#08-253

Complete

Date Full Report Received

03/04/2010

Date Abstract Report Received

03/04/2010

Investigation

Institution:
Primary Investigator:

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is of major economic significance to swine industry in the U.S.A. and worldwide. Currently, there is no effective vaccine available to combat PRRS. In previous studies, we have demonstrated that virus-neutralizing antibodies are important for protective immunity against PRRSV. These neutralizing antibodies constitute a significant correlate for evaluating the efficacy of a vaccine. Although four viral glycoproteins are present in PRRSV, their role in assembly of infectious virions, interactions with the cellular receptor, virus entry and uncoating, and immunological response in vivo remain poorly understood. Development of safe and efficacious vaccines to combat PRRSV infections requires a basic understanding of the role of these glycoproteins in virus biology. One of the objectives of the proposal is to investigate how the four glycoproteins interact with each other to form multiprotein complex and which of these proteins interact with the cellular receptor, CD163. Knowledge of these interactions will be important in developing strategies to inhibit the process of binding of the virus to the cells, so that virus infections could be blocked. Studies conducted by us and others have unambiguously demonstrated that the glycoprotein GP5 is a major inducer of protective neutralizing antibodies. By genetic manipulation of PRRSV genome, we had previously demonstrated that elimination (through a process called “hypoglycosylation”) of selected sugar moieties present on the surface of GP5 dramatically enhances the ability of a PRRSV strain to invoke a more robust response composed by PRRSV-neutralizing antibodies. As evidence indicates that other glycoproteins that make up the PRRSV may also be involved in the PRRSV-neutralizing response, another objective of the proposal is to generate PRRSVs containing hypoglycosylated forms of all the remaining glycoproteins (GP2, GP3, and GP4) and assess the effects of these changes on the ensuing PRRSV-neutralizing antibody response. To carry out the studies in the proposed objectives, we examined interactions of PRRSV glycoproteins amongst each other and as well as with the receptor CD163. Our results show that strong interaction exists between GP4 and GP5 proteins, although weak interactions among the other minor envelope glycoproteins and GP5 have been detected. Both GP2a and GP4 proteins were found to interact with all the other GPs resulting in the formation of multiprotein complex. Our results further show that GP2a and GP4 proteins also specifically interact with the CD163 molecule. The carboxy-terminal 223 residues of CD163 molecule are not required for interactions with either the GP2a or the GP4 protein, although these residues are required for conferring susceptibility to PRRSV infection in BHK-21 cells. Overall, we conclude that the GP4 protein is critical for mediating interglycoprotein interactions and along with GP2a, serves as the viral attachment protein that is responsible for mediating interactions with CD163 for virus entry into susceptible host cell. Furthermore, mutations in the glycosylation sites of GP2, GP3, and GP4 proteins were introduced individually and mutant PRRSVs were generated from infectious clones containing these mutant glycoproteins. When inoculated into pigs and antibody response in the infected pigs were analyzed, we observed that there was a general down-regulation of neutralizing antibody response in pigs infected with the glycosylation mutant viruses. These results are contrary to our expectation of obtaining higher levels of neutralizing antibody response in these infected pigs. Overall, our results suggest that hypoglycosylation of the minor glycoproteins of PRRSV does not enhance neutralizing antibody response in pigs. Since we have demonstrated interactions of GP2a and GP4 with the PRRSV receptor CD163, we are currently examining which regions of these glycoproteins interact with CD163. This will be important in obtaining specific antibodies or peptide mimetics against these regions of GP2a and GP4 that could potentially block PRRSV infections. Our studies supported by the NPB grant (#08-253) have been recently published in Journal of Virology (a copy of the paper is forwarded to B. L. Everitt). Contact information: Asit K. Pattnaik, Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, E126 Beadle Center, 1901 Vine Street, Lincoln, NE 68588, Phone: 402-472-1067; Fax: 402-472-8722; e-mail: apattnaik2@unl.edu