A Novel Motif in the 3'-UTR of PRRSV-2 Is Critical for Viral Multiplication and Contributes to Enhanced Replication Ability of Highly Pathogenic or L1 PRRSV

Abstract

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) with enhanced replication capability emerged in China and has become dominant epidemic strain since 2006. Up to now, the replication-regulated genes of PRRSV have not been fully clarified. Here, by swapping the genes or elements between HP-PRRSV and classical PRRSV based on infectious clones, NSP1, NSP2, NSP7, NSP9 and 3'-UTR are found to contribute to the high replication efficiency of HP-PRRSV. Further study revealed that mutations at positions 117th or 119th in the 3'-UTR are significantly related to replication efficiency, and the nucleotide at position 120th is critical for viral rescue. The motif composed by 117-120th nucleotides was quite conservative within each lineage of PRRSV; mutations in the motif of HP-PRRSV and currently epidemic lineage 1 (L1) PRRSV showed higher synthesis ability of viral negative genomic RNA, suggesting that those mutations were beneficial for viral replication. RNA structure analysis revealed that this motif maybe involved into a pseudoknot in the 3'-UTR. The results discovered a novel motif, 117-120th nucleotide in the 3'-UTR, that is critical for replication of PRRSV-2, and mutations in the motif contribute to the enhanced replicative ability of HP-PRRSV or L1 PRRSV. Our findings will help to understand the molecular basis of PRRSV replication and find the potential factors resulting in an epidemic strain of PRRSV.

Keywords: 3′-UTR; HP-PRRSV; epidemic; motif; replication; secondary structure.

Figure 1

Construction strategy of the full-length chimeric plasmids. (A,B) Full-length chimeric plasmids were constructed by exchanging the corresponding NSP1, NSP2, NSP3, NSP4, NSP5, NSP7, NSP9, NSP10, NSP11, NSP12, GP2, GP3, GP4, GP5, M and 3′-UTR regions between CH-1a and HuN4 infectious clone. NSP6, NSP8 and N proteins are highly homologous. The boxes represent genomic fragments of parental backbone viruses HuN4 (gray) and CH-1a (white).

Figure 2

The comparison of replication efficiency between parental and chimeric viruses in PAMs. (A,B) The viral titers of parental HuN4, CH-1a and their chimeric viruses in PAMs. The parental and mutant viruses infected PAMs at a MOI of 0.01. The data were presented as the mean standard deviation of three independent experiments. LOD: limit of detection. Asterisk (*) indicates a significant difference between HuN4 and CH-1a and HC₁ and CH₁, respectively. (***, p < 0.001). Pound (#) indicates a significant difference between HuN4 and CH-1a and HC₂ and CH₂, respectively. (###, p < 0.001). And (&) indicates a significant difference between HuN4 and CH-1a and HC₇ and CH₇, respectively. (&, p < 0.05; &&&, p < 0.001). Phi (φ) indicates a significant difference between HuN4 and CH-1a and HC₉ and CH₉, respectively. (φ, p < 0.05; φφ, p < 0.01; φφφ, p < 0.001). Delta (δ) indicates a significant difference between HuN4 and CH-1a and HC_3′-UTR and CH_3′-UTR, respectively (δ, p < 0.05; δδδ, p < 0.001).

Figure 3

Predicted 3′-UTR secondary structures of parental HuN4 and mutant viruses. (A) The alignment of 3′-UTR between HP-PRRSV HuN4 strain and classical PRRSV CH-1a strain. The 3′-UTR differential nucleotides in HuN4 and CH-1a were marked in red. (B) The 3′-UTR secondary structures of HuN4, CH-1a, HuN4-3′-UTR-C9T, HuN4-3′-UTR-17+G, HuN4-3′-UTR-A117G and HuN4-3′-UTR-A119G were predicted by mFold method. Different nucleotides between HuN4-3′-UTR and CH-1a-3′-UTR were depicted by hollow round. Mutant nucleotides in the 3′-UTR of HuN4-3′-UTR-C9T, HuN4-3′-UTR-17+G, HuN4-3′-UTR-A117G and HuN4-3′-UTR-A119G were depicted by gray round. (C) The 3′-UTR secondary structures of HuN4-3′-UTR-G120A, HuN4-3′-UTR-117+120, HuN4-3′-UTR-119+120 and HuN4-3′-UTR-117+119+120.

Figure 4

Comparison of replication efficiency of parental and mutant viruses. (A) The growth kinetics of parental HuN4 and mutant viruses (HuN4-3′-UTR-C9T and HuN4-3′-UTR-17+G). LOD: limit of detection. Asterisk (*) indicates a significant difference between HuN4 and HuN4-3′-UTR-C9T. (*, p < 0.05). Pound (#) indicates a significant difference between HuN4 and HuN4-3′-UTR-17+G (#, p < 0.05). (B) The growth kinetics of parental HuN4 and mutant viruses (HuN4-3′-UTR-A117G and HuN4-3′-UTR-A119G). LOD: limit of detection. Delta (δ) indicates a significant difference between HuN4 and HuN4-3′-UTR-A117G (δδδ, p < 0.001). Phi (φ) indicates a significant difference between HuN4 and HuN4-3′-UTR-A119G (φφφ, p < 0.001). (C) Plaque morphology of the parental PRRSV HuN4 and mutant viruses in Marc-145 cells. (D) The diameters of viral plaques were measured and analyzed. Asterisk (*) indicates a significant difference between HuN4 and its mutant viruses (***, p < 0.001).

Figure 5

The proportion and mutations in the 3′-UTR motif of different PRRSV lineages. (A) The proportion of differential lineage PRRSVs based on all available full-length sequences of PRRSV-2 strains (n = 765) in 1991–2019. (B) Different mutation patterns of 117–120th of 3′-UTR in different lineage PRRSVs. The representative strain of each lineage, together with the amount of PRRSV strains in corresponding mutation patterns, was listed on the right side of the sequences.

Figure 6

The effects of different 3′-UTR motifs on PRRSV replication. (A) Schematic diagram of luciferase plasmids used for Luciferase reporter assay analysis. (B) The effects of different 3′-UTR motifs on PRRSV replication. The 3′-UTR mini-genome system mutant plasmids and Renilla luciferase plasmid were co-transfected to Marc-145 cells. After 24 h post-transfection, cells were infected with 0.1 MOI PRRSV HuN4 for another 24 h, and then firefly luciferase activity was measured. Asterisk (*) indicates a significant difference between parental and mutant viruses. (*, p < 0.05; ***, p < 0.001).

Figure 7

The pseudoknot structure in the 3′-UTR. (A) A possible pseudoknot in the 3′-UTR was formed and involved in PRRSV replication after deleting the 49 nucleotides at the 5′-terminal of 3′-UTR. The truncated 3′-UTR secondary structures of L1-NADC30, L3-QYYZ, L5-VR-2332 and L8-HuN4 are shown. (B) The nucleotides involving in the formation of pseudoknot are depicted in gray, and base–pairing interaction is depicted by lines. The predicted pseudoknot structure was based on the 3′-UTR sequence of PRRSV HuN4.