Chimeric Virus as a Source of the Potato Leafroll Virus Antigen

Abstract

Large quantities of potato leafroll virus (PLRV) antigen are difficult to obtain because this virus accumulates in plants at a low titer. To overcome this problem, we constructed a binary vector containing chimeric cDNA, in which the coat protein (CP) gene of the crucifer infecting tobacco mosaic virus (crTMV) was substituted for the coat protein gene of PLRV. The PLRV movement protein (MP) gene, which overlaps completely with the CP gene, was doubly mutated to eliminate priming of the PLRV MP translation from ATG codons with no changes to the amino acid sequence of the CP. The untranslated long intergenic region located upstream of the CP gene was removed from the construct. Transcribed powerful tobamovirus polymerase of the produced vector synthesized PLRV CP gene that was, in turn, translated into the protein. CP PLRV packed RNAs from the helical crTMV in spherical virions. Morphology, size and antigenic specificities of the wild-type and chimeric virus were similar. The yield of isolated chimera was about three orders higher than the yield of native PLRV. The genetic manipulations facilitated the generation of antibodies against the chimeric virus, which recognize the wild-type PLRV.

Chimeric Virus Made from crTMV RNA and the Coat Protein of Potato Leafroll Virus is Targeted to the Nucleolus and Can Infect Nicotiana benthamiana Mechanically

“…Varitzev (Potato Farming Institute of the Russian Agricultural Academy) kindly provided tube potato plants infected with wild type PLRV (Canadian strain). Antiserum to chimeric PLRV obtained as described [13]. Antibodies against PLRV and the CMV (cucumber mosaic virus) purchased from DSMZ plant virus collection.…”

“…Binary vectors-The crTMV-based vector, pCambia-crTMV-CP PLRV , which produced hybrid virus particles, crTMV-CP-PLRV, of chimeric crTMV RNA encapsidated by PLRV CP, was engineered by replacing the crTMV CP gene with the PLRV 22.3 kDa CP (ORF 3) gene [13].…”

“…There are several differences between these constructions and those made by us, including different strains of TMV, deletion of regulatory sequences, absence of the P5 protein, and different movement proteins. Additionally, we employed a more efficient method of plant infection using agroinfiltration with the powerful P19 silencer [13], instead of P1/HC-Pro [7].…”

“…Surprisingly, fresh virus inoculum in plant sap but not purified virus preparations that were kept frozen at −20 • C was infectious mechanically. The likely explanation is the virus RNA degradation before or during its extraction from the frozen chimeric virus [13]. Bald [27] observed the digestion of RNA from tomato spotted wilt virus unless the whole virus genome is covered by a protein coat.…”

“…In our previous study [13], we constructed an efficient agroinfiltrable viral binary vector pCambia-crTMV-CP PLRV to overcome the problem of the low PLRV yield; its complete nucleotide sequence (14410 bp) is presented [14]. The vector contains a recombinant cDNA of the crucifer-infecting tobacco mosaic virus (crTMV) [15], in which the coat protein gene replaced by the corresponding PLRV gene.…”