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

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1031165
• Grant No.: 2023-67015-40731
• Cumulative Award Amt.: $650,000.00
• Proposal No.: 2022-08199
• Project Start Date: Sep 1, 2023
• Project End Date: Aug 31, 2027
• Grant Year: 2023
• Program Code: [A1221]- Animal Health and Production and Animal Products: Animal Health and Disease

Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801

Performing Department
(N/A)

Non Technical Summary
Porcine reproductive and respiratory syndrome virus (PRRSV) was first described in the United States in 1987 as an infectious agent responsible for a "mystery swine disease". Clinical signs after infection include reproductive failure during late gestationsuch as abortions, respiratory distress in young pigs, and poor growth performance.Furthermore, PRRSV is often complicated by secondary infections that can lead to a much more severe disease and increased mortality.PRRSV is currently endemic in most swine-producing countries and is the most economically destructive virus affecting swine worldwide. Vaccines and other control measures have not proved effective in disease control and elimination. PRRSV is divided into two distinct species, PRRSV-1 and PRRSV-2.The virus mainly infects porcine alveolar macrophages.Porcine CD163 protein serves asthe majorcellular entry receptor for PRRSV. Our previous work showed that genetically modified pigs lacking expression of CD163 on macrophages are completely resistant to infection with PRRSV. Since CD163 is critical for PRRSV infection, the goal of the proposed project is to identify specific regions and domains in CD163 implicated in viral infection.The first objective is the use of a novel in vitro system to map the CD163 peptide sequences recognized by PRRSV-1 and PRRSV-2 genotypes. The goal of this objective is to generate modifications in CD163 that prevent PRRSV infection without affecting key biological functions of the viral receptor.The second objective of the proposed project is to identifyspecific regions in viral proteins and CD163 involved in virus-receptor binding. Understanding the molecular basis for the interaction between CD163 and PRRSV creates the opportunity to explore novel control measures, such as vaccines that target putative neutralizing epitopes and antiviral drugs, which can interrupt the interaction.Our findings will provide critical information for understanding CD163-mediated PRRSV infection and enhance our comprehension of viral pathogenesis. The results obtained from this proposal will be instrumental for the development of novel strategies to prevent or block PRRSV infection and will provide new data that can be used to control infection with new vaccines and small molecules that can disrupt the virus-CD163 interaction. Furthermore, our results will provide significant information to design a new set of CD163 receptors that are resistant to PRRSV infection but structurally and functionally intact.

Animal Health Component

40%

Research Effort Categories

Basic

60%

Applied

40%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4030	1101	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4030 - Viruses;

Field Of Science
1101 - Virology;

Keywords

cd163

prrsv

pstii

srcr4-5 interdomain

srcr5

srcr7

viral glycoproteins

Goals / Objectives
Porcine reproductive and respiratory syndrome virus (PRRSV) is the most costly disease in the history of swine production. PRRSV is now endemic in most swine-producing countries. The impact of PRRSV on production continues to be approximately $500-600 million per year in losses. Vaccines have been largely ineffective in controlling the disease and possess several limitations, including reversion to virulence, absence of protection against genetically diverse field isolates, continuous shedding of vaccine virus, and transplacental infections. The next generation of vaccines designed to correct these deficiencies is several years away. Our group demonstrated that genetically modifiedpigs lacking the PRRSV receptor, CD163, are completely resistant to PRRSV. Theseresults provided the first clear demonstration that CD163 modifications provide a great opportunity to completely prevent the infection of pigs with PRRSV. The main goals of this proposed research are:Goal 1- Determine the role of CD163 inporcine reproductive and respiratory syndrome virus (PRRSV) infection. This proposed goal aims to generate modifications in CD163 receptor sufficient to preserve its biological functions while blocking PRRSV infection.This goal includes one objective:Objective 1. Define novel domains and peptide sequences in CD163, which control infection with PRRSV-1 and PRRSV-2 viruses. This objective aims to identify peptide sequences in CD163 that are involved in PRRSV-1 and PRRSV-2 infections. The purpose is to make modifications in CD163, which confer PRRSV resistance while retaining the structural conformation of CD163 and maintaining key biological functions. Goal 2-A second overall aim is the use of modifications in PRRSV glycoproteins to gain a better understanding of the interaction between PRRSV and corresponding peptide sequences on CD163 receptor. This goal includes one objective divided into three sub-objectives:Objective 2. Identify peptide sequences in viral proteins and CD163 involved in virus-receptor binding. This objective aims to identify specific regions and/or domains in viral proteins and CD163 involved in virus-receptor association.Understanding the molecular basis for the interaction between CD163 and PRRSV creates the opportunity to explore novel control measures, such as vaccines that target putative neutralizing epitopes and antiviral drugs, which can interrupt the interaction.Objective 2a.Identify peptide sequences and domains in CD163 implicated in recognition of PRRSV. The main purpose of this sub-objective is to determine which CD163 regions are important for infection, and binding to the viral envelope proteins.Objective 2b. Identify mutations that result from adaptation of PRRSV to cells expressing mutant CD163 proteins. This sub-objective aims to stablish which viral proteins are involved in receptor recognition by performing serial-passages of PRRSV on cells expressing mutant CD163 receptors.Objective 2c. Identify mutations in PRRSV glycoproteins that prevent infection. The purpose of this sub-objective is to identify amino acids in the viral envelope glycoproteins that influence infection through direct interactions with CD163.

Project Methods
Objective 1Methods: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.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience: The target audiences include fellow scientists, particularly virologists and swine geneticists, as well as swine producers and genetics companies. It also encompasses researchers working in porcine disease control and prevention, members of the general public concerned about swine diseases, journalists covering swine health issues, and graduate and undergraduate students studying viral diseases in swine. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?So far, this project has been directly associated with the training of one PhD student and two undergraduate students. For the graduate student, this project will serve as the foundation for his thesis work. Additionally, it will provide an opportunity to develop a broad range of skills necessary for a career in pathobiology, including experimental design and logistics, molecular biology, and data analysis and interpretation. The two undergraduate students involved in this project have gained a deep understanding of the scientific method and valuable experience in laboratory methodologies. How have the results been disseminated to communities of interest?The preliminary results presented in this grant were disseminated through an oral presentation at the 2023 NAPRRS/NC229 International Conference of Swine Viral Diseases (ICSVD). Additionally, preliminary results identifying peptide sequences in GP2 involved in virus-receptor binding were presented at a graduate research symposium at UIUC. What do you plan to do during the next reporting period to accomplish the goals?We are currently in the process of identifying peptide sequences or domains in viral proteins that are involved in virus-receptor binding. To achieve this, we have created different deletion mutants of the viral envelope glycoproteins. Binding experiments between CD163 and the deletion mutants will be performed to identify the peptide sequences critical for receptor binding and PRRSV infection. From this data, we also expect to identify mutations in PRRSV glycoproteins that hinder PRRSV infection.

Impacts
What was accomplished under these goals? Objective 1 - We have completed the mapping of sites in porcine CD163 that are important for PRRSV infection. Infection experiments demonstrated resistance to PRRSV following the deletion of specific regions of CD163: the SRCR4/5 interdomain region and exon 13, which encodes the first 12 amino acids of the 16-amino-acid PSTII domain. Additionally, we identified a specific pentapeptide located in the SRCR5 and SRCR7 domains that is critical for PRRSV infection. Our findings demonstrated that CD163 mutant proteins resistant to infection still retained the ability to interact with the viral enevelope glycoproteins. This suggests that while multiple domains contribute to infection, they do not necessarily influence the binding of viral glycoproteins. It may indicate that the envelope viral proteins form multiple interactions with CD163 or that the receptor regions important for infection require other cellular binding partners for PRRSV infection. In collaboration with a research group from Acceligen that generated pigs with a defined CD163 exon 13 deletion (ΔExon13 pigs), we found the following: The gene-edited pigs were completely resistant to PRRSV-2 infection; Alveolar macrophages derived from genetically modified pigs were also fully resistant to both PRRSV-1 and PRRSV-2 infections; and the deletion of exon 13 did not compromise the main biological functions associated with CD163.These results demonstrate that a modification of CD163 through a precise deletion of exon 13 provides a strategy for protection against PRRSV infection. Objetive 2. To analyze the role of N-glycosylation in PRRSV infection, we examined the specific contribution of envelope protein-linked N-glycans to the infection of permissive cells. For this purpose, we developed a novel strategy to modify these N-glycans, involving the production of monoglycosylated PRRSV and viral glycoproteins with varying glycan states. Our results indicate that the removal or alteration of N-glycans from PRRSV significantly affects viral infection. Specifically, we found that complex N-glycans are essential for efficient infection in cell cultures. Furthermore, the presence of high-mannose type glycans on the PRRSV surface is the minimal requirement for productive viral infection. Notably, PRRSV-1 and PRRSV-2 have different N-glycan structure requirements for optimal infection. We also demonstrated that the removal of N-glycans from PRRSV does not affect viral attachment, suggesting that these carbohydrates play a critical role in regulating viral entry. Bindingexperiments further revealed that N-glycans present in the viral envelope glycoproteins are not necessary for binding to the essential viral receptor CD163. Finally, we found that N-glycans in CD163 are not required for PRRSV infection.In conclusion, we found that the presence of N-glycans in the viral glycoproteins appears to be necessary for efficient infection of permissive cells. Additionally, our findings provide important insights into PRRSV infection and enhance our understanding of viral pathogenesis.

Publications

• Type: Journal Articles Status: Published Year Published: 2024 Citation: Salgado, B., Rivas, R.B., Pinto, D., Sonstegard, T.S., Carlson, D.F., Martins, K., Bostrom, J.R., Sinebo, Y., Rowland, R.R., Brandariz-Nu�ez, A., 2024. Genetically modified pigs lacking CD163 PSTII-domain-coding exon 13 are completely resistant to PRRSV infection. Antiviral Research 221, 105793
• Type: Journal Articles Status: Published Year Published: 2024 Citation: Rowland, R.R., Brandariz-Nu�ez, A., 2024. Role of N-linked glycosylation in porcine reproductive and respiratory syndrome virus (PRRSV) infection. Journal of General Virology 105, 001994
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Oral presentation at 2023 NAPRRS/NC229 International Conference of Swine Viral Diseases (ICSVD). "Specific regions in CD163 required for PRRSV infection are not necessary for binding to viral envelope proteins." Ana M.M. Stoian, Raymond R.R. Rowland, Alberto Brandariz-Nu�ez.