CD163 Porcine

What is CD163 and what is its significance in porcine research?

CD163 is a approximately 120 kDa single-pass type 1 transmembrane cell surface glycoprotein expressed on cells of the monocyte/macrophage lineage in pigs. It belongs to the scavenger receptor cysteine-rich (SRCR) superfamily and functions as a receptor for hemoglobin-haptoglobin complexes. The protein's significance in porcine research stems primarily from its role as a key receptor for PRRSV, making it a critical factor in understanding viral pathogenesis and developing strategies for disease control. CD163 contains nine SRCR domains, two linker domains (also called proline serine threonine domains), a cytoplasmic domain, and a short cytoplasmic tail, with each domain potentially contributing to different biological functions .

What cell types express CD163 in pigs and how can they be identified?

CD163 expression is restricted to cells of the monocyte-macrophage lineage, with porcine alveolar macrophages (PAMs) being the principal target cells for PRRSV infection. In normal primary PAMs, nearly all cells (approximately 85%) are CD163 positive. For identification and quantification, flow cytometric analysis using specific antibodies such as mouse anti-pig CD163 antibody (clone 2A10/11) can be employed to detect CD163 expression. For FACS analysis, approximately 10 μL of working dilution antibody can be used to label 1x10^6 cells in 100 μL .

How does CD163 expression relate to PRRSV susceptibility?

The relationship between CD163 expression and PRRSV susceptibility follows a threshold-dependent pattern. Research has demonstrated that:

• Cells with low CD163 abundance (approximately 20%) do not initiate PRRSV infection

• Cells with moderate CD163 abundance display limited susceptibility

• High levels of constitutive CD163 expression are required for effective PRRSV infection

This indicates that CD163 expression must reach or exceed a specific threshold for PRRSV to establish successful infection. This understanding is crucial for experimental design when studying viral entry and replication mechanisms .

Which domains of CD163 are essential for PRRSV infection?

Deletion and chimeric mutant studies have identified that SRCR domain 5 (SRCR 5) is essential for PRRSV infection. The four N-terminal SRCR domains (SRCR 1-4) and the cytoplasmic tail are not required for virus entry. Other CD163 protein domains need to be present but can be replaced by corresponding SRCR domains from CD163-L1, resulting in:

• Reduced infection efficiency when SRCR 6 and interdomain regions are replaced

• Unchanged infection efficiency when SRCR 7 to SRCR 9 are replaced

Additionally, antibodies specifically recognizing SRCR 5 can reduce PRRSV infection, further confirming this domain's critical role in viral entry .

How do different CD163 splice variants affect PRRSV infection?

In immortalized PAMs (iPAMs), alternative splicing of CD163 mRNA results in a short-form transcript lacking SRCR tandem repeats 1 to 5 of the functional domain, rendering these cells less susceptible to PRRSV infection. This demonstrates that specific structural features of CD163, particularly the transmembrane domain and complete SRCR domains, are essential for PRRSV receptor functionality .

What differentiates type 1 and type 2 PRRSV in their interactions with CD163?

Despite phenotypic similarities between type 1 and type 2 PRRSV at several levels, research has revealed distinct differences in how these viral genotypes recognize CD163. Studies with CD163-modified pigs have confirmed that while CD163 serves as the likely receptor for all PRRS viruses, there is a clear distinction between viral genotypes in their CD163 recognition patterns. This distinction is important when designing experiments to study receptor-virus interactions or when developing targeted interventions against specific PRRSV genotypes .

How can researchers isolate and maintain CD163-expressing cell lines for PRRSV studies?

To establish stable cell lines expressing CD163 for PRRSV studies, researchers can:

• Isolate total cellular RNA from appropriate source cells (e.g., PAMs, U937, MARC-145)

• Amplify CD163 transcripts using RT-PCR with specifically designed primers based on known CD163 sequences

• Clone the full-length CD163 cDNA into appropriate expression vectors (e.g., pRSV-Script)

• Transfect non-permissive cell lines (e.g., porcine kidney, feline kidney, or baby hamster kidney cells)

• Select stable transfectants using appropriate antibiotics (e.g., G418 sulfate at 500-1,000 μg/ml)

• Maintain cells in suitable media such as DMEM supplemented with 2-10% gamma-irradiated fetal bovine serum, antibiotics, GlutaMax, and sodium pyruvate

• Verify CD163 expression levels using flow cytometry

These stable cell lines can be used for virus susceptibility assays and studying CD163-PRRSV interactions .

What methods can be used to quantify CD163 abundance on cell surfaces?

Quantification of CD163 abundance on cell surfaces can be achieved through:

• Flow cytometric analysis using fluorochrome-conjugated anti-CD163 antibodies (e.g., PE-conjugated mouse anti-pig CD163, clone 2A10/11)

• Immunofluorescence microscopy with appropriate antibodies

• Western blotting for total protein analysis

• RT-PCR for transcript analysis, which can identify full-length versus short-form CD163 mRNA

For flow cytometric sorting technology, researchers can isolate CD163-positive single-cell-derived clones with varying CD163 abundances to study the relationship between receptor abundance and viral infection. This approach has revealed that CD163 levels remain stable during cell passage, with no significant changes in expression or viral susceptibility over at least 10 passages of high-CD163-expressing cells .

How should experiments be designed to evaluate the role of CD163 in PRRSV attachment versus entry?

To distinguish between CD163's role in PRRSV attachment versus entry, researchers should design multi-phase experiments:

• Virus attachment assay: Performed at 4°C to allow binding but prevent internalization, followed by washing and quantification of attached virions. This approach has shown that virus attachment is not influenced by CD163 abundance.

• Entry/infection assay: Conducted at 37°C to allow complete viral entry, followed by incubation and assessment of viral replication (e.g., through luciferase reporter assays).

• Control studies: Include alternative viruses (e.g., pseudorabies virus) that do not depend on CD163 for entry to validate the specificity of findings.

Research using this approach has demonstrated that while CD163 abundance significantly affects PRRSV infection, it does not influence virus attachment during the initial cell binding phase, suggesting its critical role is in the post-attachment entry process .

How does CD163 from different species affect PRRSV host range and inter-species transmission?

CD163 from various species can serve as functional PRRSV entry receptors when expressed in non-permissive cells. Transfection experiments have shown that full-length CD163 cDNAs from pig, human, African green monkey, mouse, and dog encode functional PRRSV receptors. This cross-species functionality may be critical for understanding potential inter-species transmission risks.

The ability of CD163 from multiple species to support PRRSV infection when expressed in non-permissive cells suggests that species barriers to PRRSV infection are likely determined by CD163 expression patterns or levels rather than fundamental structural incompatibilities. This has significant implications for assessing potential reservoir hosts and transmission risks in mixed agricultural settings .

What are the implications of CD163 gene modification for developing PRRSV-resistant pigs?

CD163 gene modification represents a promising approach for developing PRRSV-resistant pigs. Key findings include:

• CD163 knockout (KO) pigs are resistant to infection with genotype 2 (type 2) PRRSV

• Targeted modifications of specific domains (particularly SRCR 5) can potentially confer resistance while preserving important CD163 functions

• The threshold-dependent nature of CD163-mediated infection suggests that partial reduction of expression might provide significant protection

The challenge remains to develop refinements in the modification of CD163 with the goal of making pigs refractory to infection while retaining important biological functions such as hemoglobin-haptoglobin complex binding and the associated anti-inflammatory effects. This requires careful consideration of which domains to target for modification while preserving others .

How do alternative splicing mechanisms affect CD163 expression and function in different cell types?

Alternative splicing significantly impacts CD163 expression and function across different cell types. In immortalized PAMs (iPAMs), CD163 mRNA undergoes alternative splicing, producing two transcript forms:

• Normal full-length CD163 in primary PAMs

• A short form (~1500 bp) lacking SRCR1 to SRCR5 in transformed iPAMs

This alternative splicing pattern appears to be influenced by cellular transformation, as observed in both PAM-Tang cells and CRL-2483 cells transformed using SV40 large T antigen. The predominance of short-form CD163 transcripts explains why only approximately 20-35% of transformed cells remain CD163-positive at the protein level.

Understanding these alternative splicing mechanisms is crucial for interpreting results from immortalized cell lines and may provide insights into potential regulatory mechanisms for CD163 expression that could be targeted for therapeutic interventions .

How should researchers interpret discrepancies between CD163 mRNA and protein expression?

When confronted with discrepancies between CD163 mRNA and protein expression, researchers should:

• Examine alternative splicing: As seen in iPAMs, alternative splicing can produce short-form transcripts that do not result in functional surface CD163

• Assess post-translational modifications: CD163 is a glycoprotein, and changes in glycosylation may affect antibody detection or protein stability

• Evaluate subcellular localization: CD163 must be properly trafficked to the cell surface to function as a PRRSV receptor

• Consider protein turnover rates: Differences between mRNA and protein levels may reflect variations in protein stability and degradation

In experimental settings, researchers should employ multiple detection methods (RT-PCR for transcripts, flow cytometry for surface expression, and functional assays for receptor activity) to comprehensively assess CD163 status .

What control experiments are essential when studying CD163-mediated PRRSV infection?

Essential control experiments when studying CD163-mediated PRRSV infection include:

• Cellular controls:

  • CD163-negative parental cell lines

  • Cells expressing CD163 variants lacking specific domains

  • Cells expressing CD163 from different species

• Viral controls:

  • Different PRRSV isolates (type 1 and type 2)

  • Non-PRRSV viruses (e.g., pseudorabies virus) to verify CD163 specificity

• Experimental controls:

  • Virus attachment assays at 4°C to distinguish attachment from entry

  • Antibody blocking studies using domain-specific anti-CD163 antibodies

  • Assessment of CD163 stability across multiple cell passages

These controls help distinguish CD163-specific effects from other cellular factors and ensure experimental reproducibility and validity .

How can researchers accurately assess the threshold level of CD163 required for PRRSV infection?

To accurately assess the threshold level of CD163 required for PRRSV infection, researchers should:

• Establish cell populations with varying CD163 expression levels using:

  • Flow cytometric cell sorting to isolate single-cell-derived clones with different CD163 abundances

  • Inducible expression systems to modulate CD163 levels

• Quantify CD163 expression using:

  • Flow cytometry with appropriate calibration standards

  • Quantitative Western blotting

  • Surface protein biotinylation assays

• Correlate CD163 levels with infection parameters:

  • Viral entry efficiency

  • Viral replication kinetics

  • Progeny virus production

Research has shown that cells with approximately 20% CD163 abundance do not initiate PRRSV infection, while increasing CD163 abundance enhances susceptibility in a non-linear manner. This suggests a threshold-based model rather than a simple linear correlation between receptor abundance and infection efficiency .