Identification of dispensable nucleotide sequence in 3' untranslated region of porcine reproductive and respiratory syndrome virus

Abstract

The 3' untranslated region (UTR) of porcine arterivirus genome plays a pivotal role for virus replication, yet the properties of 3' UTR remain largely undefined. We conducted site-directed mutagenesis to the 3' UTR of the type II porcine reproductive and respiratory syndrome virus (PRRSV). Serial deletions of the 3' UTR showed that at least 40 nucleotides immediately following the ORF7 stop codon were dispensable for the viability of PRRSV in cultured cells. We then constructed a chimeric PRRSV cDNA clone using type II PRRSV as the backbone containing the 3' UTR from the type I PRRSV. The chimeric virus was viable and shared similar properties with the parental virus. Our results provided the first description of the 40nt dispensable region in type I PRRSV 3' UTR, and further predicted structure demonstrated that the high-order structure of 3' UTR might play significant roles in its function.

Fig. 1

Comparison of 3′ UTR nucleotide sequence of type I and type II PRRSV. Clustal W program was used to conduct nucleotide sequence alignment of PRRSV 3′ UTR, collected from five representative strains of both genotype I including LV (GenBank Accession# M96262), SD01-08 (DQ489311), EuroPRRS (AY366525), LV421 (AY588319), five type II strains including VR2332 (AY150564), JX143 (EF188048), P129 (AF494042), CH1a (AY032626), and APRRS (GQ330474). Dot (.)at the beginning of the type I panel denote deletion in comparison with the type II 3′ UTR, while black shaded nucleotides are inter-typically conserved domains, arbitrarily designated as CS1, CS2, LSL stands for possible long bulged stem-loop region (see Fig. 7 for detail), and VR represents variable region.

Fig. 2

Mutagenesis of PRRSV 3′ UTR. The upper panel shows the PRRSV genomic organization encoding 9 ORFs (denoted by boxed numbers), which is flanked by terminal untranslated regions (UTR) represented by gray bars. The lower panel depicts the 3′UTR mutations, in which restriction enzyme sites were inserted (Pac I and Asc I); Nucleotide deletion was engineered in p3URAD40 and p3URD40, respectively. In pSURLV, the entire 3′UTR of pAPRRS was replaced by that of type I PRRSV marked by the black-striped bar. “TGA” denotes the stop codon of the ORF7.

Fig. 3

Immunofluoresence assay of the transfected MARC-145 cells by PRRSV 3′UTR mutants. The in vitro RNAs generated from the full-length cDNA template promoted by T7 were synthesized by the T7 mRNA mMachine kit. 2 μl of each synthetic RNA was transfected into MARC-145 cells using DMRIE-C as instructed by the supplier. The cell monolayer was fixed at 72 hpi, and stained by monoclonal antibody against N and Alexa Fluor 568 goat anti-mouse IgG (H + L). The IFA patterns were shown for the parental and mutant viruses, labeled by the rescued viruses vAPRRS, v3URAD40, v3URD40, vSURLV, and the mock control.

Fig. 4

RT-PCR and nucleotide sequencing of the rescued viruses. For verification if the mutation were retained in the rescued viruses collected from P5 culture supernatant, the target region was amplified as described in the materials and methods. A) The agarose gel electrophoresis of RT-PCR products of v3URAD40 (lane 1), vSURLV (lane 2), and parental virus vAPRRS (lane 4). B) Nucleotide sequence lineup showed that all viruses retained the engineered mutations. The sequence was compared with the parental APRRS (GQ330474), identical sequence displayed as dot (.), while variation was shown the actual nucleotide, natural or artificial deletion denoted as (-). The upstream boxed sequence showed that the vSURLV is homologous with type II APRRS in ORF7, while the downstream box indicated the vSURLV 3′ UTR is identical with type I PRRSV (LV).

Fig. 5

Virological characterization of the mutant viruses. A) Viral plaque morphology. The rescued viruses at P5 were serially 10-fold diluted and inoculated in fresh MARC-145 cells in 6-well-plates. After one hour adsorption, the infected cell monolayer was overlaid with agarose and stained with crystal violet at 5 dpi. B) One step growth curves of deletion mutant viruses. MARC-145 cells were infected with the P5 mutant virus at an MOI of 1. The cell supernatants were harvested at the indicated time points. Viral titration was conducted by plaque assay on fresh MARC-145 cells, as detailed in the text.

Fig. 6

Virus RNA transcription process was identified with Northern Blot. MARC-145 cells in T-75 flasks were infected with the three mutant viruses and the intracellular RNAs were extracted at 36 hpi. The viral genomic RNA and sgmRNAs 2 to 7 were recognized only by genotype specific probe with an oligo complementary to different genotype 3′ UTR respectively, all the sample did two repeat wells, then hybridized with different probe: (A) add type II specific probe VR3 (B) add type I specific probe LVR3.

Fig. 7

The secondary structure of the PRRSV 3′ UTR predicted by Mfold: Location of the extra nucleotides in the type II virus were also shown in predicted secondary structure, the grey marked region is the highly variable region in type II PRRSV, the above region of the lines is conserved secondary structure in two type PRRSV, while the bottom region of the line is variable secondary structure. 1. A). type I, B). type II, C). 3URD40.

Fig. 8

Porcine arterivirus contains an inter-typically conserved long bulged stem-loop in 3′ UTR. Mfold program was used for analysing the predicted secondary structures of the 3′ UTR of PRRSV, followed by RNAviz drawing of the structure; (A). the structure for the entire 3′ UTR consensus sequence, obtained from comparison of the available type I PRRSV cited in Fig. 1; (B). The predicted structure from the consensus sequence of the last 111 nt of the type II 3′ UTR, generated from 100 available GenBank deposited sequences (Accession No. available upon request).