Nucleotide-binding oligomerization domain-like receptor X1 restricts porcine reproductive and respiratory syndrome virus-2 replication by interacting with viral Nsp9

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) causes one of the most economically important diseases of swine worldwide. Current antiviral strategies provide only limited protection. Nucleotide-binding oligomerization domain-like receptor (NLR) X1 is unique among NLR proteins in its functions as a pro-viral or antiviral factor to different viral infections. To date, the impact of NLRX1 on PRRSV infection remains unclear. In this study, we found that PRRSV infection promoted the expression of NLRX1 gene. In turn, ectopic expression of NLRX1 inhibited PRRSV replication in Marc-145 cells, whereas knockdown of NLRX1 enhanced PRRSV propagation in porcine alveolar macrophages (PAMs). Mechanistically, NLRX1 was revealed to impair intracellular viral subgenomic RNAs accumulation. Finally, Mutagenic analyses indicated that the LRR (leucine-rich repeats) domain of NLRX1 interacted with PRRSV Nonstructural Protein 9 (Nsp9) RdRp (RNA-dependent RNA Polymerase) domain and was necessary for antiviral activity. Thus, our study establishes the role of NLRX1 as a new host restriction factor in PRRSV infection.

Keywords: Nonstructural Protein 9 (Nsp9); Nucleotide-binding oligomerization domain-like receptor X1 (NLRX1); PRRSV-host interactions; Porcine reproductive and respiratory syndrome virus (PRRSV); Replication.

Fig. 1

PRRSV infection up-regulates NLRX1 expression in PAMs. (A) PAMs were infected at an MOI of 0.1 with PRRSV or UV-inactived PRRSV for the designated time. Total RNA was extracted, and real-time PCR was performed to determine the relative levels of NLRX1. Gene expression was normalized to that of β-actin. Significant differences from the uninfected cells are denoted by asterisks (**) for P < 0.01. (B) PAMs were infected at the indicated MOI with PRRSV for 48 h. Total RNA was extracted, and real-time PCR was performed to determine the relative levels of NLRX1. (C) PAMs were infected at an MOI of 0.1 with PRRSV or UV inactived PRRSV for the designated time. Cell lysates were blotted with the indicated antibodies.

Fig. 2

Ectopic expression of NLRX1 impairs PRRSV replication in Marc-145 cells. (A) Marc-145 cells were transfected with pCAGGS-HA vector or HA–NLRX1 for 24 h and then infected at an MOI of 0.5 with PRRSV. After the designated hour postinfection, virus titers were determined by TCID₅₀ assay. (B) Marc-145 cells were transfected with vector or HA–NLRX1 for 24 h and then infected at the indicated MOI with PRRSV for 24 h. Virus titers were determined by TCID₅₀ assay.

Fig. 3

Enhancement of PRRSV replication by NLRX1 knockdown in PAMs. (A) PAMs were transfected with a scrambled control siRNA (NC) or three different siRNA duplexes against NLRX1 for 48 h. Total RNA was extracted, and real-time PCR was performed to determine the relative levels of NLRX1. β-action level was monitored as an internal control. (B) PAMs were transfected with NC siRNA or siRNA 2^# against NLRX1. After 48 h, cell lysates were blotted with anti-NLRX1 or anti-β-actin antibody. (C) PAMs were transfected with the control siRNA or the NLRX1 siRNA for 48 h and then infected at an MOI of 0.1 with PRRSV. Virus titers were determined by TCID₅₀ assay at 12, 24, and 48 h post infection.

Fig. 4

NLRX1 suppresses the synthesis of viral subgenomic RNAs. (A) Schematic representation of full-length NLRX1 and corresponding deletion mutants is shown. Protein motifs are indicated. (B) Marc-145 cells were transfected with HA–tagged NLRX1, or its deletion mutants. After 24 h, cells were infected at an MOI of 0.5 with PRRSV for 24 h. Virus titers were determined by TCID₅₀ assay. (C) Marc-145 cells were transfected with an increasing amount of NLRX1 expression plasmid or control plasmid and then infected with PRRSV (MOI = 0.5) for 24 h. PRRSV total RNA were measured by qPCR. (D) PAMs were transfected with control siRNA or the NLRX1 siRNA for 48 h and then infected at an MOI of 0.1 with PRRSV. Viral total RNAs were measured by qPCR at 24 h post infection. (E) Marc-145 cells were transfected with the empty vector or NLRX1 expression plasmid for 24 h and then infected at an MOI of 0.5 with PRRSV. The levels of viral RNAs were monitored at 24 hpi by qPCR. The viral RNA levels in control cells were normalized to a value of 1.

Fig. 5

Interaction of the Nsp9 with NLRX1. (A) HA–tagged NLRX1 was cotransfected with Flag–tagged Nsp9 into HEK293 T cells. Cell lysates were immunoprecipitated (IP) with anti-HA antibody or anti-Flag antibody and then blotted as indicated. (B) Hela cells were transfected with Flag–tagged Nsp9 for 24 h. Cells were then fixed and incubated with anti-NLRX1 and anti-Flag antibodies. DAPI, 4′6-diamidino-2-phenylindole. (C) Flag–tagged Nsp9 was cotransfected with the indicated HA–tagged NLRX1 mutants into HEK293 T cells. Cell lysates were immunoprecipitated with anti-HA antibody and blotted with the indicated antibodies. (D) The schematics of Nsp9 and corresponding truncation constructs. Numbers indicate the residues where deletions begin or end. (E) HA–tagged NLRX1 was cotransfected with the indicated Flag–tagged Nsp9 mutants into HEK293 T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and blotted with the indicated antibodies.