Identification of 5' and 3' cis-acting elements of the porcine reproductive and respiratory syndrome virus: acquisition of novel 5' AU-rich sequences restored replication of a 5'-proximal 7-nucleotide deletion mutant.
We here demonstrate the successful engineering of the RNA genome of porcine reproductive and respiratory syndrome virus (PRRSV) by using an infectious cDNA as a bacterial artificial chromosome. Runoff transcription from this cDNA by SP6 polymerase resulted in capped synthetic RNAs bearing authentic 5' and 3' ends of the viral genome that had specific infectivities of >5 x 10(5) PFU/microg of RNA. The synthetic viruses recovered from the transfected cells were genotypically and phenotypically indistinguishable from the parental virus. Using our system, a series of genomic RNAs with nucleotide deletions in their 5' ends produced viruses with decreased or no infectivity. Various pseudorevertants were isolated, and acquisition of novel 5' sequences of various sizes, composed predominantly of A and U bases, restored their infectivities, providing a novel insight into functional elements of the 5' end of the PRRSV genome. In addition, our system was further engineered to generate a panel of self-replicating, self-limiting, luciferase-expressing PRRSV viral replicons bearing various deletions. Analysis of these replicons revealed the presence and location of a 3' cis-acting element in the genome that was required for replication. Moreover, we produced enhanced green fluorescent protein-expressing infectious viruses, which indicates that the PRRSV cDNA/viral replicon/recombinant virus can be developed as a vector for the expression of a variety of heterologous genes. Thus, our PRRSV reverse genetics system not only offers a means of directly investigating the molecular mechanisms of PRRSV replication and pathogenesis but also can be used to generate new heterologous gene expression vectors and genetically defined antiviral vaccines.
Choi
Yu-Jeong
YJ
Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, Cheongju, Korea.
Yun
Sang-Im
SI
Kang
Shien-Young
SY
Lee
Young-Min
YM
D024509:3' Flanking Region / Q000235:genetics*; D024506:5' Flanking Region / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000096:biosynthesis; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D009154:Mutation; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D012093:Replicon
D018076:DNA, Complementary; D012367:RNA, Viral; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases