Inducing Autophagic Cell Death by Nsp5 of Porcine Reproductive and Respiratory Syndrome Virus

Research Article

Austin Virol and Retrovirology. 2015; 2(2): 1014.

Inducing Autophagic Cell Death by Nsp5 of Porcine Reproductive and Respiratory Syndrome Virus

Liping Yang¹, Rong Wang1,3, Zexu Ma¹, Yu Wang¹ and Yanjin Zhang1,2*

¹VA-MD College of Veterinary Medicine, University of Maryland, USA

²Maryland Pathogen Research Institute, University of Maryland, USA

³Laboratory Animal Center, School of Medicine, Xi’an Jiaotong University, China

*Corresponding author: Yanjin Zhang, VA-MD College of Veterinary Medicine, Maryland Pathogen Research Institute, University of Maryland, College Park, USA

Received: October 04, 2014; Accepted: November 09, 2015; Published: November 10, 2015


Porcine Reproductive and Respiratory Syndrome (PRRS) leads to severe economic losses to the swine-producing industry. Many unclear questions remain on pathogenesis of PRRS virus (PRRSV), including the mechanism of PRRSV-induced cell death. In this study, we cloned and expressed a PRRSV non-structural protein, nsp5, and discovered that it induced cell death in cultured cells. The nsp5 protein localized in cytoplasm and majority of the protein concentrated in perinuclear region. Along with extension of incubation time, the nsp5 tended to form puncta and polarized besides nucleus. An interesting observation was that the nsp5 expression induced cell death. Cell viability assay showed that the cells with nsp5 expression had over 2-fold more cell death than cells with empty vector. Further study indicated that the nsp5 induced cell death via autophagy. Treatment with 3-MA, an autophagy inhibitor, blocked the nsp5- induced cell death. These results suggest that nsp5 might play an important role in PRRSV-induced cell death. Further examination on the mechanism is warranted.

Keywords: Porcine reproductive and respiratory syndrome virus; PRRSV; NSP5; Autophagy; Cell death


Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a positive-sense single-stranded RNA virus in the family of Arteriviridae, Order Nidovirales [1]. The other members of the Arteriviridae include equine arteritis virus, lactate dehydrogenaseelevating virus, and simian hemorrhagic fever virus. PRRSV causes a contagious viral disease of pigs which is characterized by reproductive failure in sows and respiratory disease in pigs of all ages, which leads to an estimated $664 million in losses per year to the swine-producing industry in the USA alone [2]. The genome of PRRSV is a little over 15 kb in length with over ten Open Reading Frames (ORFs) [3-5]. The genome is composed of replicas genes located at the 5’-end, followed by genes encoding structural proteins. ORFs 1a and 1ab encode long polypeptides that are cleaved into individual Non-Structural Proteins (nsp), nsps 1-12, including proteases, a helicase and the RNAdependent RNA polymerase. ORFs 2-7 encode structural proteins: E, GP2, GP3, GP4, 5a, GP5, M and N.

Nsp5 is predicted to be 170 amino acids encoded by ORF1a and its predicted molecular mass is 18.9 kDa [6,7]. Nsp5 and several other nsps have been implicated in the induction of interferon-? and possibly in cell-mediated immune response [8]. Nsp5-7 was shown to cause autophagy in a similar way to nsp6 of corona viruses [9]. But the exact function of nsp5 in PRRSV replication or pathogenesis is unknown. The objective of this study to determine the expression pattern of this protein and its effect on cell viability. We discovered that nsp5 locates in cytoplasm in perinuclear region. It tends to polarize besides the nucleus along with extension of culture time. Cell viability assay showed that nsp5 causes cell death. Treatment with 3-MA rescued the nsp5-induced autophagic cell death. These results indicate that nsp5 might play an important role in PRRSV pathogenesis.

Materials and Methods

Cells, transfection, and chemicals

HEK293T, HEK293 and HeLa cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). Transfection of the cells with plasmid DNA was performed by using FuGene HD (Promega, Madison, WI), according to the instructions of the manufacturer.

3-Methyladenine (3-MA), an inhibitor of autophagy, was used to treat cells with nsp5 expression at 5 mM.


The nsp5 sequence was PCR amplified and cloned to vector pCAGEN (Addgene plasmid# 11160) with HA-tag at N-terminus and VenusC1 vector as previously reported [10,11]. All primers used for plasmid construction were listed in Table 1. All constructed plasmids were subjected to DNA sequencing to confirm the inserts.