Subcellular localization and topology of porcine reproductive and respiratory syndrome virus E protein
Introduction
PRRSV belongs to the genus Arterivirus in the family Arteriviridae (Cavanaugh, 1997) and causes significant economic losses in the swine industry worldwide (Halbur et al., 1995). The viral genome consists of a positive single-stranded polyadenylated RNA molecule of approximately 15 kb in length and is composed of the 5′ untranslated region (UTR), nine ORFs (ORF1a, ORF1b, ORF2a, ORF2b, and ORF3-7) and the 3′ UTR (Meulenberg et al., 1995). Those ORFs encode 13 nonstructural proteins (ORF1a and ORF1b) and 7 structural proteins including GP2, E, GP3, GP4, GP5, M and N, respectively (Snijder and Meulenberg, 1998). Despite possessing similar virion morphology and genome organization, PRRSV is divided into two genotypes, the European genotype and the North American genotype, based on their antigenic and genetic characteristics (Meng et al., 1995, Nelsen et al., 1999).
The small envelope (E) protein is a hydrophobic protein, which is encoded by the ORF 2b starting from the +6 nucleotide position in mRNA2. The E protein consists of 73 and 70 amino acids for the North American and European type of PRRSV respectively and has an apparent molecular mass of 10 kDa (Snijder et al., 1999, Wu et al., 2001). The specific antibody against the E protein has been detected in PRRSV-infected pigs (Wu et al., 2001). It has been proposed that the E protein is essential to the virus infection, since deletion of the E protein would affect the incorporation of GP2, GP3 and GP4 into virions, possibly through forming multi-meric complex with other proteins to assemble into virions (Wissink et al., 2005). The E protein is also an ion-channel-like protein and maybe facilitates uncoating of virions (Lee and Yoo, 2006). However, although the E protein has been found exhibiting predominately perinuclear distribution when fused with enhanced green fluorescent protein (EGFP) (Wu et al., 2001), the exact subcellular localization of the PRRSV E protein in infected cells is not clear. In addition, mutation of the N-myristoylation site in the E protein could significantly reduce the growth of the virus (Du et al., 2010), but the mechanism is not known.
In the present study, the specific antibody against E protein peptide was developed to localize the E protein in the infected cells. The localization sequences of the E protein were identified by generating a series of truncated E proteins fused with EGFP in MARC-145 cells. Subsequently, ultra-centrifugation experiment with different extraction buffers was used to identify whether the E protein was an integral membrane protein embedded in the phospholipid bilayer. Then roles of the N-myristoylation site and the C-terminal domain in the subcellular localization and membrane association of the E protein were defined. Additionally, fluorescence protease protection (FPP) assay provided a useful means to establish a topology model of the E protein. Finally, colocalization between the E protein and the other viral structure proteins of the PRRSV was investigated by immunofluorescence assay.
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Cells and viruses
MARC-145 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL, Inc., Gaithersburg, MD, USA) supplemented with 10% FBS (PAA, laboratories, Linz, Austria) at 37 °C with 5% CO2. PRRSV VR-2332 strain was provided by Prof. Jiyong Zhou (Zhejiang University, Hangzhou, China) and propagated in MARC-145 cells.
Antibodies
The mouse anti-myc-tag (9E10) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), mouse anti-FLAG (M2) antibody from Sigma–Aldrich (St. Louis, MO, USA), rabbit
Sequence analysis of the PRRSV E protein
Sequence comparisons and protein structure predictions were done to determine the degree of conservation of the E sequences in 235 PRRSV isolates, locate putative transmembrane sequence and detect putative structural and functional motifs. The repertoire of amino acids deduced from the analysis of 235 PRRSV isolates revealed that amino acids were strictly conserved at most of positions (Fig. 1b). Moreover, most of the positions showing apparent variability were occupied by residues with similar
Discussion
The PRRSV genome encodes 7 structural proteins and 13 nonstructural proteins (Snijder and Meulenberg, 1998). Although biological and biochemical data regarding some PRRSV proteins have been accumulated, the biology of the E protein is not very clear yet. The E protein induces specific antibody in PRRSV-infected pigs (Wu et al., 2001) and is critical for the PRRSV infection (Lee and Yoo, 2006, Wissink et al., 2005). The E protein may function as an ion channel protein which might play an
Acknowledgements
We gratefully acknowledge Prof. Hanchun Yang (China Agricultural University) for his pertinent advice on the experiment. We thank Dr. Chunli Li (Institute of Microbiology, Chinese Academy of Sciences) for his help with the laser scanning confocal microscopy. We also appreciate the help from Prof. Quan Chen and Dr. Yingli Shang (Institute of Zoology, Chinese Academy of Sciences) for providing the Tetracycline-Regulated Expression System for Mammalian Cells.
This work was supported by the grants
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