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Altered drug sensitivity, fitness, and evolution of human immunodeficiency virus type 1 with pol gene mutations conferring multi-dideoxynucleoside resistance Presentation by: Christina Stujenske.

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  1. Altered drug sensitivity, fitness, and evolution of human immunodeficiency virus type 1 with pol gene mutations conferring multi-dideoxynucleoside resistancePresentation by: Christina Stujenske Maeda, Y., D.J. Venzon, and H. Mitsuya. 1998. Altered drug sensitivity, fitness, and evolution of human immunodeficiency virus type 1 with pol gene mutations conferring multi-dideoxynucleoside resistance. Journal of Infectious Diseases 177:1207-1213.

  2. Introduction • 5 sets of mutation in the pol gene have been identified in HIV-1 patients which have received long term combination therapy • AlaVal @ codon 62 (A62V) • ValIle @ codon 75 (V75I) • PheLeu @ codon 77 (F77L) • PheTyr @ codon 116 (F116Y) • GlnMet @ codon 151 (Q151M) • Frequency of emergence of Q151M mutation & its related mutations has been 3.5% to  19%

  3. Are Mutations Random? • Previous research has shown that multiple mutations associated with drug resistance occur in a certain order • Also, it is thought that once a certain mutation occurs the virus may acquire other mutations to improve infectivity & drug resistance

  4. What did Researchers Want to Examine? • Replication kinetics of HIV-1 variants with multidrug resistance mutations in presence of drugs • Hope is to understand & predict outcome of infection with HIV-1 mutants • Also viral population changes after termination or change of antiretroviral therapy

  5. How Did Researchers Do The Experiment? • Acquired the reagents (ddN) and cells (H9 and MT-cell) to propagate HIV-1 • Created infectious HIV-1 Clones • Exposed H9 and MT-cells to infectious HIV-1 Clones to produce infection • Determined drug sensitivity & viral replication profile

  6. Drug Susceptibility of Clones with A.A. Substitutions • Cells treated with Zidovudine, Didanosine, or Zalcitabine • Exposed to infectious clones • Sensitivity to dideoxynucleosides, ddN, was recorded (table 1) • Inhibitory concentration, IC50, values were compared • the concentration required for 50% inhibition

  7. Infectious HIV-1 Clones & Sensitivity to ddN

  8. Sensitivity of Single Mutation Clones

  9. Sensitivity of Multi Mutation Clones

  10. Amino Acid Codon Table

  11. Determined Sensitivity to ddN • HIV-175/77/116/151 > HIV-162/75/77/116/151 HIV-177/116/151 > HIV-1151 • Data shows reduced sensitivity & that observed resistance is due to mutations in pol gene • Next: to examine replication rate of clones in absence & presence of drugs

  12. HIV-1 Replication in H9 Cells in Absence of Drugs • Compare the replication rate of infectious clones in absence of drugs • Exposed H9 cells to infectious clone • Monitored production of p24 Gag protein

  13. Replication Profiles of Clones in Absence of Drugs in H9 Cells

  14. Replication Profiles of Clones in Absence of Drugs in Peripheral Blood Mononuclear Cells

  15. Replication Rates of Clones in Presence of Drug • Major driving force in clinical setting for drug resistance is survival of virus • H9 cells exposed to infectious clones & then cultured in zidovudine or didanosine • To inhibit HIV-1wt replication2 M zidovudine and 10 M of didanosine were chosen

  16. Replication Rates of Clones in Presence of Drug • 4 infectious clones (62, 75, 77, & 116) did not replicate with chosen concentrations • Significant difference in replication rates of infectious clones with certain mutations

  17. Replication Profile of Infectious Clones 2 M of Zidovudine HIV-162/75/77/116/151 > HIV-177/116/151 > HIV-175/77/116/151 HIV-1151

  18. Replication Profile of Infectious Clones 10 M of Didanosine

  19. Conclusions • Reduced sensitivity of infectious clones supports notion that mutations RT region are responsible for viral resistance to RT inhibitors • Replication kinetics of clones in the absence of drugs were comparable • Replication kinetics of clones in the presence of drugs differed significantly

  20. Viral Replication Profile Conclusion Seem to show that HIV-1 does evolve in presence of inhibitors by developing mutations and optimizing replication competence with more mutations. Before therapy the wild type clone is sensitive to the ddN and does not replicate. Once there are 1 to multiple mutations virus aquires resistance and reasonable ability to replicate in presence of drug.

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