Introduction

1. Pol gene quasispecies of human immunodeficiency virus: mutations associated with drug resistance in virus for patients undergoing no drug therapyPresentation by: Christina Stujenske Najera, I., A. Holguin, M. E. Quinones-Mateu, M. Angeles Munoz-Fernandez, R. Najera, C. Lopez-Galindez, and E. Domingo. 1995. pol gene quasispecies of human immunodeficiency virus: mutations associated with drug resistance in virus from patients undergoing no drug therapy. Journal of Virology. Jan 1995:23-31.

2. Introduction • Explore pol gene region of HIV-1 genome in lymphocytes of infected individuals • codons 41 to 108 and 181 to 219 • pol gene • code for reverse transcriptase • Reverse transcriptase • enzyme in retrovirus that can construct double stranded DNA from single stranded RNA template

3. Why Explore pol Gene? • Explore nature of pol quasispecies in lymphocytes to: • detect presence of mutations • mutations related to drug resistance • mutation in two populations: patients undergoing no drug therapy & patients undergoing drug therapy

4. What is a Quasispecies? • Viral quasispecies • termed so because they replicate as complex distributions of mutant genomes • Quasispecies represent a large reservoir of genetic variation • infected individuals have 109 to 1012 infectious particles • mutation frequencies (clonal viruses) average 10-3 to 10-5 substitutions per nucleotide

5. HIV-1 Quasispecies • Can reach high levels of heterogeneity in both infected individual & organs • Influenced by • prolonged replication of virus in diseased individuals • tropism of virus for a variety of cells • expression of provirus triggered by T-cell activation

6. Importance of HIV-1 Quasispecies • Obstacle in control of viral disease • drug therapy • Main concern • resistance to antiretroviral agents from patients treated with reverse transcriptase inhibitors

7. What are Mutations? • Some alterations in DNA due to DNA base sequence changes • Frame shift Mutations • Inertion or Deletion Mutation

8. Techniques • Viruses • Prep of nucleic acids from lymphocytes • PCR amplification of viral sequences • Molecular cloning • Nucleotide sequencing

9. Origin of Lymphocyte Samples: Table 1 • Group A vs. Group B • Hom./Het. • Homosexual or Heterosexual • Transf • Transfusion • Vertical • Mother to Baby • IVDA • Intravenous drug abuser

10. Nucleotide Sequence and Mutations: Fig 1 • Nucleotide sequence at residues 121 to 324 of HIV-1 reverse transcriptase • Encoding amino acids 41 to 108 of reverse transcriptase • Two groups • A: patients not subjected to antiretroviral therapy (25) • B: patients subjected to antiretroviral therapy (35)

11. Mutations related to drug resistance in pol gene: Fig. 1 • Nucleotide sequences at residues 121 to 324 of HIV-1 reverse transcriptase genomic region • Group A: 7 samples w/mutations A-209G • Group B: THF17/+4 w/mutations G-223A and A-225G; mutations A-205G & C-206A • Group B: D16/+52 w/mutations A-301G

12. Figure 1: Group A

13. Original Codon Codon with Mutation Individual Samples: Group A

14. Figure 1: Group B

15. Original Codon Mutations

16. Relation to drug resistance: Fig 1 • Some mutations affected codons related to resistance to reverse transcriptase inhibitors • A-209G: AZT resistance • G-223A & A-225G: Loss of sensitivity to D4T • A-205G & C-206A:DDC resistance • A-301G: Non-nucleoside analog resistance

17. Mutant spectra of individual quasispecies • Sample mutation composition of quasispecies by cloning & sequencing individual genomes • Four viral samples cloned • Viral samples: 137, V75-5, D17/+20, & 49 • Viral samples cloned 20-21 times

18. Analysis of Mutant Spectra of Cloned Species: Fig. 4 • 137: A-209G AZT resistance • 49:9 clones showed A & 11 clones showed G at 552 • association w/resistance to FTC & 3TC • V75-5: 8 C-206G • C-206G w/A-205G associated w/resistance to DDC • D17/+20: complete dominance of L-41 & Y-215, also A-309C Pyridinones resistance

19. Mutation Frequency • Defined as • the proportion of mutation positions relative to the consensus nucleotide sequence for each sample group • Calculated by • dividing the number of mutations by the total number of nucleotides sequenced in the corresponding group

20. Mutation Frequencies: Patient Samples and Clones • Explain occurrence of mutations related to drug resistance in absence of positive selection by drugs • Patient samples: range from 0.8x10-2 to 3.4x10-2 substitutions per nucleotide • Clones: range from 3.6x10-3 to 1.1x10-2 substitutions per nucleotide

21. Mutation Frequency Tables

22. What do the Results of Mutation Frequency Mean? • Reverse transcriptase coding region shows high mutation frequency in natural HIV-1 quasispecies • Substitutions related to resistance to antiretroviral agents occur in viral sequences from patients not exposed to relevant inhibitors • Presence of mutations related to drug resistance is expected consequence of high mutation frequency in pol gene

23. Comparison of Frequencies & Meaning • Mutant spectra of individual quasispecies averaged four fold lower than independent isolates • Mutation frequency of Group A between codon 181 and 195 lower than 196 to 219 • implication: HIV-1 in presence of inhibitor may fix mutations to preserve functionality of domains from 181 to 195

24. Figure 2: Mutation Frequency Comparison Group A vs. Group B Few mutations in this Area compared to group B More mutations in this area compared to group A

25. Infection & Resistance • Could individuals in group A been infected with a mutant strain and mutant strain a result of further amplification? • Highly unlikely • no evidence from clinical history of patient points to possibility

26. Conclusion • Mutations associated with drug resistance were found in infected individuals who had not undergone drug therapy • Tolerance of reverse transcriptase to a.a. substitutions is supported by data • Out of about 300 residues, only about 3 were invariant • Unless fitness of virus in danger, one to a few a.a. substitutions will appear during replication with high probability

27. Questions?