The Role of Glycosylation in the Control of Processing and Cellular Transport of the Functional Amyloid PMEL17

Abstract

Two full-length genomic cDNA clones, pTA/FMDV and pCA/FMDV, were constructedthat contained three point-mutants [A174G and A308G (not presentin pTA/FMDV); T1029G] in the genome compared with the wild type A/AKT/58strain of foot-and-mouth disease virus. These two viruses were rescuedby co-transfection of pCA/FMDV with pCT7RNAP, which can express T7RNA polymerase in BHK-21 cell-lines, or by transfection of the invitro transcribed RNA. Their biological properties were analyzedfor their antigenicity, virulence in suckling-mice (LD50) and growthkinetics in BHK-21 cells. The in vivo rescued viruses showed highpathogenicity for 3-day-old unweaned mice (LD50=10(-7.5)). However,the in vitro transcribed RNA derived from pTA/FMDV had lower pathogenicityfor suckling-mice (LD50=10(-6)), and the in vivo transcribed RNArecovered from pCA/FMDV co-transfected with pCT7RNAP showed no significantdifferences from the wild type virus. These data showed that recoveryof the infectious foot-and-mouth disease virus directly from theuse of in vivo techniques was better than from in vitro methods.Furthermore, the reverse genetic procedure technique was simplifiedto a faster one-step procedure based on co-transfection with pCT7RNAP.These results suggest that in vivo RNA transcripts may be more valuablefor engineering recombinant foot-and-mouth disease virus than invitro RNA transcripts, and may contribute to further understandingof the biological properties, such as replication, maturation andquasispecies, of the foot-and-mouth disease virus.