LTR R U5 PBS. 5' LTR : weak promoter [Inr, TATA-box, and three Sp1 sites] + enhancer elements.

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Valentine Nichols
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1 Foreword HIV has a very complex system of transcription regulation and mrna splicing. This produces something that approximates "life-cycle" stages. Expression from the promoter always occurs but at first it is at a reduced level and at first the transcripts are very short. The shortness is because the pre-initiation complex that forms is of low quality. Polymerase begins transcription in a low processivity state. This early stage is critical for HIV. Later on the transcription complexes become better quality and polymerase can make full length transcripts. We have noted before that many transcription factors have multiple functions - kind of like Swiss Army Knifes. HIV amplifies this to the point of absurdity. 1

2 LTR 5' LTR provides acts as a very weak promoter. 3' LTR provides a polyadenylation signal. This is very clever considering that they are the same sequences! R U5 U3 R U5 PBS U3 U3 R U5 PBS U3 R U5 5' LTR : weak promoter [Inr, TATA-box, and three Sp1 sites] + enhancer elements. One enhancer binds a host transcription factor called NF-kappa-B. This transcription factor is typically activated when cells of the immune system respond to an infection (cellular response). The immune response involves proliferation of immune cells and NF-kappa-B is turned on when these cells make the decision to divide. The HIV promoter is also activated by NF-kappa-B. Therefore, when the immune system is stimulated the expression of HIV is also stimulated!! HIV has subverted a defensive host response system for its own use. Taube R, Fujinaga K, Wimmer J, Barboric M, Peterlin BM Tat transactivation: a model for the regulation of eukaryotic transcriptional elongation. Virology. Nov;264(2):245-53

3 LTR -new R U5 U3 R U5 PBS U3 U3 R U5 PBS U3 R U5 One enhancer binds a host transcription factor called NF kappab (nuclear factor [kappa]b) AND NFAT (nuclear factor of activated T cells) and Ets family members. These transcription factors are typically activated when cells of the immune system respond to an infection (cellular response). The immune response involves proliferation of immune cells and NF-kappa-B is turned on when these cells make the decision to divide. The HIV promoter is also activated by NF-kappa-B. Therefore, when the immune system is stimulated the expression of HIV is also stimulated!! HIV has subverted a defensive host response system for its own use. NF [kappa]b and NFAT relocalize to the nucleus after lymphocyte activation. In the cytoplasm NF-[kappa]B is associated with a cytoplasmic inhibitor, I[kappa]B. Lymphocyte activation causes the phosphorylation, ubiquitination, and proteosomal degradation of the I[kappa]B. NFAT is dephosphorylated by calcineurin (a reaction inhibited by cyclosporin A) and, after its nuclear import, assembles with AP1 to form the fully active transcriptional complex.

4 0 þ þ þ Miguel Stevens, Erik De Clercq, Jan Balzarini Medicinal Research Reviews, 26:

5 Skip NFK-b In cytoplasm bound to I-kappa-B alpha Phosphor of IkBa causes its destruction NFK-b goes into nucleus NFK-b gets phosphorylated It can recruit CBP or HDAC which means that it can stimulate or repress. 5

6 Skip How NF-kappa-b is activated. p50/p65 dimers are NFkB 6

7 Nucleosome 0 and 1 are reproducibly positioned (remodeled). Skip p50 dimers are NFkB p50/p65 dimers are NFkB 7

8 Skip p50 dimers are NFkB p50/p65 dimers are NFkB nuc-1 has to be moved (remodeled) TFIIH - ATP-dep helicase TFIIH - kinase 8

9 Stages of HIV replication Why are the transcripts stuck in the nucleus during the rev minus stage?

10 REV + / - RRE exon 1 intron exon 2 RRE nuclear membrane REV + / - splice factors RRE RRE REV - RRE REV + RRE

11 Experiment which first shows a function of rev. B-globin transcripts are very efficiently spliced. Skip

12 Rev Rev/RRE binds host cell factor called CRM-1. This guy causes nuclear export of the Rev/RRE complex. CRM-1 exports it using the exportin 1 pathway. Rev then goes back into the nucleus.

13 Tat 13

16 Tat does more than you think 101 amino acids in clinical isolates In addition to regulating transcription it can leave the cell and enter uninfected cells! It remains able to regulate transcription in these cells. Extracellular Tat can stimulate production of cytokines and expression of cytokine receptors, modulate survival, proliferation and migration of different cell types and has angiogenic effects. Miguel Stevens, Erik De Clercq, Jan Balzarini Medicinal Research Reviews, 26:

17 Kaposi s sarcoma Human herpesvirus 8 HHV8 Found in all KS Much more commonly found in males Becoming less common Also seen in those at risk for but without HIV Old paper showed that Tat expression in mouse epithelia would induce something that looked like Kaposi s sarcoma. Cells that are in the tumor do not express Tat. Hengge et al The Lancet Infectious Diseases 2:344

18 Accessory Proteins So called because they are not required for replication in culture. BUT they are required for successful persistence in a real host. Nef Vpu Vif Vpr

19 Nef Helps keep the infected cell alive. blocks multiple apoptosis signals that would cause the infected cell to die. down regulates CD4 from cell surface, reduces chance of autofusion or syncytial cell fusion down regulates surface levels of MHCI proteins. This prevents cell from telling other immune cells that it is infected. activates the intercellular signaling system that causes bystander apoptosis. activates the infected T cell which helps get HIV genome expressed. expressed before integration.

20 Nef deletion A cohort of HIV positive men who were infected with a virus with a deletion in the Nef gene were identified in Australia. They have been mostly asymptomatic since They seem to be able to live with the virus - very, slow progression of the disease. Learmont JC, et al N Engl J Med. Jun 3;340(22): Immunologic and virologic status after 14 to 18 years of infection with an attenuated strain of HIV-1. A report from the Sydney Blood Bank Cohort. Birch MR, et al J Clin Virol. Oct;22(3): An examination of signs of disease progression in survivors of the Sydney Blood Bank Cohort (SBBC).

21 Vif, Vpu and Vpr Vif Cell puts CEM15, a cytidine deaminase (cell defense system), into the maturing virus particle to tries to damage the viral genome. HIV puts Vif into the particle and these inhibits the activity of CEM15. Vpu Stimulates degradation of CD4 within the endoplasmic reticulum. This prevents the Env polyprotein from being bound up in the endoplasmic reticulum. Vpr gives HIV the ability to enter nuclei of non-dividing cells. Connects the pre-initiation complex to the importin 7 nuclear import pathway.

22 99 bp DNA flap common for Lentiviruses Important for entering non-dividing cells RTC = reverse transcription complex. PIC = preintegration complex. Zennou et al HIV-1 Genome nuclear import is miediated by a central DNA flap Cell 101:

24 Vpr gives HIV the ability to enter nuclei of nondividing cells. Connects the preinitiation complex to the importin 7 nuclear import pathway.

25 What possible advantage to the virus is a 2 stage life cycle? Rev > Rev+ Provides time for Nef to down regulate CD4 and to adjust MHCI so that the cell does not appear to be infected. Other reasons may exist that await discovery.

26 Polyproteins HIV gag and pol regions shown. LTR gag pol mrna gag pol The unspliced mrna contains the coding regions for both the gag and pol genes. However, they overlap one another and are in different reading frames. Translation in the gag reading frame produces a 55 kda protein. Pr55 a polyprotein HIV protease cleaves Pr55 to produce: p17 p24 p7 p6

27 The polycistronic mrna can also be translated in another way. frame shift region gag pol mrna Translation begins in the gag reading frame but then a ribosome frame shift occurs! This now causes the pol gene to be read in the correct reading frame. Now a bigger protein is made: polyprotein pr160 First part is in the gag reading frame. Then a translational frameshift occurs and the second part is in the pol reading frame. Proteolysis produces the following from the polyprotein. p11 this is the protease itself this is reverse transcriptase p66/51 this is the integrase p32

28 Quasi-species Reverse transcription error rate is 10-4 to Daily in vivo virus production rate is > per day Viral recombination Copy-choice recombination --> Strand switching Sources of diversity when 2 variants are in same cell. Inherent in mechanism of reverse transcription to allow this. Superinfection oddly enough appears common. Ideas have been prev covered. Read for clarification if you wish. Malim & Emerman Cell 104:

29 Quasispecies

30 Quasispecies

31 Quasispecies

32 This has been discussed. So what? The diversity generated in a single infected individual can be greater than the worldwide diversity of influenza A virus during an epidemic 36.1 million people are currently estimated to be infected. Weiss RA NATURE 410:

33 What is possible with such diversity? Many of the variants are produced invisible to some or ALL components of the immune system - true. Restriction - Genetic bottleneck associated with the mode of transmission. <-- Helps restrict things a bit. What if mode of transmission changed!!!!???? Weiss RA Nature 410: Rambaut, A., Posada, D., Crandall, K. A. & Holmes, E. C. (2004) Nat Rev Genet 5,

34 HIV2 differs from HIV 1. A large number of small changes HIV 2 lacks vpu but has vpx (dupl of vpr). Not presented in class. Read for clarification if you wish.

35 Origin HIV 1 is related to SIVcpz simian immunodeficiency virus, cpz = chimpanzees HIV 2 is related to SIVsmI smi = mangabeys HIV1 and 2 entered humans at different times and probably multiple time. Humans hunt primates for food. It is believed that during the hunt or during preparation of the meat that humans were contaminated by blood transfer. When? A number of early samples from infected humans have been found from the 1959-the mid 1960 s. These do not represent the original entry. Evolutionary studies suggest that the original tntry time was between 1915 to 1941.

36 Current therapies Only 16 drugs in use. Nucleoside analogs (Nucleoside Reverse Transcriptase Inhibitor - NRTI) Non-nucleoside Reverse Transcriptase Inhibitors (NNRTI) HIV protease inhibitors Fusion inhibitors such as Enfuvirtide these inhibit gp41 <-- a viricide. HAART - highly active antiviral therapy (used to be called Combination Therapy).

37 Reverse Transcription Inhibitors Inhibitors of reverse transcriptase nucleoside analogs that work just like the dideoxynucleotides used to sequence DNA. Basically, the trick with these is to find one that the reverse transcriptase accepts readily but which the host's DNA polymerases tend to reject. AZT or azidothymidine has been used since N3 is an azido group which is attached to the 3' position of the deoxyribose. Reverse transcriptases use AZT preferentially over TTP bout our cellular DNA polymerases prefer TTP. It takes an additonal 10 to 20 times more TTP to block DNA synthesis by DNA polymerases. The use of AZT along prolongs life by about 2 years. Unfortunately, AZT resistant mutants appear after about 6 months of treatment. Fortunately, these mutants are still sensitive to other analogs (ie. ddc, ddi & 3TC).

38 Combination therapy... Now called HAART $15,000 / year for drugs alone

39 Other ideas Parts of life cycle that differ most greatly from how cells do things are the good candidates for targets. RNAs that look like TAR or RRE can soak up all of the TAT and REV. RNAi Ribozymes Vaccines small molecule inhibitors Transgenically modify person s immune cells in culture and reintroduce them ARGs, make them more likely to kill themselves if infected, express anti-hiv ribozymes, 39

40 Viricides gp41 When it promotes fusion, gp41 exposes a hydrophobic peptide. A peptide matching another part of gp41 can block fusion. This one is called T20 and it may represent a part of gp41 that normally binds the hydrophobic peptide. Approved for use in March 2003.

41 gp41 41

42 Reservoirs Quiescent T-cells - remember past infections. Half live 4 years Monocytes CD14&CD16 + CCR5 bind HIV. Can go into the brain & other places. Wait months or decades. Drugs may not get there. Mature into macrophages - turn up anywhere. Macrophages make a lot of virus probably more on a per cell basis than a T-cell. Monocytes also make dendritic cells. Show up at every entry point into the body where they interact with T-cells and B-cells. Fat cells! May, M. Playing Hide and seek the deadly way The Scientist vol 18: 16-19

43 Shock & Kill

44 CDC site Aids Knowledge Base Jama HIV/AIDS Information Center National Institute of Allergy and Infectious Diseases Combination Antiretroviral Therapy June 1998 Am.Acad.Family Physicians The Body: Gene Therapy for HIV/AIDS HIV Infection and AIDS, FDA related treatment, testing, fraud, prevention,clinical trials

Fayth K. Yoshimura, Ph.D. September 7, of 7 HIV - BASIC PROPERTIES

Fayth K. Yoshimura, Ph.D. September 7, of 7 HIV - BASIC PROPERTIES 1 of 7 I. Viral Origin. A. Retrovirus - animal lentiviruses. HIV - BASIC PROPERTIES 1. HIV is a member of the Retrovirus family and more specifically it is a member of the Lentivirus genus of this family.