GP3 is a structural component of the PRRSV type II (US) virion

Authors

M. de Lima, University of Nebraska - Lincoln
I. H. Ansari, University of Nebraska - Lincoln
P. B. Das, University of Nebraska - Lincoln
B. J. Ku, National Veterinary Research and Quarantine Service, Busan, Republic of Korea
F. J. Martinez-Lobo, Universidad Complutense, Madrid, Spain
Fernando A. Osorio, University of Nebraska - LincolnFollow

Document Type

Article

Date of this Version

7-2009

Comments

Published in Virology 390:1 (July 20, 2009), pp. 31-36; doi: 10.1016/j.virol.2009.04.017 Copyright © 2009 Elsevier Inc. Used by permission. http://www.elsevier.com/locate/yviro

Abstract

Abstract Glycoprotein 3 (GP3) is a highly glycosylated PRRSV envelope protein which has been reported as being present in the virions of PRRSV type I, while missing in the type II PRRSV (US) virions. We herein present evidence that GP3 is indeed incorporated in the virus particles of a North American strain of PRRSV (FL12), at a density that is consistent with the minor structural role assigned to GP3 in members of the Arterivirus genus. Two 15aa peptides corresponding to two different immunodominant linear epitopes of GP3 derived from the North American strain of PRRSV (FL12) were used as antigen to generate a rabbit monospecific antiserum to this protein. The specificity of this anti-GP3 antiserum was confirmed by radioimmunoprecipitation (RIP) assay using BHK-21 cells transfected with GP3 expressing plasmid, MARC-145 cells infected with FL12 PRRSV, as well as by confocal microscopy on PRRSV-infected MARC-145 cells. To test if GP3 is a structural component of the virion, ³⁵S-labelled PRRSV virions were pelleted through a 30% sucrose cushion, followed by a second round of purification on a sucrose gradient (20–60%). Virions were detected in specific gradient fractions by radioactive counts and further confirmed by viral infectivity assay in MARC 145 cells. The GP3 was detected in gradient fractions containing purified virions by RIP using anti-GP3 antiserum. Predictably, the GP3 was less abundant in purified virions than other major structural envelope proteins such as GP5 and M. Further evidence of the presence of GP3 at the level of PRRSV FL12 envelope was obtained by immunogold staining of purified virions from the supernatant of infected cells with anti-GP3 antiserum. Taken together, these results indicate that GP3 is a minor structural component of the PRRSV type II (FL12 strain) virion, as had been previously described for PRRSV type I.