Performing Department
Veterinary Medicine
Non Technical Summary
Porcine reproductive and respiratory syndrome (PRRS) causes significant economic losses to the swine industry. The current strategies to control PRRS are inadequate due to the limited efficacy of the current vaccines. An improved vaccine and cost-effective antivirals are needed for PRRS prevention and control. PRRS virus (PRRSV) binds receptors to initiate infection; and CD163, a scavenger receptor on macrophages and monocytes, is the validated entry receptor for PRRSV. Other receptors have also been found to contribute to PRRSV infection. Recently, the neonatal Fc receptor (FcRn) was identified to be a receptor for multiple arteriviruses, including PRRSV. However, it is not known whether FcRn plays a role in the PRRSV entry of primary cells from pigs and what the molecular mechanism is for the PRRSV binding FcRn. This project aims to characterize the PRRSV interaction with FcRn, identify the viral protein binding FcRn, and study the molecular basis of the virus-cell interaction. Completing this project will provide insights into the PRRSV interaction with FcRn and facilitate the development of novel antiviral strategies against PRRSV.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The previous study showed that FcRn is a receptor for PRRSV in MA-104 cells (Shaw et al., 2024) but did not verify the findings in primary cells from pigs. We plan to determine if FcRn is needed for PRRSV infection of primary pulmonary alveolar macrophages (PAMs) and characterize the molecular basis for the PRRSV interaction with FcRn. The objectives of the proposed research are listed below:Determine if FcRn plays a role in PRRSV infection of primary pulmonary alveolar macrophages (PAMs). PRRSV targets PAM cells in pigs to initiate infection (Lunny et al., 2016). The role of FcRn in PRRSV entry of PAMs needs to be examined. Antibodies against porcine FcRn will be added to the PAM cells before PRRSV inoculation to determine if the FcRn blocking inhibits PRRSV replication. Also, RNAi-mediated depletion of FcRn will be conducted to verify the findings. PRRSV attachment and entry assay will be performed to determine whether FcRn assists PRRSV initial binding or the following entry. Completing these experiments will yield information on FcRn roles in PRRSV infection of PAMs.Identify the PRRSV protein that interacts with FcRn. The virion surface proteins are expected to interact with FcRn for attachment and entry. We will identify the PRRSV proteins that interact with FcRn. Immunoprecipitation (IP) will be performed to identify the presence of the viral proteins in FcRn precipitates. Co-transfection of cells with plasmids encoding FcRn and individual PRRSV proteins GP2a, GP3, GP4, GP5, and M will be done. IP with protein G beads will be done to precipitate any binding proteins. Western blotting will be done to reveal the binding proteins. Further study will be conducted to verify the interaction between FcRn and the PRRSV protein identified. Completing these experiments will reveal the PRRSV protein that is responsible for the FcRn interaction.Characterize the molecular basis of the interaction between the PRRSV protein and FcRn. Protein-protein interaction modeling will be first conducted to determine the potential binding domain and amino acid residues in the binding interface. Based on the analysis, truncations of the viral protein will be designed and constructed. IP will be used to determine the interaction. After the critical domain/motif in the interaction is identified, we will conduct alanine scanning to find the stretch of residues that are important for the interaction with FcRn. Then site-directed mutagenesis will be conducted to identify the individual critical residues in the interaction. Finally, the key residues will be verified in full-length protein and PRRSV cDNA clone. Virus recovery from the mutant PRRSV cDNA clone will be done. We anticipate that the mutant virus will replicate much less than the wild-type in PAM cells if FcRn is needed for the virus entry. Completing these experiments will reveal the critical residues in the FcRn-interaction viral protein, further verifying the role of FcRn in PRRSV infection.
Project Methods
Objective 1. Determine if FcRn plays a role in PRRSV infection of PAM cells.Assess the effect of FcRn antibodies on PRRSV infection of PAMs. PAM cells will be cultured for adaptation 24 h before virus inoculation. Antibodies against FcRn will be added to the culture before the virus inoculation, as described. IgG isotype control will be included in the experiment. The cells with the antibodies will be incubated at 4°C for 1 h, and then inoculated with PRRSV VR-2385 at an MOI of 0.01, 0.1 and 1. The culture supernatant will be harvested 12, 24, and 48 h post-infection (hpi), followed by titration in MARC-145 cells. If FcRn is needed for PRRSV entry, we expect the virus titers from the antibody-treated wells will be much lower than the control.Deplete FcRn in PAM cells and assess the effect on PRRSV infection. PAM cells will be treated with siRNA against FcRn for 2 days before inoculation with PRRSV. Non-target random siRNA will be included as a control. The cells will be harvested to determine FcRn expression and PRRSV yield at 24 and 48 hpi. We anticipate that the siRNA treatment will reduce FcRn expression and PRRSV replication if FcRn is needed for PRRSV infection in PAM cells.Determine whether the attachment or entry of PRRSV is affected by the FcRn depletion. The PAM cells treated with the siRNA against FcRn will be used to test PRRSV attachment and entry. Non-target random siRNA will be included as a control. The cells will be incubated with PRRSV at an MOI of 1 and 10 for 1 h at 4°C, followed by removal of the inoculum and rinse with PBS. The cells will be lyzed for RNA isolation to determine the PRRSV RNA level for attachment assay. For entry assay, the cells will be incubated at 37°C for 1 h, followed by treatment with protease K to remove binding virions, rinse, and lysis for RNA isolation. If FcRn has any role in PRRSV attachment, we expect that the cells treated with FcRn siRNA will have less PRRSV RNA than the control in the attachment assay. If FcRn has any role in PRRSV entry, we expect that the PRRSV RNA in the cells treated with FcRn siRNA will be much lower than the control. This experiment will reveal the role of FcRn in PRRSV entry.Objective 2. Identify the PRRSV protein that interacts with FcRn.Identify the viral protein with immunoprecipitation (IP). MARC-145 cells will be used in this study for the robustness and accessibility of the cell line. PRRSV strain VR-2385 will be used for the experiment.MARC-145 cells will be inoculated with PRRSV VR-2385 at an MOI of 0.01, 0.1, and 1. The cells will be harvested 24 hpi for IP with antibodies against FcRn. Protein G magnetic beads will be used to precipitate the FcRn, followed by SDS-PAGE and Western blotting with antibodies against PRRSV. Convalescent pig serum with antibodies that recognize PRRSV structural proteins will be used for initial blotting. Based on the protein size of the potential binding partners, we will use monoclonal antibodies to determine the protein identity.Screen the viral proteins with co-transfection and IP. This experiment is designed to identify the individual viral proteins that potentially interact with FcRn in transiently transfected cells. Co-transfection of HEK293T cells with plasmids encoding porcine FcRn and individual PRRSV proteins GP2a, GP3, GP4, GP5, and M will be done. Plasmids encoding E and ORF5a will also be used. These viral proteins are virion surface proteins. We have the plasmid encoding porcine FcRn and the plasmids encoding individual PRRSV proteins as Myc-tagged fusion. IP and immunofluorescence assay (IFA) will be done to determine the potential interaction. Bimolecular fluorescence complementation (BiFC) will be performed to confirm the interaction.IP. The cells will be analyzed at 24 h post-transfection, followed by IP with FcRn antibodies. Protein G beads will be used to precipitate any binding proteins. Western blotting with Myc-tag antibody will be done to reveal the binding proteins. Reciprocal IP will be done to verify the interaction. It is known that PRRSV GP2a, GP3, and GP4 form a trimer and that GP5 and M form a dimer. In case FcRn binds to the conformational epitopes of the multimers, we will also conduct co-transfection with FcRn and three plasmids encoding GP2a, GP3, and GP4 or two plasmids encoding GP5 and M separately. IP with FcRn antibodies and blotting with Myc-tag antibody will be done.IFA. HeLa cells will be transfected with plasmids encoding FcRn and the individual viral proteins GP2a, E, GP3, GP4, GP5, ORF5a, and M. The cells will be fixed for IFA with antibodies against FcRn and Myc-tag, followed by confocal microscopy to observe the subcellular localization of these proteins. We anticipate that some of these viral proteins might co-localize with FcRn, which would indicate potential interaction.Objective 3. Characterize the molecular basis of the interaction between PRRSV protein and FcRn.Protein-protein interaction modeling will be first conducted to determine the potential binding domain and amino acid residues in the binding interface. Based on the analysis, truncations of the viral protein will be designed and constructed. IP will be used to determine the interaction. After the critical domain/motif in the interaction is identified, we will conduct alanine scanning to find the stretch of residues that are important for the interaction with FcRn. The next step will be site-directed mutagenesis to identify the critical residues in the interaction. Finally, the key residues will be verified in the full-length protein and the PRRSV cDNA clone.Determine the domains of FcRn and the viral protein in their interaction. Protein-protein interaction analysis will be performed with online programs such as AlphaFold. Based on the predictions of potential interacting domains and the protein structure analysis, truncations of FcRn and the viral gene will be constructed. Co-transfection and IP will be done as described above. We anticipate that some truncations of the viral protein and FcRn interact.Identify the critical residues for the interaction. Due to the limited time, we will focus on the viral protein in this experiment. Based on the AlphaFold analysis, we will conduct alanine scanning to determine the residues that are critical to the interaction with FcRn. Site-directed mutagenesis will be done for the alanine substitutions in the viral protein's truncation. Co-transfection and IP will be done as described above. We anticipate that some mutants of the viral protein will lose interaction with FcRn.Verify the role of the residues in the full-length protein. Site-directed mutagenesis will be performed to substitute the critical residues in the full-length viral protein with alanine. Co-transfection and IP will be performed as described above. We anticipate that some mutants of the viral protein will lose the interaction with FcRn.Verify the role of the residues in the PRRSV cDNA clone. Site-directed mutagenesis will be performed to substitute the critical residues in the PRRSV infectious cDNA clone. Virus recovery and titration will be done afterward. We anticipate that mutation of the residues will impair the viral replication.