HAVCR1 Mouse

What is HAVCR1 and its mouse ortholog (mHavcr1)?

HAVCR1 (also known as CD365, TIM1, or KIM1) was initially discovered as the cellular receptor for Hepatitis A Virus (HAV). The mouse ortholog, mHavcr1, shares significant homology with human HAVCR1 and performs similar functions. Both proteins contain an immunoglobulin-like (IgV) domain that is critical for HAV binding, followed by mucin and transmembrane domains. Research has demonstrated that both human HAVCR1 and mHavcr1 function as phosphatidylserine (PS) receptors that bind apoptotic cells and modulate immune responses .

Recent knockout studies have conclusively demonstrated that HAVCR1 is a functional HAV receptor, with evidence showing that transfection of the mouse ortholog (mHavcr1) into HAVCR1 knockout cells restores susceptibility to HAV infection . This conservation of function across species highlights the evolutionary significance of this receptor.

How do HAVCR1 haplotypes affect experimental outcomes in mouse models?

HAVCR1 exhibits significant genetic polymorphism in both humans and mice, which can substantially influence experimental outcomes. The gene contains single nucleotide polymorphisms (SNPs) and insertion/deletion variants throughout its sequence, particularly in exon 4 encoding the functional mucin domain .

In human studies, specific HAVCR1 haplotypes have been associated with differential susceptibility to viral infections. For instance, haplotype B has been linked to higher mRNA expression levels and increased susceptibility to rheumatoid arthritis . When working with mouse models, researchers must consider:

• Different mouse strains may carry distinct mHavcr1 haplotypes that affect receptor function and expression

• These haplotype differences can influence susceptibility to HAV infection and immune responses

• Polymorphisms in the mucin domain can alter receptor shedding and viral binding characteristics

Research has demonstrated that HAVCR1 haplotypes affect susceptibility to specific viral genotypes. For example, haplotype C was associated with significantly different distribution patterns when comparing patients infected with HCV genotype 1 versus non-genotype 1 (48.83% vs. 28.42%, P = 0.024, OR = 2.40) . This suggests that similar haplotype-dependent effects may exist for HAV strains in mouse models.

What CRISPR/Cas9 strategies are most effective for HAVCR1 knockout in mouse models?

Successful CRISPR/Cas9 knockout of HAVCR1 requires careful design considerations to ensure complete functional elimination of the receptor. Based on published research, the most effective strategies include:

• Targeting the IgV domain in exon 2, which is critical for HAV binding

• Using dual sgRNAs to create substantial deletions (100-101 bp) rather than small indels

• Ensuring the targeting strategy creates frameshift mutations that prevent expression of downstream domains

• Avoiding designs that might result in truncated but potentially functional receptor fragments

One successful approach involved introducing deletions of 100 bp in one copy and 101 bp in the other copy of the HAVCR1 gene in AGMK cells, resulting in complete knockout with reading frame shifts that prevented expression of all domains . This clean knockout strategy is superior to approaches that might leave soluble forms of HAVCR1 containing the functional IgV binding domain.

Validation of the knockout should include genomic sequencing to confirm the expected deletions, protein expression analysis to verify absence of the receptor at the cell surface, and functional testing to demonstrate resistance to HAV infection .

What cell systems are optimal for studying mHavcr1 function in viral infection?

The selection of appropriate cell systems is critical for studying mHavcr1 function in viral infection. Based on receptor dependency patterns, the following considerations should guide cell system selection:

• African Green Monkey Kidney (AGMK) cells:

  • Demonstrate high dependence on HAVCR1 for HAV infection

  • Show 72-96% protection against HAV infection when HAVCR1 is blocked by monoclonal antibodies

  • Provide the cleanest experimental system for isolating HAVCR1-specific effects

• Vero E6 cells (monkey):

  • Express alternative HAV receptors in addition to HAVCR1

  • Show only partial protection (37-45%) when HAVCR1 is blocked

  • Less suitable for studying HAVCR1-specific effects due to alternative receptor usage

• Huh7 cells (human hepatoma):

  • Express multiple HAV receptors

  • Show differential receptor usage for different forms of HAV (exosome-enclosed HAV versus naked virus particles)

  • Anti-HAVCR1 antibodies partially protect against exo-HAV (40%) but not vpHAV infection

For definitive studies of mHavcr1 function, AGMK cells provide the most straightforward system, as HAVCR1 knockout renders these cells resistant to HAV infection, and susceptibility can be restored by transfection with either human HAVCR1 or mouse Havcr1 cDNA .

How should researchers quantitatively assess HAV infection in HAVCR1 mouse models?

Quantitative assessment of HAV infection in HAVCR1 mouse models requires sensitive and specific assays. The following methodological approaches are recommended:

• CFU-based selection assays:

  • Utilize recombinant HAV containing antibiotic resistance genes (e.g., HAV-Bsd)

  • Infected cells gain resistance to antibiotics (blasticidin) and form colonies

  • Provides quantitative measurement of infection efficiency through colony counting

• Quantification methods for different HAV forms:

  • Separately assess infection by naked virus particles (vpHAV) and exosome-enclosed HAV (exo-HAV)

  • Purify virus preparations using established protocols for consistency

  • Compare infection rates between wild-type and HAVCR1-knockout cells

• Validation through multiple detection methods:

  • Immunofluorescence detection of viral proteins

  • RT-qPCR for viral RNA quantification

  • Flow cytometry for infected cell enumeration

• Statistical analysis:

  • Calculate protection percentages when using receptor-blocking antibodies

  • Apply appropriate statistical tests (P values below 0.05 considered significant)

  • Report odds ratios (ORs) and 95% confidence intervals (CIs) for comparative analyses

This multi-faceted approach ensures robust quantification of infection rates and receptor dependencies.

How does the mouse HAVCR1 ortholog compare to human HAVCR1 in mediating HAV infection?

Comparative analysis of mouse Havcr1 and human HAVCR1 reveals important insights into their receptor function:

• Functional conservation:

  • Transfection of mouse Havcr1 cDNA into HAVCR1 knockout AGMK cells restores susceptibility to both vpHAV and exo-HAV infection

  • This demonstrates that the mouse ortholog is a functional HAV receptor despite species differences

• Binding domain conservation:

  • The IgV domain, critical for HAV binding, shows high conservation between species

  • Both receptors utilize this domain for viral recognition and attachment

• Species-specific considerations:

  • Despite functioning as HAV receptors, mouse liver cells may contain alternative HAV receptors distinct from mHavcr1

  • This suggests species-specific differences in the repertoire of HAV receptors

• Experimental evidence:

  • Rescue experiments in knockout cells provide the strongest evidence for functional equivalence

  • Both human HAVCR1 and mHavcr1 restore HAV susceptibility when transfected into receptor-negative cells

This cross-species functionality makes mouse models valuable for studying HAV-receptor interactions, though researchers should remain aware of potential species-specific differences in receptor expression patterns and alternative receptor usage.

How can contradictory findings about HAVCR1 function be reconciled in experimental design?

The HAV receptor field has seen contradictory findings regarding HAVCR1 function. These contradictions can be reconciled through rigorous experimental design:

• Knockout strategy considerations:

  • Complete deletion of the IgV domain is essential for functional studies

  • Strategies leaving the IgV domain intact or creating only small indels may produce misleading results

  • Reading frame shifts that prevent expression of all receptor domains should be confirmed

• Cell line selection impact:

  • Different cell lines show variable dependence on HAVCR1 for HAV infection

  • Using cells with robust alternative receptor expression (like Vero E6 or Huh7) may mask HAVCR1-specific effects

  • AGMK cells provide the clearest system for isolating HAVCR1-dependent phenotypes

• Validation through rescue experiments:

  • Definitive evidence comes from restoring susceptibility to HAV in knockout cells

  • Both human HAVCR1 and mouse Havcr1 should be tested in rescue experiments

  • Domain mutants can help map functional regions required for receptor activity

• Virus preparation considerations:

  • Separate testing of naked virus particles (vpHAV) and exosome-enclosed HAV (exo-HAV)

  • Different HAV forms may show distinct receptor dependencies

  • Standardized virus preparations should be used across experiments

What is the role of HAVCR1 in immune modulation and how does this affect HAV pathogenesis?

Beyond its function as a viral receptor, HAVCR1 plays significant roles in immune modulation that may influence HAV pathogenesis:

• B-cell regulation:

  • In vitro stimulation of activated B cells with anti-TIM-1 monoclonal antibodies enhances proliferation and immunoglobulin production

  • This suggests HAVCR1 involvement in modulating humoral immunity during infection

• Phosphatidylserine receptor function:

  • HAVCR1 binds phosphatidylserine (PS) on apoptotic cells

  • This function modulates immune responses and may contribute to viral apoptotic mimicry

  • HAV may exploit this immune-regulatory function to establish persistence

• Implications for HAV pathogenesis:

  • The long incubation period of HAV (up to 4 weeks) remains poorly understood

  • HAVCR1's immunomodulatory functions may help explain how HAV evades the immune response during this period

  • The subsequent necroinflammatory process that clears infection likely involves HAV-HAVCR1 interactions

• Research approaches:

  • Study HAV infection in mice with selective immune cell-specific HAVCR1 knockout

  • Examine how HAVCR1 haplotypes affect immune responses to HAV

  • Investigate the timing of immune activation in relation to HAVCR1 engagement

These immunomodulatory properties may explain why HAV evolved to use HAVCR1 as its receptor, potentially allowing the virus to manipulate host immune responses during infection.

What controls are essential when validating HAVCR1-mediated effects in mouse models?

Robust validation of HAVCR1-mediated effects requires comprehensive controls:

• Genetic controls:

  • Wild-type cells with endogenous HAVCR1 expression

  • HAVCR1 knockout cells generated with the same methodology

  • Rescue cell lines with reintroduced wild-type HAVCR1

  • Cross-species validation using both human HAVCR1 and mouse Havcr1

• Antibody-based controls:

  • Blocking experiments with anti-HAVCR1 monoclonal antibodies (e.g., mAb 1D12)

  • Isotype-matched control antibodies

  • Dose-response series to establish specificity

• Infection assay controls:

  • Parallel testing of vpHAV and exo-HAV preparations

  • Quantitative infection measurement (e.g., Bsd-resistant colony formation)

  • Multiple cell lines with varying HAVCR1 dependence

• Statistical validation:

  • Report P values with appropriate corrections for multiple comparisons

  • Calculate odds ratios (ORs) and 95% confidence intervals (CIs)

  • Use statistics software like Statcalc (Epi Info) for consistency

These controls ensure that observed effects are specifically attributable to HAVCR1 function rather than experimental artifacts or alternative pathways.

How should researchers interpret variable receptor dependency across different cell types?

Variability in HAV receptor dependency across cell types represents an important biological phenomenon that requires careful interpretation:

• Quantitative assessment:

  • HAVCR1 blocking antibodies protect AGMK cells against HAV infection by 72-96%

  • The same antibodies protect Vero E6 cells by only 37-45%

  • For Huh7 cells, protection varies by virus form: 40% for exo-HAV but ineffective against vpHAV

• Interpretation framework:

  • Complete protection (>90%) suggests primary dependence on HAVCR1

  • Partial protection (30-70%) indicates significant alternative receptor usage

  • Differential protection between virus forms suggests form-specific receptor pathways

• Cell type considerations:

  • Primary hepatocytes may display different receptor dependencies than cell lines

  • Species-specific differences may exist in receptor expression patterns

  • Tissue context can influence receptor availability and function

• Experimental approach:

  • Test multiple cell types when characterizing receptor dependencies

  • Quantify protection percentages rather than binary protected/not protected outcomes

  • Consider both virus form (vpHAV vs. exo-HAV) and cell type in experimental design

This variability highlights the complexity of HAV-receptor interactions and underscores the importance of studying multiple cell types when characterizing viral entry mechanisms.

What statistical approaches are recommended for analyzing HAVCR1 haplotype associations with infection outcomes?

Analysis of HAVCR1 haplotype associations with infection outcomes requires rigorous statistical approaches:

• Haplotype frequency comparison:

  • Compare haplotype distributions between different infection outcomes

  • Analyze differences between viral genotype groups (e.g., HCV genotype 1 vs. non-genotype 1)

  • Include appropriate control populations

• Statistical methods:

  • Calculate P values and apply corrections for multiple comparisons

  • P values below 0.05 after correction (Pc) are considered statistically significant

  • Report odds ratios (ORs) and 95% confidence intervals (CIs) according to the Woolf method

• Data representation:

  HAVCR1 Haplotype	Patient Group	Frequency (%)	Statistical Comparison
  Haplotype C	HCV G1	48.83%	Base comparison
  Haplotype C	HCV non-G1	28.42%	Pc=0.024, OR=2.40, 95% CI=1.30-4.43
  Haplotype C	Controls	33.75%	Pc=0.048, OR=1.87, 95% CI=1.16-3.02

• Interpretation guidelines:

  • Strong associations: Pc < 0.01, OR > 2.0

  • Moderate associations: Pc < 0.05, OR > 1.5

  • Weak associations: Pc < 0.1 or OR < 1.5

  • Consider biological plausibility alongside statistical significance

These statistical approaches provide a framework for identifying meaningful associations between HAVCR1 genetic variants and infection outcomes in mouse models.

What approaches can identify alternative HAV receptors in mouse models?

Identifying alternative HAV receptors in mouse models requires systematic approaches:

• Cell-based screening strategies:

  • Compare HAV susceptibility in HAVCR1 knockout versus wild-type cells

  • Focus on cell types showing partial protection with anti-HAVCR1 antibodies

  • Develop receptor competition assays with soluble HAVCR1

• Genomic and proteomic approaches:

  • Conduct comparative transcriptomics between susceptible and resistant cells

  • Perform genome-wide CRISPR screens in HAVCR1-knockout backgrounds

  • Use affinity purification with viral particles to identify interacting proteins

• Functional validation:

  • Express candidate receptors in HAVCR1 knockout cells resistant to HAV

  • Test whether candidate receptors restore HAV susceptibility

  • Develop blocking antibodies against candidate receptors to assess protection

• Tissue-specific considerations:

  • Mouse liver cells appear to contain alternative HAV receptors distinct from mHavcr1

  • Tissue-specific receptor expression may influence HAV tropism

  • Compare receptor profiles across tissues with differential HAV susceptibility

Identification of these alternative receptors will enhance understanding of HAV's cellular tropism and species-specific infection patterns.

How might HAVCR1 polymorphisms be leveraged for personalized HAV prevention strategies?

HAVCR1 polymorphisms offer potential for developing personalized HAV prevention strategies:

• Haplotype-based risk stratification:

  • Specific HAVCR1 haplotypes associate with differential susceptibility to viral infections

  • Haplotype B has been linked to higher mRNA expression and disease susceptibility

  • Individuals with high-risk haplotypes might benefit from prioritized vaccination

• Genotype-specific considerations:

  • HAVCR1 haplotype C shows differential distribution between HCV genotype 1 (48.83%) and non-genotype 1 (28.42%) infections

  • Similar genotype-specific associations may exist for HAV strains

  • Tailored prevention strategies based on circulating viral genotypes and host genetics

• Quantitative risk assessment:

  • Haplotype C carriers showed 2.30-fold increased susceptibility (OR 2.30, 95% CI 1.51-3.47)

  • This allows for numeric risk assessment and prioritization of preventive measures

• Translational research priorities:

  • Develop rapid genotyping assays for HAVCR1 haplotypes

  • Conduct prospective studies correlating haplotypes with infection outcomes

  • Create mouse models expressing different human HAVCR1 haplotypes for vaccine testing

These approaches could lead to more efficient allocation of preventive resources by targeting individuals with genetic susceptibility to HAV infection.