J Vet Sci.  2013 Sep;14(3):307-313. 10.4142/jvs.2013.14.3.307.

The signal sequence of type II porcine reproductive and respiratory syndrome virus glycoprotein 3 is sufficient for endoplasmic reticulum retention

• Kim DG
• Song CS
• Choi IS
• Park SY
• Lee JB
• Lee SS

• KMID: 1705561
• DOI: http://doi.org/10.4142/jvs.2013.14.3.307

Abstract

The glycoprotein 3 (GP3) of type II porcine reproductive and respiratory syndrome virus has the characteristic domains of a membrane protein. However, this protein has been reported to be retained in the endoplasmic reticulum (ER) rather than transported to the plasma membrane of the cell. In this study, we performed confocal laser scanning microscopy analysis of variants of GP3 and foundthat the signal sequence of the GP3 led to confinement of GP3 in the ER, while the functional ortransmembrane domain did not affect its localization. Based on these results, we concludedthat the signal sequence of GP3 contains the ER retention signal, which might play an important role in assembly of viral proteins.

Keyword

endoplasmic reticulum; glycoprotein 3; porcine reproductive and respiratory syndrome virus; retention signal; signal sequence

MeSH Terms

Animals
Cell Line
Cell Membrane/*metabolism/virology
Cricetinae
Endoplasmic Reticulum/*metabolism/virology
Microscopy, Confocal/veterinary
Plasmids/genetics/metabolism
Porcine respiratory and reproductive syndrome virus/*genetics/metabolism
*Protein Sorting Signals
Sequence Analysis, Protein/veterinary
Viral Envelope Proteins/chemistry/*genetics/metabolism
Protein Sorting Signals
Viral Envelope Proteins

Figure

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  Fig. 1 Schematic pictures of GFP fusion construct of GP3 variants. The constructs were designed based on computational analysis of the amino acid sequence of GP3. Constructs containing original GP3 and mutants with deletion of the signal sequence or transmembrane domain fused with GFP were created. Additionally, to confirm the effects of the signal sequence on localization of the GP3, constructs containing signal sequence fused with GFP were also built. Each construct was designated based on the domains that the fusion protein contained. SS and TM represent signal sequence and transmembrane domain, respectively.

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  Fig. 2 Confocal microscopic examination of BHK-21 cells transfected with GP3-GFP construct. (A) The GP3-GFP construct showed an evenly distributed GFP localization signal (green) in the perinucleus area excluding the nucleus. No rim-like signals at the nuclear or plasma membrane were observed, which is a typical sign of a transmembrane protein. (B) Endoplasmic reticulum (ER) was also counter stained with ER-Tracker (red) and visualized. (C) When these two signals were overlaid, strong colocalization signal was observed. Scale bars = 10 µm.

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  Fig. 3 Confocal microscopic examination of cells transfected with variants of GP3-GFP. (A~C) GP3ΔTM2-GFP shows distribution of the GFP signal (green) in the cytoplasm excluding the nucleus and its colocalization with ER counterstaining (red). (D~F) GP3ΔTM1,2-GFP showed similar distribution to that of GP3ΔTM2-GFP. However, the GFP signal showed granular accumulation. (G~I) GP3ΔS.S.-GFP, GP3ΔS.S.TM2-GFP, and GP3ΔS.S.TM1.2-GFP showed distribution of the GFP signal in both the nucleus and cytoplasm. Characteristically, the GFP signals of GP3ΔS.S.TM2-GFP and GP3ΔS.S.TM1,2-GFP were observed to be granular and this tendency was increased in GP3ΔS.S.TM1,2-GFP. Additionally, weak signals were observed in the nuclear membrane (arrow) in both the construct and the plasma membrane (dotted-arrow) in GP3ΔS.S.TM1.2-GFP. (J) Cells transfected with EGFP-N1 showed even distribution in the nucleus and cytoplasm. Scale bars = 10 µm.

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  Fig. 4 Signal sequence of GP3 was fused with GFP to confirm its effect on localization. (A) GFP signal (green) was observed in the cytoplasm excluding the nucleus. No signal was observed in the plasma membrane and nuclear membrane. This distribution pattern is similar to that of GP3ΔTM1,2-GFP (Fig. 3D). (B and C) ER was counterstained with ER Tracker (red) and overlaid with GFP signal, which revealed that S.S.-GFP colocalized with ER. Scale bars = 10 µm.

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