EFFECT OF MODIFICATIONS ON CD163 AND VIRAL ENVELOPE GLYCOPROTEINS ON VIRUS-HOST INTERACTIONS

Methods:The hypothesis under this objective is that specific amino acids within the SRCR4/5 interdomain, thePSTII domain and thepentapeptide (ADHFGE) participate in the resistance of CD163-transfected cells to PRRSV infection.The first step is to generatemutations in the CD163 regions described above to find the minimal requirements of CD163 to support viral infection.Plasmids expressing porcine CD163 fused to enhanced green fluorescent protein (EGFP) or to a FLAG epitopewill be used.Mutations will be prepared by usinga Q5 site-directed mutagenesis kit.Correct orientation of the mutant constructs will be confirmed by sequencing and by measuring cell surface expression using anti-CD163 antibody.The CD163 constructs fused to GFP or FLAG will be transfected into PRRSV nonpermissive cells.Transfected cells will be incubated for 24h,and EGFP expression will be monitoredunder a fluorescence microscope. At 48h post-transfection, the cells will be infected with a PRRSV-2 isolateexpressing RFP (red fluorescence protein). The PRRSV infected cells will be detected by the co-localization of green and red fluorescence in the same cell.Colocalization analyses will be performed using ImageJ software. To detect PRRSV-1 virus infection, cells will be immunostainedwith a PRRSV N protein antibody. To detect CD163 mutants fused to FLAG, cells will be immunostained with FLAG antibodies.All the data will be represented as three independent experiments in triplicate and analyzed using student’s t test.CD163 has a variety of important biological functions, including the clearance of cell-free hemoglobin (Hb) and Hb/haptoglobin (Hb-Hp) complexes from the blood. For future antiviral therapies it is crucial to refine the genomic editing of CD163 sufficient to preserve its biological functions while blocking PRRSV infection. To confirm whether the CD163 variants are still able to take up Hb-Hp complexes, we will be evaluated the uptake of Hb-Hp by labelling Hb with Alexa Fluor 488 (AF488). Cells expressing different CD163 variants will be incubated with HbAF488-Hp complexes and uptake will be analyzed by FACS and immunofluorescence (IF).To evaluate whether antibodies that recognize the ADHFGE pentapeptide exposed on the SRCR5 surface can prevent infection, cells expressing different CD163 mutant variants will be incubated with different dilutions of specific antibodies targeting ADHFGE for 1h at 37°C, followed by inoculation with PRRSV for 1h at 37°C. After inoculation, cells will be washed and further incubated for 24-48h at 37°C. The PRRSV infected cells will be detected as indicated above.Objective 2aMethods:We will perform immunoprecipitation (IP) and colocalization studies to confirm that the CD163 regions required for PRRSV infection are not necessary for their association with the viral glycoproteins. We will test the interaction of SRCR4-5 interdomain fused to SRCR5, and PSTII domain with viral glycoproteins.The results of this experiments will confirm that the CD163 domains required for infection are not necessary for the binding to individual viral glycoproteins or the existence of “Multi-Domain” model for describing the interaction between PRRSV and CD163.To determine whether there is a direct interaction between CD163 and the viral glycoproteins, we will perform pulldown assays using purified proteins. Due to viral envelope proteins are heavily glycosylated, to purify CD163 and the viral glycoproteins, we will use thebaculovirus expression system.The recombinant baculovirus will be generated using the Bac-to-Bac system.For construction of the transfer vector, the viral glycoproteins fused to HA epitope or CD163-FLAG will be cloned into the pFastBac-1 vector containing a One-Strep tag. The correctness of the constructs will be confirmed by sequencing. The pFast-Bac1 vectors containing the genes of interest will be recombined into the bacmid bytransformation of DH10 Bac cells. The presence of the different genes in the bacmidswill be confirmed by sequencing. The bacmids will be then used to generate the corresponding recombinant baculovirus.The bacmids will be transfected into Sf9 cells. The cells will be incubated at 28°C for 3 days, and recombinant viruses expressing the proteins of interest will be harvested.To purify CD163-FLAG and the viral glycoproteins, Sf9 insect cells growing in suspension will be infected with recombinant baculovirus and incubated at 28°C for 72 h.Cells will be collected by centrifugation and resuspended in lysis buffer. The lysate will be clarified by centrifugation, and the recombinant proteins will be purified by affinity chromatography, followed by ion-exchange chromatography. The purity of the recombinant proteins will be analyzed by SDS-PAGE and Western blotting using anti-FLAG and anti-HA antibodies.Pulldown assays to determine if there is a direct interaction between CD163-FLAG and the viral glycoproteins will be performed byusing anti-FLAG agarose beads. In these experiments, we will also use CD163 mutant variants to determine which modifications in CD163 affect to its association with the viral proteins.Objective 2bMethods:Adaption will be performed by the multiple serial passage of PRRSV-1 and PRRSV-2 isolates on cells stably expressing CD163 variants. Adaptation is defined as an increase in virus infection during passage. The approach will be to grow a mutant cell line on a 24 well plate.We possess PRRSV-2-RFP and PRRSV-1-EGFP isolates. The virus stock will be titrated on the confluent cells using serial 1/10 dilutions. After 1hincubation, medium will be removed, and wells washed to remove excess virus. Growth of virus will be monitored under a fluorescence scope. After 3-5 days, the titer will be recorded and the medium from the last well showing substantial growth will be recovered and used for the next passage round. These steps will be repeated until the titration endpoint of the virus on a mutated CD163 is the same as the WT virus propagated on a WT CD163 expressing cell.At the end of the adaptation process, the structural genes will be sequenced, and the peptide sequences compared to the parent virus sequence. To confirm that an amino acid change is involved in adaptation, we will make the same amino acid changes in an infectious clone and test the virus for infection. IP and colocalization studies will be performed to determine if a particular mutation affects the interaction between CD163 and the viral glycoproteins.Objective 2cMethods:We will perform IP and colocalization studies to identify specific sequences in the viral glycoproteins that interacts with CD163.Mutations in the viral glycoproteins will be prepared as indicated above. The role of N-glycosylation of the viral glycoproteins on the binding to CD163 will be also evaluated with the same methods.The approach is to remove individual N-sites by replacing Asn with Gln by mutagenesis.A second approach will be used to confirm the peptide sequences in PRRSV involved in the recognition of CD163. To this end, we will generateinfectious clones harboring the same mutations that affect the association with CD163. The infectious clones used for mutations under this objective are the same viruses used in Objective 1.Theinfectious clone plasmidswill be transfected into a PRRSV-nonpermissive cell line, and the virions collected. The mutated virions will be tested for infection on cells expressing CD163.The ORFs of the structural viral genes overlap. Therefore, the mutations performed for these experiments will mostly incorporate alanine substitutions. Making deletions would be more difficult, since a deletion in one gene may produce a deletion in the overlapping gene. Any loss of infection will be followed up by IP/ colocalization experiments to determine if a mutation in the viral glycoproteins results in the loss of binding to CD163.