Identification of amino acid residues important for anti-IFN activity of porcine reproductive and respiratory syndrome virus non-structural protein 1

Abstract

The non-structural protein 1 (nsp1) of porcine reproductive and respiratory syndrome virus is partly responsible for inhibition of type I interferon (IFN) response by the infected host. By performing alanine-scanning mutagenesis, we have identified amino acid residues in nsp1α and nsp1β (the proteolytic products of nsp1) that when substituted with alanine(s) exhibited significant relief of IFN-suppression. A mutant virus (16-5A, in which residues 16-20 of nsp1β were substituted with alanines) encoding mutant nsp1β recovered from infectious cDNA clone was shown to be attenuated for growth in vitro and induced significantly higher amount of type I IFN transcripts in infected macrophages. In infected pigs, the 16-5A virus exhibited reduced growth at early times after infection but quickly regained wild type growth properties as a result of substitutions within the mutated sequences. The results indicate a strong selection pressure towards maintaining the IFN-inhibitory property of the virus for successful propagation in pigs.

Fig. 1

Mutant nsp1α with reduced ability to antagonize IRF3 mediated gene induction. ISG56 luciferase assay was performed with nsp1α alanine scanning mutant (A), nsp1α single amino acid substitution mutant (B) and nsp1α protease active mutant (C). HEK-293TLR3 cells were co-transfected with indicated nsp1α mutant/wt expression plasmids (1 μg) or empty vector (vector), ISG56-Luciferase plasmid (0.4 μg), and pRLTK (0.01 μg). At 40 h post-transfection, cells were treated with 5 μg/ml dsRNA for 6 h and assayed for luciferase activity. The panel below the bar graph shows the protein expression of respective mutant nsp1α detected using anti-FLAG antibody. Bars represent average (n=3) relative ISG56 promoter activities compared to vector control (converted to 100%). Mutants whose ISG56 promoter activities are significantly different from wt nsp1α are indicated with an asterisk ‘^⁎’ (p<0.05). Beta-actin served as loading control.

Fig. 2

Mutant nsp1β with reduced ability to antagonize IRF3 mediated gene induction. ISG56 luciferase assay was performed with nsp1β alanine scanning mutant (A) and nsp1β protease active site mutants (B). HEK-293TLR3 cells were co-transfected with indicated nsp1β mutant expression plasmids (0.5 μg) or empty vector (vector), ISG56-luciferase plasmid (0.4 μg), and pRLTK (0.01 μg). At 40 h post-transfection, cells were treated with 5 μg/ml dsRNA for 6 h and assayed for luciferase activity. The panel below the bar graph shows the protein expression of respective mutant nsp1β detected using anti-FLAG antibody. Bars represent average (n=3) relative ISG56 promoter activities compared to vector control (converted to100%). Mutants whose ISG56 promoter activities are significantly different from wt nsp1β are indicated with an asterisk ‘^⁎’ (p<0.05). Beta-actin served as loading control.

Fig. 3

Characterization of 16-5A mutant virusin vitro. (A) Hela cells were transfected with FLAG-tagged wt nsp1β or 16-5A mutant expression plasmid. Nsp1β's (in green) localization was determined 24 h post-transfection by indirect immunofluorescence using anti-FLAG antibody. Position of nucleus is indicated by DAPI (4,6-diamidino-2-phenylindole) (blue) by staining in the merge image (right). (B) Multiple step growth kinetics of 16-5A virus in comparison to parental wt virus. Monocyte-derived macrophages were infected with 0.1 MOI of both viruses and culture supernatant were collected at indicated time post-infection. They were titrated in MARC-145 cells and titers are expressed in Log₁₀TCID₅₀/ml. Error bars indicate SEM values derived from three independent experiments (‘^⁎’ indicates p<0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

The 16-5A mutant induces higher level of type I IFN mRNAs. Porcine monocyte- derived macrophages were mock-infected/infected with 1 MOI of 16-5A mutant or wt FL12 virus. Total RNA isolated from cells was reverse transcribed and real time PCR was done for detection of porcine IFN-α (A), IFN-β (B) mRNA. The mRNA copy numbers were calculated after normalization with porcine β-actin copy number and expressed relative to mock control. Bars show average of mRNA copy numbers±SEM from three independent experiments (‘^⁎’ indicates p<0.05). (C) MARC-145 cells infected with 16-5A/wt virus and 12 h post-infection were super-infected with sendai virus (SeV) 50 HA (hemagglutinating) Units/ml to induce IFN synthesis for another 12 h. ISG56 protein level was detected by immunoblotting. The β-actin served as loading control.

Fig. 5

Growth attenuation and instability of 16-5A virusin vivo. (A) The in vivo growth property of 16-5A virus. Wt FL12 or 16-5A mutant viruses were inoculated into growing pigs and serum was collected at indicated days post-infection. After isolation of total RNA from serum, 4 μl of RNA was used in a single step real-time PCR reaction to detect viral RNA copy numbers. The bars represent mean of the viral RNA copy number from 4 different animals in each group. Error bars indicate SEM values. (^⁎ indicate p<0.05 and ns is not significant). (B) Mutations in the nsp1β in 16-5A infected pigs. The nsp1β region was amplified by reverse transcription PCR from the serum of 16-5A mutant virus infected pigs at indicated days post-infection (dpi). The amino acid sequences from number 16 to 20 of nsp1β of different dpi were aligned. The 0 dpi indicates the inoculated mutant sequence and dot (.) indicates no change in amino acid. The wt nsp1β sequence is mentioned in bottom for comparison. (C) ISG56 luciferase assay to determine the level of suppression of IRF3 dependent activation by nsp1β wt and nsp1β TVmut. Luciferase assay was performed and the results are presented as mentioned earlier (‘^⁎’ indicates p<0.05).