Engineering the PRRS virus genome: updates and perspectives

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is endemic in most pig producing countries worldwide and causes enormous economic losses to the pork industry. Infectious clones for PRRSV have been constructed, and so far at least 14 different infectious clones are available representing both genotypes I and II. Two strategies have been taken for progeny reconstitution: RNA transfection and DNA transfection. Mutations, insertions, deletions, and replacements of the viral genome have been employed to study the structure function relationship, foreign gene expression, functional complementation, and virulence determinants. Essential regions and non-essential regions for viral replication have been identified in both the coding regions and non-encoding regions. Foreign sequences have successfully been inserted into the nsp2 and N regions and in the space between ORF1b and ORF2a. Chimeras between member viruses in the family Arteriviridae have also been constructed and utilized to study cell tropism and functional complementation. This review discusses the advances and utilization of PRRSV reverse genetics and its potential for future research.

Keywords: Arterivirus; Genetic manipulation; Infectious clones; PRRSV; Porcine reproductive and respiratory syndrome; Reverse genetics.

Fig. 1

Transcription and translation of PRRSV genome. (A) PRRSV genome organization. PRRSV possesses a single-stranded positive-sense RNA genome of 15 kb in length with a 3′-polyadenylated tail and the 5′-cap (gray). The viral genome is polycistronic, harboring ORF1a and ORF1b, and structural genes of ORF2a, ORF2b, and ORFs 3 through 7, plus ORF5a within ORF5. (B) Viral gene expression. Non-structural proteins (black) are produced from pp1a and pp1ab after proteolytic processing. The PRRSV replicase-processing scheme involves the rapid auto-proteolytic release of nsp1α, nsp1β, and nsp2 (yellow boxes), mediated by papain-like proteinase (PLP) domains residing in each of them. The remaining polyproteins are processed by nsp4, resulting in a set of 14 individual nsps. The cleavage sites by PLPs and nsp4 are annotated by curved arrows and blue triangles, respectively. Structural proteins (color-coded) are expressed from the subset of sg mRNA. The 3′-co-terminal nested set of minus-strand RNAs is produced as a template for plus-strand sg mRNA synthesis. TRS, transcription regulatory sequence. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Engineering of infectious clones for nsp2 region. (A) Schematic presentation of the nsp2 protein. The nsp2 protein consists of five regions: hypervariable region I (HV-I), PLP2 cysteine protease core, hypervariable region II (HV-II), transmembrane regions, and the C-terminal tail. White areas indicate natural deletions. A triangle indicates the position of natural insertion. (B) Location of experimental sequence deletions (Orange). (C) Foreign gene insertion sites. Triangles indicate the position of insertion. GFP, green fluorescent protein; HV, hypervariable region; PLP, papain-like proteinase. Numbers indicate amino acid positions of nsp2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Engineering of infectious clones in N gene. (A) Schematic presentation of the nucleocapsid (N) protein. NLS and RBD overlap each other. (B) Deletion tolerance regions (yellow) in N protein. NLS, nuclear localization signal; NES, nuclear export signal; P, phosphorylation site; S, disulfide bridge, RBD, RNA-binding domain; NoLS, nucleolar localization signal. NCI, non-covalent interaction motif. Numbers indicate amino acid positions of N. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

PRRSV infectious clone for foreign gene expression. A copy of TRS (Black bar) is inserted between ORF1b and ORF2. Two kinds of sg mRNAs are produced from this construction. The GFP or other foreign genes is inserted between the synthetic TRS (Black bar) and the original TRS (Brown bar), and the original TRS leads to generation of mRNA for GFP expression. The inserted TRS drives the synthesis of sg mRNA for ORF2 expression. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)