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Covariation Analysis of HIV-1 Protease Cleavage Sites

Explore co-adapted natural polymorphisms, compensatory mutations, and drug resistance in HIV-1 protease cleavage sites using covariation analysis. Learn how mutations affect viral fitness and drug efficacy.

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Covariation Analysis of HIV-1 Protease Cleavage Sites

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  1. Júlia Kornai1, Jochen Bodem2&Viktor Müller1 Covariation Analysis of the Amino Acid Sequence of HIV-1 Subtype B Protease and its Gag-Pol Cleavage Sites 1Institute of Biology, Eötvös Loránd University, Budapest, Hungary 2Institute of Virology and Immunobiology, University of Würzburg, Germany

  2. MA CA p2 NC p6pol PR RTp51 RTp66 INT MA CA p2 NC p6pol PR RTp51 RTp66 INT Protease function is essential for the virus HIV genome Gag-Pol gene several proteins are transcribed into a single polypeptide product TFP the polypeptide must be cleaved by the viral protease to obtain functional proteins TFP

  3. Protease recognises cleavage sites protease Functional viral proteins

  4. Protease inhibitors and drug resistance Mutations can alter the active site of the protease and inhibit the drug effect Drugs block the protease produced virions will be noninfectious drug cannot bind impaired recognition of cleavage sites The price of resistance reduced viral fitness

  5. Compensatory mutations in the cleavage sites Mutations in the cleavage sites can restore the efficiency of cleavage.

  6. How to recognise compensatory mutations from the amino acid sequence? protease TFP MA CA p2 NC p6pol PR RTp51 RTp66 INT cleavage sites = mutation Look at a large number of sequences: Is the occurrence of mutations at a given position of the protease associated with the occurrence of mutations at a given position of a cleavage site? STATISTICAL TEST

  7. Data selection • We downloaded nucleotide sequences containing PR from the Los Alamos HIV Sequence Database (www.hiv.lanl.gov): 14868 sequences • Alignment, translation, quality control: 14172 seqs • Restrict analysis to positions present in >100 sequences: Gag-Pol positions 407-1145

  8. 7 cleavage sites Available data ≥100 sequences TFP Gag-Pol MA CA p2 NC p6pol PR RTp51 RTp66 INT Gag MA CA p2 NC p1 p6gag ≥100 sequences

  9. Polymorphisms in the cleavage sites

  10. Covariation analysis STEP 1: select a position pair 1 position in a cleavage site 1 position in the protease NC TFP p6pol PR RTp51 RTp66 STEP 2: count AA pairs

  11. Covariation analysis STEP 1: select a position pair 1 position in a cleavage site 1 position in the protease NC TFP p6pol PR RTp51 RTp66 STEP 2: count AA pairs

  12. . . . CTERQANFLREDLAFLQ IGGFIKVRQYDQISIEICG . . . Covariation analysis STEP 2: count AA pairs calculate consensus vs. non-consensus IGGFIKVRQYDQIPIEICG IGGFIKVRQYDQIPIEICG IGGFIKVKQYDQILIEICG IGGFVKVRQYDQIPIEICG IGGFIKVRQYDQILIEICG IGGFVKVRQYDQIPIEICG IGGFIKVRQYDQILIEICG IGGFIKVRQYDQILIEICG CTERQANFLREDLAFLQ CTERQANFLREDLAFLQ CTERQANFLRKNLAFPQ CTERQANFLREDLAFPQ CTERQANFLREDLAFPQ CTERQANFLREDLAFPQ CT-RQVNFLRENLAFPQ CAERQANFLRENLAFPQ

  13. Covariation analysis STEP 3: test independence of mutations in the position pairs Significance: Strength of the correlation:

  14. Covariation analysis • Consider all position pairs consisting of a protease and a cleavage site position. • Restrict the analysis to position pairs that were present in at least 100 sequences and yielded an expected value 5 for all combinations  322 protease-cleavage site position pairs • Repeat on sequences containing drug resistant protease (2 or more resistance-associated mutations) • Report correlations with p≤0.05 and |φ|>0.2  41 pairs involving 7 CS and 18 PR positions

  15. Shading indicates PR positions associated with drug resistance + correlation indicates preferential co-mutation (23 position pairs) - correlation indicates avoidance of co-mutation (18 position pairs)

  16. Interpretation • Positive covariation: • Co-adapted natural polymorphism • Compensatory mutation • Negative covariation: • The mutant PR tolerates the CS mutant less than the wild type PR • May also be affected by treatment (directly or indirectly) • Almost all covariations could be attributed to the pattern of the consensus AA and the most prevalent mutation at the given positions.

  17. Summary of results • Candidate compensatory mutations in cleavage sites • e.g. p6pol/PR P5 • Candidate resistance mutations in PR • e.g. PR positions 16, 37, 41 • Co-adapted natural polymorphisms (hints for structural studies) • e.g. PR positions 12, 13, 62, 64 and TFP/p6pol P4, P1’, p1/p6gag P1’

  18. Further directions • Implement control for phylogeny to detect non-functional associations that may have arisen through common descent. • Experimental tests to validate the relevance of detected associations.

  19. Acknowledgements • People • Júlia Kornai (ELTE, Hungary) • Jochen Bodem (University of Würzburg, Germany) • Funding • EU FP5 grant QLK2-CT-2001-02360 (HIV PR) • EU FP6 grant IST-027446 (Virolab) www.virolab.org Poster P-36

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