Fig. 1.

Nucleotide sequence alignment and prediction of the secondary structure of the 5′ UTR of type 1 and type 2 PRRSV strains, and diagram of mutant construction. (A) The Clustal V program was used to conduct nucleotide sequence alignment of the 5′ UTR of PRRSV strains including 5 type 1 strains and 11 type 2 strains. Nucleotides of inter-genotypically different domains were shaded gray and nucleotides in inter-genotypically conserved domains are shaded black. The 5′ UTR regions were designated artificially as the CS I, CS II, CS III, and CS IV domains. E and N denote type 1 and 2, respectively. SL1-5 denotes the stem–loop structures that exist in the 5′ UTR of the two genotypes, according to Mfold. LTH was contained in SL5, see Fig. 1B for details. TRS-L was in white and black shaded. (B) The RNA secondary structures of the 5′- proximal region of PRRSV genomes as predicted by Mfold program with the RNAviz modification. The displayed consensus type 1 (i), consensus type 2 (ii) represent the 5′-proximal 280 bp and 250 bp sequences, respectively. The TRS-L is shaded gray and the ORF1 translational initiation codon is shown in a box. The prominent stem–loops in dashed frames containing the LTH structures were inter-genotypically conserved, although the primary sequence identity of the two genotypes was low. (C) Schematic representation of mutation insertions between TRS, the initiation codon of ORF1 and PRRSV 5′ UTR. The sequences of PRRSV 5′ UTR, 3′ UTR, and the poly (A) tail are indicated. The mutants pTLNd4 and pPa2 were constructed from the PRRSV full length infectious cDNA clone pAPRRS using PCR-based mutagenesis. Nde I and Pac I were inserted between the TRS and the translational initiation codon of ORF1. Nucleotide sequences at the end of the 5′ UTR are shown in uppercase and the inserted nucleotides are shown in lowercase. The translational initiation codon of ORF1 (ATG) is in bold. The chimeric clone pTLV8 was obtained by replacing type 2 5′ UTR with type 1 5′ UTR, while pSHSP5 was contructed by replacing type 1 5′ UTR with type 2 counterpart, based on type 1 backbone. White bars denoted the sequence of type 2 and the hatched bar denoted the sequence of type 1. The parental pAPRRS was used as a control in all experiments. The top loops of the LTH in pTLNd4 and pPa2 were shown in (i) and (ii).