the virion lengths of PVA chimera with various numbers of inserts were directly proportional to their genome lengths.