PLSCR1 Human

How is PLSCR1 expression regulated in human cells?

PLSCR1 expression is strongly induced by both type I (α, β) and type II (γ) interferons, serving as an interferon-stimulated gene (ISG) . Viral infections also significantly upregulate PLSCR1 expression, as demonstrated in studies with HCMV where expression was drastically induced from 24 hours post-infection . The basal expression levels vary between cell types; for example, OUMS-36T-3 (36T-3) cells show significantly higher constitutive PLSCR1 expression compared to human embryonic lung (HEL) cells . This differential expression may contribute to varied antiviral responses across tissue types.

What experimental approaches are commonly used to study PLSCR1 function?

Common approaches include:

• CRISPR-Cas9 genome editing to create PLSCR1 knockout cell lines, allowing comparison of viral replication between wild-type and PLSCR1-deficient cells .

• Genome-wide CRISPR screens in various cell types (e.g., lung epithelia and hepatocytes) with and without interferon stimulation to identify PLSCR1 as an antiviral factor .

• Viral plaque assays to quantify the impact of PLSCR1 on viral replication capacity .

• Protein-protein interaction studies to identify PLSCR1's binding partners during viral infection .

• Reporter gene assays to assess PLSCR1's impact on viral promoter activity .

• Advanced imaging techniques such as whole-cell 4Pi single-molecule switching nanoscopy to visualize PLSCR1's interaction with virus-containing vesicles .

How does PLSCR1 mechanistically restrict SARS-CoV-2 infection?

PLSCR1 restricts SARS-CoV-2 through a specific mechanism targeting viral entry. According to recent studies, IFNγ-induced PLSCR1 interferes with SARS-CoV-2 uptake in both endocytic and TMPRSS2-dependent fusion routes . Advanced imaging using whole-cell 4Pi single-molecule switching nanoscopy revealed that PLSCR1 directly targets SARS-CoV-2-containing vesicles to prevent spike-mediated fusion and viral escape into the cytoplasm .

Mechanistically, the C-terminal β-barrel domain of PLSCR1—rather than its lipid scramblase activity—is essential for this fusogenic blockade . This represents a late entry inhibition step that prevents viral RNA release into the host-cell cytosol. This mechanism allows PLSCR1 to exhibit broad antiviral activity against multiple coronavirus lineages, including Delta and Omicron variants, though recent variants may show signs of adaptation to this restriction .

What differences exist in PLSCR1's antiviral activity against different SARS-CoV-2 variants?

Research indicates that PLSCR1's restriction activity varies across SARS-CoV-2 variants. While it effectively restricts the original USA-WA1/2020 strain, Delta B.1.617.2, and Omicron BA.1 lineages, newer variants like Omicron (XBB.1.5) appear to have developed resistance to PLSCR1-mediated restriction .

Time course experiments revealed that parental SARS-CoV-2 replicated with faster kinetics upon PLSCR1 depletion, whereas Omicron (XBB.1.5) replication was not significantly affected by PLSCR1 depletion in Huh-7.5 cells . This suggests that recent variants may have adapted to either directly antagonize PLSCR1 or utilize alternative entry pathways that evade PLSCR1-mediated restriction. This evolutionary adaptation highlights the ongoing virus-host arms race and represents an important consideration when designing experiments to test PLSCR1's antiviral activity against emerging variants.

How does PLSCR1 restrict HCMV replication at the molecular level?

PLSCR1's mechanism of action against HCMV differs from its anti-SARS-CoV-2 activity. For HCMV, PLSCR1 specifically interacts with HCMV immediate early proteins 1 (IE1) and 2 (IE2) in vivo . This interaction results in reduced levels of the cAMP-responsive element (CRE)-binding protein (CREB)- IE2 and CREB-binding protein (CBP)- IE2 complexes .

Since these complexes play critical roles in IE2-mediated transactivation of viral early promoters through interactions with CREB, CBP, and IE2, PLSCR1 effectively suppresses viral replication by interfering with transcriptional activation . Evidence for this mechanism includes:

• PLSCR1 expression represses both CRE- and HCMV major immediate early (MIE) promoter-regulated reporter gene activities.

• HCMV plaque formation and MIE gene expression are significantly increased in PLSCR1-knockout human fibroblast cells.

• Viral titers in PLSCR1-knockout cells are substantially higher (>13-fold) than in cells expressing PLSCR1.

This demonstrates that PLSCR1 can employ distinct mechanisms to target different viral families, highlighting its versatility as an antiviral factor .

What are the advantages of arrayed vs. pooled CRISPR screens for identifying PLSCR1's antiviral properties?

The identification of PLSCR1 as an antiviral factor has benefited from arrayed CRISPR screens, which offer distinct advantages over pooled approaches:

Arrayed CRISPR Screen Advantages:

• Enables direct observation of single gene perturbation effects without competition among cells with different gene knockouts

• Shorter culture time requirements minimize confounding effects

• Allows identification of crucial cellular functions that may be co-opted by viruses or essential for viral replication

• Better captures genotype-phenotype correlations

• Enables observation of cell-autonomous effects without confounding intercellular interactions

These advantages were demonstrated in a genome-wide arrayed CRISPR knockout screen in Huh-7.5 cells infected with SARS-CoV-2, which successfully identified PLSCR1 as a prominent antiviral factor . The arrayed format was particularly valuable for revealing PLSCR1's role, which might have been obscured in a pooled screening approach due to competition effects or longer culture requirements.

What experimental controls are essential when evaluating PLSCR1's impact on viral replication?

When designing experiments to assess PLSCR1's antiviral activity, several critical controls should be implemented:

• PLSCR1 Expression Verification: Western blot analysis comparing basal and IFN-induced PLSCR1 expression levels in experimental cell lines. This is particularly important as basal PLSCR1 expression varies significantly between cell types (e.g., 36T-3 cells versus HEL cells) .

• Matched PLSCR1-KO and Parental Cell Lines: Using gene-edited knockout lines alongside their unmodified parental cells ensures direct comparison of viral replication effects .

• Multiple Viral Strains/Variants: Testing across different viral strains or variants, as PLSCR1's effectiveness varies (e.g., against parental SARS-CoV-2 versus Omicron XBB.1.5) .

• Time Course Analysis: Assessing viral replication at multiple timepoints post-infection, as PLSCR1's effects may be more pronounced at specific stages of the viral life cycle .

• IFN Stimulation Controls: Including conditions with and without IFN treatment to distinguish between constitutive and IFN-induced PLSCR1 antiviral effects .

• Viral Input Normalization: Standardizing the multiplicity of infection (MOI) across experiments to ensure comparable viral challenge conditions.

• Complementation Studies: Reintroducing wild-type or mutant PLSCR1 into knockout cells to confirm specificity and identify critical functional domains .

What PLSCR1 genetic variants exist in human populations and how might they affect antiviral immunity?

Analysis of human population genomic data from gnomAD has revealed several PLSCR1 variants, with p.His262Tyr being particularly notable as the most common nonsynonymous variant in this gene . This variant has a minor allele frequency (MAF) of 0.07, with a maximum MAF of 0.1, and is found in homozygous form in 4,348 individuals in the gnomAD database .

Functional studies suggest that the p.His262Tyr variant may alter PLSCR1's antiviral function, potentially affecting its ability to restrict SARS-CoV-2 infection. Cell culture experiments have shown increased SARS-CoV-2 infection in cells expressing this variant, suggesting it may confer increased susceptibility to infection . Unlike some other PLSCR1 variants that affect nuclear localization, p.His262Tyr appears to influence antiviral activity through alternative mechanisms.

The prevalence of this variant in human populations raises important questions about differential susceptibility to viral infections, particularly for coronaviruses and herpesviruses against which PLSCR1 provides protection.

How do PLSCR1 variants correlate with COVID-19 severity in genome-wide association studies?

Genome-wide association studies (GWAS) have identified associations between PLSCR1 variants and severe COVID-19 outcomes . The p.His262Tyr variant has been implicated as a potential contributor to COVID-19 severity, consistent with functional studies showing its impact on SARS-CoV-2 restriction.

• Non-coding variants identified in GWAS studies might influence the regulation of PLSCR1 expression, contributing to functional outcomes beyond coding changes.

• GWAS studies might have missed other nonsynonymous variants besides p.His262Tyr that could potentially impact PLSCR1 function.

• The p.His262Tyr variant might affect PLSCR1 through mechanisms distinct from nuclear localization alterations.

The association between PLSCR1 variants and COVID-19 outcomes highlights the importance of this protein in human antiviral defense and suggests that genetic variation in this gene may contribute to individual differences in susceptibility to viral infections .

What are the critical knowledge gaps in understanding PLSCR1's role in antiviral immunity?

Several important knowledge gaps remain in our understanding of PLSCR1's antiviral functions:

• Mechanism of Variant-Specific Restriction: While PLSCR1 restricts multiple SARS-CoV-2 variants, newer variants like Omicron XBB.1.5 appear less affected . The molecular basis for this differential restriction requires further investigation.

• Interplay with Other ISGs: How PLSCR1 cooperates with or complements other interferon-stimulated genes in antiviral defense remains poorly understood.

• Tissue-Specific Functions: PLSCR1 expression and function may vary across tissue types, potentially explaining differential susceptibility of tissues to viral infection.

• Structural Determinants of Antiviral Activity: While the C-terminal β-barrel domain is essential for SARS-CoV-2 restriction , the specific structural features and interactions required for antiviral activity need further characterization.

• Viral Antagonism Mechanisms: How viruses evade or counteract PLSCR1 restriction, particularly in the context of evolved viral variants, remains to be fully elucidated.

Addressing these gaps will require integrative approaches combining structural biology, virology, immunology, and human genetics.

What experimental approaches might reveal new aspects of PLSCR1 biology?

To advance our understanding of PLSCR1, several innovative experimental approaches could be employed:

• Cryo-EM Studies: Structural analysis of PLSCR1 interactions with viral components could reveal the molecular basis of its antiviral activity.

• In Vivo Models with Human PLSCR1 Variants: Generating animal models expressing human PLSCR1 variants would allow assessment of their impact on viral infections in complex physiological contexts.

• Single-Cell Analysis: Examining PLSCR1 expression and function at the single-cell level could reveal cell-type specific roles and heterogeneity in antiviral responses.

• Systems Biology Approaches: Network analysis integrating transcriptomic, proteomic, and functional data could position PLSCR1 within broader antiviral response networks.

• CRISPR-Based Screens for PLSCR1 Modulators: Identifying genes that enhance or suppress PLSCR1's antiviral activity could reveal new therapeutic targets.

These approaches would complement existing research and potentially identify new therapeutic strategies leveraging PLSCR1's antiviral properties.