HCV NS3 Genotype-3a
Description
Recombinant Hepatitis C Virus NS3 Genotype-3a produced in E. coli, containing 236 amino acids. Recombinant Hepatitis C Virus NS3 Genotype-3a is fused to a 6xHis tag at its C-terminus and purified by proprietary chromatographic technique.
Product Specs
Escherichia Coli.
Q&A
Basic Research Questions
How do structural features of HCV NS3 Genotype-3a differ from other genotypes?
HCV NS3 Genotype-3a exhibits distinct structural characteristics compared to genotypes 1 and 2. Circular Dichroism (CD) spectroscopy reveals a combination of α-helical (30%) and β-sheet (25%) structures in NS3, while NS5A shows nearly equal proportions of both . Phylogenetic analysis of NS3 sequences demonstrates <70% amino acid identity with genotypes 1a/1b and 2a/2b, with critical mutations (e.g., D168Q in the protease domain) altering inhibitor binding efficiency .
Methodological insight:
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Use CD spectroscopy and Fourier Transform Infrared Spectroscopy (FTIR) to analyze secondary structures.
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Perform ClustalX-based phylogenetic alignment with sequences from GenBank (e.g., JQ676838 for NS3) to identify genotype-specific motifs .
What methodologies are optimal for detecting HCV NS3 Genotype-3a in clinical samples?
Genotype-specific PCR and 5’ noncoding region sequencing are gold standards. In a study of 161 HCC patients, genotype 3a was identified in 40.96% of tissues using type-specific primers, with mixed genotypes (e.g., 3a+3b) detected in 28.91% of cases .
Key steps:
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Extract RNA from liver biopsies or serum.
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Amplify NS3 regions using genotype-3a-specific primers.
Why is HCV NS3 Genotype-3a prevalent in South Asia and the UK?
Epidemiological drivers include intravenous drug use and migration patterns. In the UK, genotype-3a accounts for >50% of new infections, linked to immigrant communities from South Asia . A study in Lahore, Pakistan, found 4.9% of the population infected, with higher rates in adults aged 20–39 .
Advanced Research Questions
How do NS3 Genotype-3a mutations confer resistance to protease inhibitors?
The D168Q substitution disrupts salt bridging with R155, reducing inhibitor binding. Comparative studies show:
| Inhibitor | Genotype-1 IC₅₀ (nM) | Genotype-3a IC₅₀ (nM) | Fold Difference |
|---|---|---|---|
| BILN-2061 | 1.2 | 360 | 300× |
| VX-950 | 10 | >1000 | >100× |
| ITMN-191 | 0.8 | 240 | 300× |
Data sourced from enzymatic assays using purified NS3/4A .
Experimental design:
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Express NS3/4A at 25°C (optimal for genotype-3a stability) .
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Use surface plasmon resonance (SPR) to quantify RNA-binding affinity changes.
How does NS3 Genotype-3a influence oncogenesis in hepatocellular carcinoma (HCC)?
In a cohort of 161 HCC patients, 40.96% had NS3 Genotype-3a, with 15.66% co-infected with genotype-3b . NS3’s protease activity promotes oncogenesis by cleaving host tumor suppressors (e.g., retinoblastoma protein).
Validation approach:
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Perform immunohistochemistry on liver biopsies to localize NS3 expression.
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Correlate viral load (<10 IU/mL vs. >10⁶ IU/mL) with histopathological severity .
Why are T cell responses to NS3 Genotype-3a weaker than to genotype-1?
CD8+ T cells in genotype-3a target non-structural (NS) proteins (detected in 12/31 patients), whereas genotype-1 elicits CD4+ responses to core proteins (23/44 patients) . Limited cross-reactivity between genotypes is attributed to:
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Sequence divergence: <30% homology in immunodominant epitopes.
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HLA restriction: Genotype-3a epitopes bind poorly to HLA-A*02, common in European populations .
Assay optimization:
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Use genotype-3a-specific peptide panels in IFN-γ ELISpot assays.
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Conduct longitudinal CD4+/CD8+ subset analysis during interferon/ribavirin therapy .
Can NS3 Genotype-3a be detected in extrahepatic tissues like lymphoma cells?
Yes. HCV RNA was identified in 8.69% of Hodgkin’s lymphoma (mixed cellularity subtype) and 6.89% of diffuse large B-cell lymphoma tissues . NS3 promotes lymphomagenesis via chronic B cell activation.
Protocol:
Product Science Overview
Hepatitis C virus (HCV) is a significant global health concern, infecting over 71 million people worldwide. It is a leading cause of chronic liver diseases, including cirrhosis and hepatocellular carcinoma. HCV is a positive-sense single-stranded RNA virus belonging to the Flaviviridae family. The virus exhibits considerable genetic diversity, classified into seven major genotypes and numerous subtypes. Genotype 3a is one of the most prevalent and clinically significant genotypes, particularly in South Asia and parts of Europe.
The non-structural protein 3 (NS3) of HCV is a multifunctional enzyme with protease and helicase activities. The NS3 protease is responsible for cleaving the HCV polyprotein into functional units, essential for viral replication. The helicase activity of NS3 unwinds RNA, facilitating the replication process. Due to its critical role in the viral life cycle, NS3 is a prime target for antiviral drug development.
Recombinant NS3 proteins are produced through genetic engineering techniques, where the NS3 gene from HCV genotype 3a is cloned and expressed in a suitable host system, such as Escherichia coli. These recombinant proteins are used for various research purposes, including the development of diagnostic assays, vaccine candidates, and antiviral drugs.
The preparation of recombinant NS3 genotype-3a involves several steps:
- Gene Amplification: The NS3 gene is amplified using polymerase chain reaction (PCR) from the viral RNA extracted from infected patients.
- Cloning: The amplified gene is inserted into a plasmid vector, which is then introduced into a host cell, typically E. coli.
- Expression: The host cells are cultured under conditions that induce the expression of the NS3 protein.
- Purification: The expressed NS3 protein is purified using techniques such as affinity chromatography.
Recombinant NS3 genotype-3a proteins have several important applications:
- Diagnostic Assays: These proteins are used to develop enzyme immunoassays (EIAs) for the serological diagnosis of HCV infection.
- Vaccine Development: The antigenic properties of NS3 make it a potential candidate for vaccine development.
- Antiviral Drug Screening: Recombinant NS3 proteins are used to screen for inhibitors that can block the protease or helicase activities, leading to the development of new antiviral drugs.
