Recombinant Gorilla gorilla gorilla C-C chemokine receptor type 5 (CCR5)

How does the expression system affect the functional properties of recombinant gorilla CCR5?

The expression system significantly impacts the functional properties of recombinant CCR5. When expressed in E. coli, the protein lacks post-translational modifications that would be present in mammalian cells, which may affect proper folding and function. Research comparing CCR5 from different expression systems (Pichia pastoris versus cell-free expression) has demonstrated that the lipid environment substantially impacts receptor-ligand interactions. For example, reconstitution studies show that cholesterol levels considerably decrease the binding affinity of antagonists like maraviroc to the CCR5 receptor. Therefore, researchers should consider these factors when designing functional assays with recombinant gorilla CCR5 .

How does gorilla CCR5 compare structurally and functionally to human CCR5?

Gorilla CCR5 shares high sequence homology with human CCR5, with nucleotide similarities ranging from 98.3-99.3% within the Pongidae family (which includes humans, chimpanzees, and gorillas). Amino acid substitutions between human and gorilla CCR5 tend to cluster in specific regions: the amino and carboxy termini, the first transmembrane domain, and the second extracellular loop. Despite this high homology, there are species-specific changes that characterize CCR5 homologues from primates within a given family .

A notable difference is that all nonhuman primates in the suborder Anthropoidea, including gorillas, have amino acid substitutions at positions 13 (N to D) and 129 (V to I) compared to humans. The substitution at position 13 is particularly significant as it is critical for CD4-independent binding of SIV to CCR5 .

What evidence exists for natural selection acting on CCR5 across primate species?

Multiple lines of evidence indicate that CCR5 has been subject to natural selection across primate species:

These findings suggest that CCR5 adaptations in response to pathogen pressure have been ongoing throughout primate evolution, likely predating HIV emergence .

How do evolutionary rates of CCR5 compare between primates and rodents?

Evolutionary rate analysis reveals significant differences between primates and rodents regarding CCR5 evolution:

What are the optimal conditions for reconstituting and storing recombinant gorilla CCR5?

For optimal reconstitution and storage of recombinant gorilla CCR5:

• Reconstitution protocol:

  • Centrifuge the vial briefly before opening to bring contents to the bottom

  • Reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (recommended 50%)

  • Aliquot for long-term storage

• Storage conditions:

  • Store at -20°C/-80°C upon receipt

  • Aliquoting is necessary for multiple use

  • Avoid repeated freeze-thaw cycles

  • Working aliquots can be stored at 4°C for up to one week

• Important considerations:

  • The protein is typically stored in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0

  • Repeated freezing and thawing is not recommended as it can compromise protein integrity

What methodologies are used for functional analysis of gorilla CCR5 in HIV/SIV entry studies?

Several methodologies are employed for functional analysis of gorilla CCR5 in HIV/SIV entry studies:

• Cell-cell fusion assays: This approach uses cells expressing CD4 and either human or nonhuman primate chemokine receptors to test their ability to support virus entry and membrane fusion. HOS.CD4 cell lines expressing the entire CCR5 homologue protein from various Anthropoidea species (including gorilla) can be constructed and tested with different HIV-1 and SIV env proteins .

• Chimeric receptor analysis: Researchers use CCR5/CCR2b chimeras to determine the structural requirements for CCR5 utilization by different virus strains. This approach has revealed that M-tropic and T-tropic SIV strains have different requirements for CCR5 utilization .

• Binding competition assays: These assays measure the binding affinity of ligands (such as maraviroc) to CCR5 reconstituted in different lipid environments. Methods include plasmon waveguide resonance and fluorescence anisotropy. For example, competition between a fluorescent CCR5 agonist and maraviroc can be measured to assess binding properties .

• Coarse-grained molecular dynamics simulation: This computational approach investigates how factors such as cholesterol impact receptor conformational flexibility and dynamics .

How can researchers amplify and sequence the full-length gorilla CCR5 gene?

To amplify and sequence the full-length gorilla CCR5 gene, researchers can follow this methodological approach:

• DNA isolation:

  • Extract total cellular DNA using a commercial DNA isolation kit

  • Quantify the nucleic acid content and use directly for PCR amplification

• PCR amplification:

  • Design primers based on conserved 5' and 3' untranslated regions that flank the entire CCR5 gene

  • Example primers: 5'-GGAGGGCAACTAAATACATTCTAGG-3' (forward) and 5'-GACTGGTCACCAGCCCACTTGAGTCC-3' (reverse)

  • If these don't work for gorilla samples, try primers located within the CCR5 coding sequence: 5'-ATGGATTATCAAGTGTCAAG-3' (forward) and 5'-TCACAAGCCCACAGATATTT-3' (reverse)

• PCR conditions:

  • Use 500 ng of input cell DNA per reaction

  • Program thermal cycler for: 5 cycles at 98°C for 10s, 55°C for 30s, and 72°C for 45s; followed by 35 cycles at 98°C for 10s, 60°C for 30s, and 72°C for 45s

• Cloning and sequencing:

  • Resolve PCR products on a 1.0% agarose gel

  • Extract the appropriately sized band using gel extraction

  • Insert purified PCR product into a cloning vector (e.g., pCR2.1 by TA cloning)

  • Select at least 6 clones to ensure a 97% probability of analyzing both alleles

  • Sequence inserts in both directions using dye-terminator chemistry

  • Assemble chromatograms and compute a consensus sequence for analysis

How do gorilla CCR5 structural differences affect its function as a coreceptor for SIV compared to HIV-1?

The structural differences in gorilla CCR5 significantly impact its function as a viral coreceptor. While human CCR5 serves as the principal coreceptor for M-tropic HIV-1 strains (with T-tropic strains using CXCR4), both M-tropic and T-tropic SIV strains can use gorilla CCR5. This functional difference is attributed to specific structural elements:

• Key amino acid substitutions: The amino acid substitution at position 13 (N to D) in gorilla CCR5 is critical for CD4-independent binding of SIV to CCR5, facilitating a different mode of viral interaction .

• Domain-specific interactions: Studies using CCR5/CCR2b chimeras show that M-tropic and T-tropic SIV strains have different structural requirements for CCR5 utilization. T-tropic SIV env proteins are more dependent on the second extracellular loop of CCR5, while M-tropic SIV env proteins interact with CCR5 in a manner similar to M-tropic HIV-1 env proteins .

• Multiple domain recognition: SIV strains can recognize multiple domains of gorilla CCR5, allowing them to use this receptor despite variations in specific regions. In contrast, HIV-1 strains have more specific requirements for receptor recognition .

These differences have evolved through host-pathogen co-evolution and help explain the different patterns of viral tropism observed across primate species .

What insights do evolutionary studies of gorilla CCR5 provide for understanding HIV resistance mechanisms?

Evolutionary studies of gorilla CCR5 offer valuable insights into HIV resistance mechanisms:

These evolutionary patterns provide a framework for understanding natural resistance mechanisms that could inform therapeutic approaches for HIV infection .

How can lipid composition affect gorilla CCR5 function in reconstituted systems?

Lipid composition significantly impacts gorilla CCR5 function in reconstituted systems, with important implications for experimental design and data interpretation:

• Cholesterol effects: Research shows that cholesterol considerably decreases the binding affinity of antagonists like maraviroc to the CCR5 receptor. In detailed competition binding assays between a fluorescent CCR5 agonist and maraviroc, CCR5 reconstituted with cholesterol showed markedly different binding properties compared to CCR5 without cholesterol .

• Membrane dynamics influence: Coarse-grained molecular dynamics simulations demonstrate that cholesterol impacts receptor conformational flexibility and dynamics. This affects not only ligand binding but potentially also the receptor's interaction with G proteins and other signaling partners .

• Expression system considerations: The choice of expression system (Pichia pastoris versus cell-free expression) can result in different lipid environments for the receptor, affecting its functional properties. These differences should be accounted for when comparing results across studies using different expression systems .

For accurate assessment of gorilla CCR5 function, researchers should carefully control the lipid composition in reconstituted systems and consider how this might differ from the native cellular environment .

What are the implications of CCR5 polymorphisms in gorillas for understanding natural SIV resistance?

CCR5 polymorphisms in gorillas provide important insights into natural SIV resistance mechanisms:

• Comparative analysis with other primates: Studies of CCR5 across primate species reveal that natural hosts of SIV have developed various adaptations to control CCR5-mediated entry pathways. While specific CCR5 polymorphisms in gorillas have not been as extensively characterized as in some other species, the comparative analysis highlights multiple adaptive mechanisms .

• Alternative co-receptor usage: In some natural hosts of SIV, the virus bypasses CCR5 restrictions by utilizing alternative co-receptors. For example, SIVrcm in red-capped mangabeys uses CCR2b, while SIVsmm in sooty mangabeys can use CXCR6. Understanding these alternative pathways in gorillas could reveal additional resistance mechanisms .

• Cell-specific expression patterns: The pattern of CCR5 expression in different T cell subsets is critical for understanding SIV pathogenesis. In natural SIV hosts, central memory T cells (Tcm) are relatively protected from SIV-mediated depletion through the downregulation of CCR5. Similar mechanisms may operate in gorillas, affecting their susceptibility to SIV infection .

These findings suggest that multiple genetic and regulatory mechanisms may contribute to SIV resistance in gorillas, with potential applications for understanding HIV resistance in humans .

How do mismatch distribution analyses inform our understanding of CCR5 evolution in great apes?

Mismatch distribution analyses provide valuable insights into the evolutionary history of CCR5 in great apes:

• Multimodal distribution pattern: The distribution of pairwise haplotype distances between CCR5 haplotypes in great apes (including gorillas) is multimodal rather than unimodal. This pattern contrasts with most other loci studied in continental populations, which typically show a unimodal distribution under population growth or directional selection .

• Evidence of balancing selection: The multimodal or ragged mismatch distribution observed in CCR5 is consistent with balancing selection or a stationary population size. The level of raggedness in this distribution is highly significant (P<0.001), supporting the hypothesis that two or more divergent haplotypes are being maintained within populations by balancing selection .

• Rare-allele advantage mechanisms: The balancing selection acting on CCR5 necessarily involves some type of rare-allele advantage, which could operate through:

  • Generalized over-dominance: Heterozygotes maintain a selective advantage over homozygotes

  • Negative frequency-dependent selection: The fitness of a genotype decreases as it becomes more common in the population

These analyses support the hypothesis that CCR5 diversity in great apes, including gorillas, has been shaped by long-term balancing selection rather than neutral processes or recent selective sweeps .