Recombinant Human immunodeficiency virus type 1 group M subtype A Gag-Pol polyprotein (gag-pol)
Description
Overview of Recombinant Human Immunodeficiency Virus Type 1 Group M Subtype A Gag-Pol Polyprotein
The recombinant Human immunodeficiency virus type 1 (HIV-1) group M subtype A Gag-Pol polyprotein is a genetically engineered version of the viral precursor protein critical for HIV-1 replication. This polyprotein comprises the structural Gag (group-specific antigen) and enzymatic Pol (polymerase) domains, which are post-translationally cleaved by the viral protease (PR) to generate mature virion components. While most studies focus on subtype B, subtype A retains similar structural and functional features, with potential variations in residue composition affecting interactions or drug susceptibility . Recombinant expression enables detailed biochemical and structural analyses without handling live virus, advancing therapeutic and diagnostic research.
Virion Assembly and Budding
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Gag-Pol drives viral particle formation by multimerizing at the plasma membrane, guided by MA-membrane and CA-CA interactions .
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p6 domain recruits ESCRT complexes (e.g., Tsg101) via PTAP motifs, enabling membrane scission .
Proteolytic Processing
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PR embedded in Gag-Pol undergoes autoprocessing to liberate mature enzymes. Initial cleavage at the p6/PR site is rate-limiting .
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Suboptimal processing produces noninfectious particles, highlighting PR’s role as a key drug target .
Genome Packaging and Translation Regulation
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NC binds the Ψ-packaging signal in viral RNA, ensuring selective genome encapsidation .
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Gag-Pol bimodally regulates translation: Low concentrations enhance translation via MA-domain interactions, while high concentrations inhibit it by sequestering RNA .
Functional Complementation Studies
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RT and IN delivered in trans (e.g., fused to Vpr) restored infectivity to Gag-Pol-deficient virions, proving their modular functionality .
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Truncated Gag-Pro (lacking RT/IN) still produced infectious particles when complemented, indicating Pol domains are dispensable for assembly but critical for replication .
Mutational Analyses
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M50I/V151I mutations in IN disrupted Gag-Pol autoprocessing and virion release, rescued by compensatory mutations in RT’s RNase H domain .
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p6* deletions impaired PR activation but retained residual infectivity, underscoring its regulatory role .
Applications in Research and Therapeutics
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Drug Target Screening: Recombinant Gag-Pol enables high-throughput assays for PR/RT/IN inhibitors .
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Vaccine Development: Virus-like particles (VLPs) generated from Gag-Pol are explored as immunogens .
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Structural Biology: Recombinant proteins facilitate cryo-EM and NMR studies to map drug-binding pockets .
Challenges and Future Directions
Product Specs
Target Background
Q&A
FAQs for Researchers: Recombinant HIV-1 Group M Subtype A Gag-Pol Polyprotein
What expression systems are optimal for producing functional recombinant HIV-1 subtype A Gag-Pol polyprotein?
Recombinant adenovirus (rAd26) and modified vaccinia Ankara (MVA) vectors are widely used due to their high transduction efficiency and capacity for large antigen inserts. For subtype A Gag-Pol:
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rAd26 vectors enable simultaneous expression of mosaic antigens (e.g., mos1GagPol fused as SEQ ID NO: 28 ).
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MVA vectors (e.g., MVA-BN derivatives) allow insertion of Gag-Pol fusion antigens into specific intergenic regions (IGR 44/45 and IGR 88/89) under Pr13.5 promoters for balanced expression .
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Critical considerations: promoter selection (PrHyb for Env antigens), codon optimization for mammalian systems, and avoidance of premature polyprotein cleavage .
How do researchers validate the structural integrity of recombinant Gag-Pol polyproteins?
Methodological steps include:
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Mass spectrometry to confirm molecular weight and post-translational modifications.
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Western blotting with clade-specific antibodies targeting conserved domains (e.g., Gag-p6 or Pol-integrase regions ).
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Functional assays: Measure protease (PR) activity in vitro using fluorogenic substrates to ensure embedded PR cleaves Gag-Pol at correct sites (e.g., MA/CA or RT/RNase H junctions) .
What challenges arise when analyzing Gag-Pol processing kinetics in subtype A isolates?
Key issues include:
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Premature cleavage: Subtype-specific polymorphisms in PR cleavage sites (e.g., P1/P6 regions) can alter processing rates .
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Experimental mitigation: Use in vitro processing assays with full-length GagPol to mimic natural constraints. For example, embedded PR cleaves MA/CA 5x faster than RT/IN in subtype A .
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Data normalization: Compare cleavage rates to reference strains (e.g., NL4.3) using densitometry of SDS-PAGE gels .
How do subtype-specific variations in Gag-Pol affect viral replicative capacity (VRC) in primary cells?
Subtype A Gag-Pol exhibits distinct VRC profiles compared to subtypes D or recombinant A/D:
| Feature | Subtype A | Subtype D | A/D Recombinant |
|---|---|---|---|
| Gag-p6 insertions | Rare | Common | Intermediate |
| Mean VRC | 65% ± 12 | 82% ± 9 | 74% ± 11 |
| Methodology: |
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Clone Gag-Pol sequences from early infections into NL4.3 backbones via single-genome amplification (SGA) .
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Quantify VRC using TZM-bl luciferase assays over 7–14 days .
What experimental approaches resolve contradictions in Gag-Pol’s role in genomic RNA packaging?
Conflicting data arise from Gag’s bimodal effect on translation:
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Low Gag concentrations stimulate translation via MA domain interactions with HIV-1 5′ UTR.
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High Gag concentrations inhibit translation through NC domain binding to packaging signals .
Resolution strategy: -
Use ribopuromycylation assays to quantify polysome loading under varying Gag levels .
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Employ in vitro packaging systems with fluorescently labeled RNA to disentangle translation-packaging competition .
How do frameshift efficiency modulators impact Gag-Pol stoichiometry in subtype A?
The natural Gag:Gag-Pol ratio (20:1) is critical for virion infectivity. To study this:
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Ribosomal frameshift mutagenesis: Introduce synonymous mutations in the gag-pol slippage site (e.g., U UUA AAC → U UUU AAC) to alter ratios .
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Functional outcomes: Ratios <15:1 impair RNA dimerization, while >25:1 reduce PR-mediated maturation .
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Quantification: Use dual luciferase reporters (e.g., Gaussia-Pol and Cypridina-Gag) in transfected 293T cells .
What structural determinants in Gag-Pol regulate ordered processing of subtype A polyproteins?
Domain-swapping experiments reveal:
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PR context: Embedded PR in GagPol cleaves MA/CA 3x faster than mature PR due to N-terminal proline constraints .
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Gag-p6 domain: Insertions (>4 residues) enhance replication by delaying PR activation (e.g., PTAP duplications in subtype D improve VRC by 18% vs. subtype A ).
Method: Solve cryo-EM structures of full-length GagPol in complex with PR inhibitors (e.g., darunavir) to map cleavage site accessibility .
Why do subtype A Gag-Pol chimeras show lower VRC in some studies despite conserved functional domains?
Discrepancies stem from:
