Gene Technology

1. HIV Infection - AIDS Gene Technology Human immunodeficiency virus (HIV-1)- Retrovirus with ssRNA genome Acquired immunodeficiency syndrome (AIDS) Lecture 9 – Chapter 25 (2nd edition)

2. Size and Shape of Viruses Gene Technology

3. Retroviruses Gene Technology Retrovirus has reverse transcriptase -> to write RNA into DNA -> integration of DNA into host genome

4. Life Cycle of RNA Viruses Non-integrative RNA viruses (Influenza virus, Polio virus, …) Integrative RNA viruses (Retroviruses) (HIV, some oncoviruses) Gene Technology

5. RNA Viruses Gene Technology

6. Gene Technology Absorption of Viruses • Virus coincidentally collides with a susceptible host cell and adsorbs specifically to receptor sites on the cell membrane • Spectrum of cells a virus can infect – host range -> normally narrow host range • hepatitis B – human liver cells • poliovirus – primate intestinal and nerve cells • rabies – various cells of many mammals Influenza virus

7. Gene Technology Penetration of the host cell • Flexible cell membrane is penetrated by the whole virus or its nucleic acid by: • endocytosis – entire virus is engulfed and enclosed in a vacuole or vesicle • fusion – envelope merges directly with membrane resulting in nucleocapsid’s entry into cytoplasm Endocytosis Fusion

8. Gene Technology Release of viruses • Assembled viruses leave host cell in one of two ways: • budding – exocytosis; nucleocapsid binds to membrane which pinches off and sheds the viruses gradually; cell is not immediately destroyed • lysis – nonenveloped and complex viruses released when cell dies and ruptures HIV

9. Gene Technology HIV Infections and AIDS • First emerged in 1981 (CDC received reports of 2 rare conditions increasing -> form of pneumonia; Pneumocystis carninii + unusual skin cancer; Karposi’s sarcoma -> patients had impaired immune system) • 1982 CDC recognized AIDS as new disease • 1983 HIV recognized as source for AIDS • HIV-1 may have originated from a chimpanzee virus. • 1959 first documented case of AIDS • In 2006, the number of infected individuals worldwide was estimated to be 45 million • 6th most common cause of death among people aged 25-44 years in the U.S.

10. Gene Technology HIV Virus • Retrovirus, genus Lentivirus • Encode reverse transcriptase enzyme which makes a double stranded DNA from the single-stranded RNA genome • Viral genes permanently integrated into host DNA • HIV-1 and HIV-2 • Infects white blood cells • HIV can only infect host cells that have the required CD4 receptor plus a co-receptor. (some people miss that receptor -> cannot be infected with HIV)

11. Gene Technology HIV Infection • Transmission occurs by direct and specific routes: mainly through sexual intercourse and transfer of blood or blood products; babies can be infected before orduring birth, and from breast feeding. • HIV does not survive long outside of the body. • HIV enters through mucous membrane or skin and travels to dendritic phagocytes beneath the epithelium, multiplies and is shed. • Virus is taken up and amplified by macrophages in the skin, lymph organs, bone marrow, and blood.

12. Gene Technology HIV Infection • Primary effects of HIV infection: • extreme leukopenia (decrease in white blood cells) – lymphocytes in particular • formation of giant T cells and other syncytia (fused cells with more than 1 nucleus) allowing the virus to spread directly from cell to cell • Infected macrophages release the virus in central nervous system, with toxic effect, inflammation. • Secondary effects of HIV: • Destruction on CD4 lymphocytes allows for opportunistic infections and malignancies.

13. Gene Technology Signs and Symptoms of HIV Infections and AIDS • Symptoms of HIV are directly related to viral blood level and level of T cells. • Initial infection – mononucleosis-like symptoms that soon disappear • Asymptomatic phase 2-15 years (avg. 10) • Antibodies are detectable 8-16 weeks after infection. • HIV destroys the immune system. • When T4 cell levels fall below 200/mL AIDS symptoms appear including fever, swollen lymph nodes, diarrhea, weight loss, neurological symptoms, opportunistic infections and cancers.

14. Gene Technology AIDS

15. Gene Technology AIDS related diseases

16. Life cycle of HIV HIV attaches to CD4 and coreceptor; HIV fuses with cell membrane. Reverse transcriptase makes a DNA copy of RNA. Viral DNA is integrated into host chromosome (provirus). Can produce a lytic infection or remain latent Gene Technology

17. Function of viral proteins Gene Technology

18. The retroviral genome Gene Technology

19. Function of viral proteins –Tat protein Gene Technology Tat -> regulates transcription of HIV Tar sequence -> binding of tat protein -> Tar positions tat protein in correct location for its function -> induction of high-level expression

20. Function of viral proteins – Tat protein Promoter Gene Technology Tat -> regulates transcription of HIV Tar sequence -> binding of tat protein -> Tar positions tat protein in correct location for its function -> induction of high-level expression CAT -> Chloramphenicol acetyl transferase (reporter gene) Cp -> coat protein of phage R17

21. Prevention of HIV infection by ”mopping up” Tar and Tat as target for drug therapy!!! Gene Technology Cell expressing a high level of tRNA-Tar -> infected with HIV -> Tat protein interacts with tRNA-Tar sequence -> prevents HIV genes from being transcribed

22. Function of viral proteins – Ref protein Gene Technology Ref protein regulates transfer of unspliced mRNA through nucleus membrane If Ref not present -> just fully spliced mRNA transfered to cytoplasm -> just regulatory genes expressed (tat, nef, ref) If Ref present -> binds to RRE (Rev respond element) -> unspliced mRNA can go through -> all proteins can be made to assembly virus particles + genomic RNA for packing

23. Function of viral proteins – Ref protein Gene Technology Experiment on β-globin protein: β-globin mRNA is highly spliced -> no unspiced mRNA in cyctoplasm -> mutation in 5’ or 3’ splice site -> accumulation of unspliced mRNA in nucleus -> fusion between mutated protein and RRE site -> + Ref protein -> unspliced mRNA transported to cytoplasm -> Ref protein possible target for drug therapy !!!!

24. Function of viral proteins – Nef protein Nef protein is involved in the development and maintenance of an active onfection in vivo Nef-open (open reading frame -> active protein) Nef-deletion (nonactive protein) Nef-stop (gives truncated protein) Cells cultures -> no effect In vivo experiment (simian immunodeficiency virus in rhesus monkeys): Nef-stop -> viruses recovered from monkey -> stop lost (reverted) Nef-deletion -> virus concentration was much lower (100 times lower) + animals remained healthy -> Nef protein possible target for drug therapy !!!! Gene Technology

25. Gene Technology

26. Gene Technology Preventing and Treating HIV • No vaccine available • monogamous sexual relationships • condoms • universal precautions • No cure; therapies slow down the progress of the disease or diminish the symptoms • inhibit viral enzymes: reverse transcriptase, protease, integrase • inhibit fusion • inhibit viral translation • highly active anti-retroviral therapy

27. Treatment – Interfering with the viral DNA synthesis Gene Technology Azido group in 3’ end -> chain termination of DNA synthesis by reverse transcriptase Severe side effects -> damages bone marrow

28. Treatment – Interfering with the viral DNA synthesis Gene Technology In activate reverse transcriptase by binding to active site Problem: Transcritase has a very high mutation rate

29. Treatment – Interfering with making active proteins HIV protease: p11 Cuts precursors of proteins -> Pr55 and Pr160 -> into structural proteins (p17, p24, and p15) and functional proteins (p11, p66/51, and p32) Gene Technology

30. Treatment – Interfering with making active proteins HIV protease inhibitor: -> Small peptides binding to active site of protease Gene Technology Problem: HIV protease has a very high mutation rate

31. CD4 - Possible targets for therapy HIV virus needs a CD4 receptor to attack a cell Experiment: HeLa cells (human cancer cell line not producing CD4 on the surface) Recombinant HeLa cells produce CD4 on the surface -> can be infected with HIV Gene Technology

32. CD4 - Therapy HIV virus needs a CD4 receptor to attack a cell Experiment: Produce a high amount of soluble CD4 receptor protein -> binds to HIV gp120 and blocks infection with cells Unfortunatly not that effect in vivo gp120 is changing with high mutation rate Gene Technology

33. HIV Therapy Gene Technology

34. HIV Therapy Gene Technology

35. HIV Therapy Gene Technology

36. HIV Therapy Gene Technology

37. HIV Therapy – Some Protease Inhibitors Gene Technology EC50 -> 50% effective concentration (plasma concentration required for obtaining 50% of a maximum effect in vivo) IC50 -> 50% inhibitory concentration

38. Drugs used for HIV Therapy Gene Technology

39. Immuno - Therapy Gene Technology

40. Natural Immune to HIV Infection Some people and animals have natural immunity against HIV infections – Why? Defensins are small (20-50 AA) gene encoded peptides -> antimicrobial peptides (a lot of AMPs are active against bacteria, some eukaryotes, viruses, cancer cells, immune stimulants) -> rich in S-S bonds -> produced by mamaliens (as part of the innuate immune system) -> α, β, and θ-defensins (cyclic peptides) -> genes are available in all primates Gene Technology θ-defensins of the rhesus monkey

41. Natural Immune to HIV Infection • Some people and animals have natural immunity against HIV infections – Why? • People with a defective CD4 receptor • θ- defensins prevent infection of HIV (prevent HIV to attack CD4 receptor) • -> genes are available in all primates • -> orang utans, gibbons, macaques (rhesus) -> active peptide -> cannot be infected with HIV-1 • -> humans, chimps, and gorillas have a STOP codon in the gene -> pseudogene -> inactive peptide • -> first attemts to design synthetic drugs (θ- defensins) for therapy Gene Technology θ-defensins of the rhesus monkey preventing HIV-1 infection