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Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the genus Arterivirus in the family Arteriviridae, is the most important viral pathogens in swine industry worldwide. Here, we have investigated 5′ and 3′ cis-acting RNA elements required for PRRSV genome replication. Using the infectious PRRSV cDNA, we have manipulated the genomic RNA to generate mutant genomic RNAs, transfected these mutants into susceptible MARC-145 cells, and examined the competence of RNA replication. We found three genetic factors that were essential for viral replication. First, the cap structure present at the 5′-end of the genome was absolutely required for RNA replication. Secondly, polyadenylation of the genomic RNA at the 3′-end was also essential for RNA replication. Thirdly, approximately 100-nucleotide region just upstream of the N protein-coding region was crucial for genomic RNA replication. Taken together, our findings indicate that replication of PRRSV genomic RNA requires three important cis-acting RNA elements: 5′ cap structure, 3′ poly(A) motif, and an internal sequence of about 100 nucleotides. Further investigation is needed to elucidate the molecular mechanism(s) of how these elements act on PRRSV genome replication.
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Figure 1.
Requirement of the cap structure for PRRSV RNA replication. (A) Schematic presentation of the 5′-end region of three synthetic RNA transcripts derived from WT/GAcap, WT/GGcap, and WT/cap- cDNAs. (B) Determination of the specific infectivity of synthetic RNAs transcribed from the corresponding constructs. N.D, not detected.
Figure 2.
Requirement of the poly(A) tract for PRRSV RNA replication. (A) Schematic presentation of the 3′-end region of WT and Mutant/NPA that does not contain the poly(A) tail. (B) Determination of the specific infectivity of synthetic RNAs transcribed from WT and Mutant/NPA constructs. N.D, not detected.
Figure 3.
Schematic depiction of luciferase-expressing PRRSV viral replicons. The five viral replicons Mutant/nt14500, Mutant/nt14600, Mutant/nt14700, Mutant/nt14800, and Mutant/nt14900 have large internal deletions of nt 12163∼14500, nt 12163∼14600, nt 12163∼14700, nt 12163∼14800, and nt 12163∼14900, respectively. An expression cassette consisting of the EMCV IRES-driven LUC gene was inserted at the deletion site to facilitate the monitoring of viral replication.
Figure 4.
Expression of LUC gene. Naïve BHK-21 cells were transfected with the PRRSV RNAs transcribed from each cDNA template and seeded on 6-well plates at a density of 4×105 cells per well. After 36 hr incubation, cell lysates were prepared for LUC assays. The experiments were performed in duplicate; mean values are shown.
Table 1.
Oligonucleotides used for mutagenesis in this study.
| Oligo-nucleotides | Sequencea | Polarity |
|---|---|---|
| PR-F | 5′-TCCCGGCCAAAGCTTCATGATTTT | sense |
| PRnpa-R | 5′-AGGGCGGCCGCAACGTCTAGAATTTCGGCCGCATGGTT | antisense |
| PR14600-F | 5′-GCAGACGCGTGGAGCAGTAGTTGCACTCCTTTGG | sense |
| PR14700-F | 5′-GCAGACGCGTTTCTGGCCCCTGCCCACCACGTTG | sense |
| PR14800-F | 5′-GCAGACGCGTGGTCAACGGCACATTGGTGCCCGG | sense |
| PR14900-F | 5′-GCAGACGCGTCAACGGCAAGCAGCAGAAGAGAAA | sense |
| PR-R | 5′-CCTGCAGGGCGGCCGCAACGTTTTTTTTTTTTTT | antisense |
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