A 5'-proximal stem-loop structure of 5' untranslated region of porcine reproductive and respiratory syndrome virus genome is key for virus replication

Abstract

Background: It has been well documented that the 5' untranslated region (5' UTR) of many positive-stranded RNA viruses contain key cis-acting regulatory sequences, as well as high-order structural elements. Little is known for such regulatory elements controlling porcine arterivirus replication. We investigated the roles of a conserved stem-loop 2 (SL2) that resides in the 5'UTR of the genome of a type II porcine reproductive and respiratory syndrome virus (PRRSV).

Results: We provided genetic evidences demonstrating that 1) the SL2 in type II PRRSV 5' UTR, N-SL2, could be structurally and functionally substituted by its counterpart in type I PRRSV, E-SL2; 2) the functionality of N-SL2 was dependent upon the G-C rich stem structure, while the ternary-loop size was irrelevant to RNA synthesis; 3) serial deletions showed that the stem integrity of N-SL2 was crucial for subgenomic mRNA synthesis; and 4) when extensive base-pairs in the stem region was deleted, an alternative N-SL2-like structure with different sequence was utilized for virus replication.

Conclusion: Taken together, we concluded that the phylogenetically conserved SL2 in the 5' UTR was crucial for PRRSV virus replication, subgenomic mRNA synthesis in particular.

Figure 1

An inter-genotypically conserved RNA secondary structure models of the 5'-proximal genomic region of PRRSV, based on the consensus sequences generated by sequence lineup (Lasergene Package). (A) Predicted RNA structure by MFold for different genotypes and chimeric sequences. (i) RNA secondary structure prediction of the consensus 5'-proximal 280 nt of type I PRRSV genome from five available type I PRRSV sequences. (ii) Predicted RNA secondary structure of the consensus 5'-proximal 246 nt generated by comparison of nine type II PRRSV genomes. Stem-loop 2 (SL2) in both models are highlighted by the dashed box. The leader TRS and start codon for ORF1a in two models are shown by gray shading and solid boxes separately. Stem-loop structures are designated as E-SL1-5 for type I PRRSV and N-SL1-5 for type II, respectively. (iii) Predicted secondary structure of the 5'-proximal 246 nt of mutant EX generated by substituting N-SL2 with E-SL2. The mutant region in EX is highlighted by solid box and lowercase. (B) Schematic drawing sequence location of type II PRRSV stem-loops, represented by black boxes. Parental (WT) N-SL2 sequence from nt 46-70 (GQ330474) was shown, based on which mutations (lowercase) were made. Dashed lines represent stem base-pair deletions in the mutant plasmids D1-D6.

Figure 2

Mutational analysis of the predicted stem-loop structure in the N-SL2. (A) Strategic representation of RT-PCR used to detect (-) gRNA, (+) sg mRNA7 and (-) sg mRNA7. The positions are according to APRRSV stain (GenBank: GQ330474) and all primer sequences are listed in Table 1. pAS was a non-replicative control which was absence of gene ORF1a and ORF1b (1688-13118) in full-length cDNA clone. (B) Schematic representation of the mutations introduced into the N-SL2 structure. The loop was enlarged as described in Figure 1, and mutants L-LL and L-RR were generated by overlapping PCR mutagenesis. L-RL was generated by combining the right and left arm sequences of the L-LL and L-RR, respectively, such that the overall structure of N-SL2 was restored. All the mutated nucleotides (lowercase) are highlighted in gray shading. The stem mutants, S-LL and S-RR, were generated by overlapping PCR such that one arm sequence was replaced with that of the opposite arm. The double mutant, S-RL, was generated by combining the mutations in the left and right arms such that the overall structure was restored. All mutant sequences are shown as lowercase. (C) RT-PCR of RNAs extracted from pAS and WT transfected cells at 24 hours after transfection. DNase I and RNase A were used to omit template DNA and the reverse transcriptase. The primers were nested RT-PCR primers as same as (-) gRNA detection. A 2-kbp ladder was used as a molecular size marker. The numbers indicated the lane No. (D) RT-PCR analysis of the mutants. Total cellular RNAs were extracted from mutant plasmids-transfected from BHK-21 cells at 24 hours post-transfection. β-actin is a marker for the level of intracellular RNA isolation, and pAS is a non-replicative control.

Figure 3

Structural protein expression and phenotypic properties of the mutant viruses. (A) Northern blot analysis of mutant RNAs isolated at 48 hours post transfection from MARC-145 cells transfected with WT, EX, S-LL, S-RR and S-RL plasmids. (B) PRRSV N protein expression of WT and mutants in transfected cells. Expression of N protein was visualized by immunofluorescence staining with anti-N antibody at 24 hours post-transfection. (C) Viral plaque morphology assay. 0.01 MOI of P1 supernatants were inoculated in fresh MARC-145 cells and covered by MEM containing 2% FBS and 1% low melting agarose, and the plaques were visualized at 5 days post infection by crystal violet staining. The plaque sizes of the WT (■) and mutant EX (▲), L-LL(*), L-RR(▏), L-RL(╳), and S-RL (+), were measured by a millimeter ruler after monolayers were stained with crystal violet. The bars represent the average plaque diameters. (D) Viral multi-step growth curves. MARC-145 cells infected at an MOI of 0.01 with the P1 passage parental virus and mutant viruses and harvested at the indicated time points. The virus titers were determined by plaque assay and the results were mean values from three independent experiments. Viral titers were expressed as log₁₀ PFU/ml.

Figure 4

Mutagenesis of the serial deletion of base pairs in the N-SL2 stem. (A) RNA secondary structure prediction of the mutants. (i) Schematic representation of the predicted secondary structure of the mutants, D1-D4. The dashed boxes indicate the increasing base pair deletions from the bottom of N-SL2. (ii) RNA secondary structure prediction of the mutant D5. The remaining nucleotides of N-SL2 are highlighted in gray. N-SL1' and N-SL2' represent the regenerated stem-loop structures after the deletions. (iii) Predicted secondary structure of mutant D6. The remaining nucleotides of N-SL2 are indicated by gray shading, which disappeared and became linear nucleotides. (B) RT-PCR analysis of the mutants D1-D6 as described in Figure 2.

Figure 5

Structural protein expression and phenotypic properties of the stem deletion mutant viruses. (A) Intracellular N protein expression of WT and mutant viruses. BHK-21 cells were transfected with plasmids of WT and mutants D1-D6 as indicated in Figure 3. Expression of N protein was visualized by immunofluorescence staining at 24 hours post-transfection. (B) Viral plaque morphology and growth curves of the mutant viruses D1-D6 as described in Figure 3.

Figure 6

Genetic stability of the rescued mutant viruses. DNASTAR v7.1 program (Lasergene Package) was used to conduct nucleotide sequence alignment of the rescued mutant viruses. Dots indicated that the residues match APRRSV (GQ330474) exactly. All mutant sequences were shown as lowercase. Short lines meant the deletions in D5 mutant viruses comparing with APRRSV. The numbers indicated the sequence positions of APRRSV.

Figure 7

Similar N-SL2 RNA secondary structure can be predicted from arteriviruses and coronaviruses. Arteriviruses presented are PRRSV strains APRRSV (GenBank: GQ330474) and Lelystad (M96262), SHFV strain LVR 42-0/M6941 (NC_003092), and LDV strain Plagemann (NC_001639). For coronaviruses, presented strains were PEDV strain CV777 (NC_003436), MHV strain A59 (NC_001846) and BCoV strain Quebec (AF220295).