CMV Pp65, 561 a.a.
Description
Functional Roles in Viral Pathogenesis
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Immune Evasion: Inhibits interferon response factor 3 (IRF-3) activation, blocking host antiviral defenses .
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Nuclear Trafficking: Enters host nuclei post-infection, interacts with polo-like kinase 1 (PLK-1) to manipulate cell cycle progression .
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Viral Replication: Facilitates transcription of viral immediate-early promoters .
Immunodominance and T-Cell Responses
CMV Pp65 is a primary target for CD8+ and CD4+ T-cell responses in HCMV-infected individuals:
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HLA-A*0201-Restricted Epitopes:
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CD4+ T-Cell Help: Required for robust e6-specific CD8+ responses, highlighting nested epitope dependencies .
Table 2: Key Immunogenic Epitopes
| Epitope | Amino Acid Range | HLA Restriction | Immunogenicity |
|---|---|---|---|
| e6 | 495–503 | HLA-A*0201 | Dominant (IFN-γ high) |
| e3 | 320–328 | HLA-A*0201 | Subdominant |
| e8 | 522–530 | HLA-A*0201 | Subdominant |
Research Applications and Mutant Constructs
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Vaccine Development:
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Diagnostic Tools:
Biochemical and Recombinant Variants
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Recombinant Proteins:
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Stability: Lyophilized formulations retain activity at -18°C; urea buffers prevent aggregation .
Implications for Autoimmunity and Disease
Product Specs
Q&A
What are the critical structural and functional domains within CMV pp65 (561 a.a.)?
CMV pp65 contains two bipartite nuclear localization signals (NLSs) at residues 415–438 and 537–561, which govern its nuclear translocation. A phosphate-binding site at lysine-436 (K436) is essential for kinase activity. Mutational studies (e.g., K436N substitution combined with NLS deletions) demonstrate that disrupting these regions reduces nuclear localization and kinase function while enhancing cytoplasmic retention and antigen presentation efficiency .
Methodological Insight:
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Mutagenesis: Targeted deletions (e.g., residues 537–561) combined with fluorescent tagging enable visualization of subcellular localization via confocal microscopy .
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Kinase Assays: Radiolabeled ATP incorporation assays quantify enzymatic activity in wild-type vs. mutant pp65 .
How is the pp65 antigenemia assay optimized for clinical diagnostics?
The pp65 antigenemia assay detects CMV-infected leukocytes using immunofluorescence (IF) or immunoperoxidase staining. A modified protocol with direct erythrocyte lysis (DL) using 0.8% NH4Cl reduces processing time to 3 hours (vs. 5 hours for dextran sedimentation) while maintaining sensitivity (94% concordance with conventional methods) .
Key Parameters:
| Parameter | DL-IF Method | DS-APAAP Method |
|---|---|---|
| Assay Time | 3 hours | 5 hours |
| Cost per Test | $15 | $45 |
| False-Positive Rate | 2% | 3% |
Data Contradiction: While DL-IF is faster and cheaper, some studies report lower leukocyte viability compared to dextran sedimentation, potentially affecting quantitation in immunocompromised patients .
How does CMV pp65 contribute to autoimmune pathogenesis in systemic lupus erythematosus (SLE)?
The pp65 336–439 fragment induces cross-reactive antibodies against nuclear antigens (e.g., dsDNA, chromatin) in BALB/c mice, correlating with glomerular immunoglobulin deposition. Yeast two-hybrid assays reveal interactions between pp65 336–439 and cellular proteins (e.g., centriolar components), suggesting molecular mimicry as a mechanism .
Experimental Design:
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Animal Models: Immunize lupus-prone NZB/W mice with pp65 fragments + C3d adjuvant to accelerate autoantibody production .
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Serological Profiling: Use ELISAs and Crithidia luciliae staining to detect anti-dsDNA antibodies in patient sera .
Contradiction: Only 36.8% of SLE patients with suspected CMV infection show pp65 antigenemia positivity, yet 73% of these patients exhibit antibodies to the 336–439 fragment, implying heterogeneity in immune recognition .
What methodologies resolve discrepancies in pp65 antigenemia vs. PCR-based CMV detection?
In kidney transplant recipients, pp65 antigenemia (≥10 positive cells/200,000 leukocytes) correlates with viral culture positivity (sensitivity: 85.7%; specificity: 90%) but diverges from PCR in timing: antigenemia peaks 7–14 days earlier during reactivation .
Resolution Strategy:
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Parallel Testing: Combine antigenemia with quantitative PCR to capture early replication (antigenemia) and late viremia (PCR) .
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Threshold Adjustment: Lower antigenemia cutoffs (<5 cells) for preemptive therapy in high-risk SLE patients with lymphopenia .
How does CMV pp65-specific T-cell polyfunctionality evolve during aging?
In adults aged ≥70, pp65-reactive CD8+ and CD4+ T cells exhibit cytotoxic polyfunctionality (e.g., IFN-γ, perforin, CD107a co-expression) proportional to response magnitude. Higher CMV-IgG titers correlate with larger T-cell expansions (r=0.62, p<0.01), suggesting chronic antigenic drive refines effector phenotypes .
Technical Approach:
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Flow Cytometry Panels: Use 10-color panels to assess 5+ effector functions simultaneously .
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Hierarchical Clustering: Group polyfunctional subsets (e.g., IL-2+ vs. cytotoxicity-dominant) to stratify patients by immune risk .
How are conflicting results in pp65’s immunomodulatory roles reconciled?
Case Study: While pp65 knockout (pp65mIINLSKO) enhances cytotoxic T lymphocyte (CTL) killing in vitro , its immunogenicity in HHDII mice matches wild-type pp65, implying redundant epitopes outside the NLS regions .
Resolution:
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Epitope Mapping: Use overlapping 15-mer peptides to identify CTL epitopes retained in mutants .
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Transcriptomic Analysis: RNA microarrays reveal pp65mIINLSKO upregulates immune signaling pathways (e.g., NF-κB) more robustly than wild-type, compensating for epitope loss .
What innovations improve pp65 antigenemia’s reliability in non-transplant settings?
In SLE, antigenemia’s sensitivity falls to 36.8% due to leukopenia. Solutions:
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Leukocyte Enrichment: Use Polymorphprep density gradients to recover granulocytes .
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Serial Monitoring: Test weekly during immunosuppressive therapy to detect delayed positivity .
Can pp65-targeted vaccines avoid autoimmune cross-reactivity?
Hypothesis: Masking the 336–439 epitope via structural redesign could reduce anti-dsDNA responses. Approach:
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Computational Modeling: Use RosettaDesign to engineer pp65 variants with disrupted mimicry sites .
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Preclinical Testing: Compare autoantibody induction in BALB/c mice immunized with wild-type vs. redesigned pp65 .
How does pp65 regulate CMV latency-reactivation cycles?
Unresolved Mechanism: pp65’s kinase activity phosphorylates viral/cellular targets (e.g., UL44), but its role in latency is unclear. Experimental Tools:
Product Science Overview
Cytomegalovirus (CMV) is a member of the Betaherpesvirinae subfamily of Herpesviridae, which also includes herpes simplex viruses types 1 and 2, varicella-zoster virus, and Epstein-Barr virus . CMV is known for its ability to remain latent in the body for long periods. It has the largest genome among the herpesviruses, ranging from 230-240 kilobase pairs . CMV is a double-stranded linear DNA virus with 162 hexagonal protein capsomeres surrounded by a lipid membrane .
The CMV Pp65 (UL83) protein is one of the most immunodominant antigens of the virus. It plays a crucial role in the immune response against CMV infections. The recombinant form of this protein, consisting of 561 amino acids, is derived from Escherichia coli (E. coli) and has a molecular weight of approximately 62.8 kDa .
The recombinant CMV Pp65 protein is produced in E. coli and purified using proprietary chromatographic techniques . The protein is formulated in a buffer containing 25mM Tris-HCl (pH 8), 8M urea, and 5mM β-mercaptoethanol (βMe) . It is highly pure, with a purity level greater than 95% as determined by SDS-PAGE .
The CMV Pp65 antigen is suitable for use in enzyme-linked immunosorbent assays (ELISA) and Western blots. It is an excellent antigen for detecting CMV with minimal specificity problems . The protein is immunoreactive with sera from CMV-infected individuals, making it a valuable tool for diagnostic and research purposes .
