HIV/AIDS: Molecular Biology and pathogenesis. George N. Pavlakis National Cancer Institute, USA

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1 HIV/AIDS: Molecular Biology and pathogenesis George N. Pavlakis National Cancer Institute, USA

2 Infection RNA Export, packaging Virion formation RNA Reverse transcription DNA RNA Integration Transcription DNA DNA old new

3 HIV-1 gag pol vif vpr vpu tat env nef

4 HIV-1 mrnas Export mechanism: Specialized Default

5 gag pol env HIV-1 PR RT RNAseH IN gp120 gp41 MA CA NC p6 VIRION

6 Env Env And HIV-1 Entry

7 HIV-1 vif tat gag pol env vpr vpu nef

8 HIV-1 Promoter NFkB Sp1 TATA HIV DNA Assembly Clearance

9 HIV-1 Promoter NFkB Sp1 TATA HIV DNA Assembly Clearance Pausing Elongation

10 HIV uses > Alternative splicing > Bicistronic mrnas > Poly-protein production and processing > frameshifting to express many proteins from one primary transcript

11 HIV-1 vif tat gag pol env vpr vpu nef vif vpr tat1 vpr tat rev Vpu/env nef

12 Nuclear export signal (NES) Rev Nucleoporins AAA Transport nuclear pore complex

13 LTR gag pol env LTR unspliced Specialized export spliced Default export Specialized nuclear export mechanisms are necessary to export the unspliced transcripts that serve as virion RNA and also encode the essential Gag/Pol polyproteins

14 HIV-1 Regulatory and Accessory Proteins Tat transcriptional transactivator Rev viral mrna export factor Vif prevents Innate immunity by APOBEC3G Vpr multifunctional, Transactivator, G2 arrest, nuclear import Nef multifunctional, CD4, MHC-1 downregulation Vpu CD4 degradation in ER

15 HIV-1 Proteins as Molecular Adaptors Interactors Tat Cyclin T viral RNA Rev CRM1 (exportin1) viral RNA Nef CD4, MHC-1 AP1, AP2 Vif APOBEC3G PROTEASOME Vpr TFIIB, hgr, others p300/cbp Vpu CD4 h-βtrcp

16 The Importance Of CD4 Regulation Several HIV proteins modulate CD4 > Env > Nef > Vpu

17 HIV Research Has Benefited Many Fields Control of transcriptional elongation (Tat) Macromolecular export from the nucleus (Rev) Innate Immunity against retroviruses (Vif) Intracellular trafficking of proteins (Gag, Rev, Tat)

18 HIV-1 Genome Compact Highly regulated Adapted for high and rapid expression in permissive cells Able to integrate, to cause a chronic active infection and to persist indefinitely Multiple interactions with cellular factors through viral multifunctional adaptors

19 Effects of Rev on HIV-1 mrna and Protein Expression HIV-1 vpr gag tat pol env vif vpu nef J. Virol. 63: 1265 (1989); PNAS 86: 1495 (1989)

20 Identification of RNA Elements Responsible for Poor Expression of HIV vif gag pol tat vpr vpu env INS (inhibitory/instability elements) INS are negatively acting RNA elements > Are transferable to non-hiv reporter like CAT, GFP > Act when placed outside of open reading frame Rev-RRE interaction counteracts the negative-acting INS RNA/codon optimization results in highly efficient expression of gag, pol, env > Changing RNA without affecting coding potential > Key methodology to generate expression vectors for HIV vaccine approaches J. Virol. 66: 150 (1992); ibid 66: 7176 (1992;) ibid 68: (994); ibid 71: 4892 (1997)

21 50kDa Protein PSF hnrnpa1 polya Binding protein AAA X Transport nuclear pore complex Afonina et al. J Biol Chem. 1997;272: Najera et al J Mol Biol. 1999;285: Zolotukhin et al Mol Cell Biol. 2003;23:

22 Nuclear export signal (NES) Rev GTP Ran Nucleo porins AAA Transport 50kDa Protein PSF polya Binding protein nuclear pore complex hnrnpa1

23 Generation of Efficient Vectors for HIV/SIV Protein Expression by RNA/Codon Optimization gag vif pol tat vpr vpu env INS (inhibitory/instability elements) Two ways to eliminate inhibitory/instability elements (INS): Bypass the effect of INS by co-expression of Rev protein Provide alternative post-transcriptional control elements CTE, RTE Elimination of INS is achieved by introducing multiple point mutations in the coding regions (RNA optimization/codon optimization) > RNA optimization leads to dramatically increased protein expression and is an essential component of DNA vaccines [Schwartz, et al. (1992) J Virol. 66: ; J Virol. 66: ] (first generation of DNA vaccine vectors)

24 Posttrancriptional (RNA) Optimization: Stable mrna=better Protein Expression AAA AAA TAT AAA TTA AAA CAT ATA.. AAG AAG TAC AAG CTA AAG CAC ATC.. Lys Lys Tyr Lys Leu Lys His Ile WT optimized Changes in multiple codons result in stable mrna, efficiently exported and translated in the ribosome

25 Inhibitory Sequences In Coding Regions of HIV-1 Gag pa M1 M1 M2 M3 M4

26 Optimization of HIV-1 gag: 94.4% nt identity (84/1503 nt changed) M7 M8 M1 M2 M10 M3 M4 M11 M12 M13 M6

27 AU- and GC-profile of HIV gag WT GC = 43.9% AU = 56 % OPT GC = 48.9% AU = 51 % Optimized gag mrna has increased GC-content

28 p17 p24 p15 p55bm13p5 protease (Schneider R. et al J Virol (1997): 71, )

29 Posttranscriptional Regulation Is Essential for HIV Expression RNA export via: LTR gag pol env LTR Viral Rev protein prt 1. Elimination gag of negative acting env sequences (RNA optimization) LTR pol LTR in gag/pol and env results in Rev-independent efficient expression plasmids Development of optimized DNA vectors Retroelements: RTE for in vivo DNA delivery pol Cellular Factors prt gag IAP LTR LTR 2. Replacement of the Rev regulation by posttranscriptional regulatory system of SRV-1 results musd in live-attenuated element non-pathogenic virus prt pol gag LTR LTR model to study mechanisms mediating protective immunity against SIV musd

30 We Keep Learning from Retroviruses Retroviruses provide important clues to understand distinct transport pathways used to export cellular proteins and mrnas Discovery of novel RNA export elements and RNA export factors Discovery of mechanisms of function of cellular factors involved in retroviral posttranscriptional regulation

31 Posttranscriptional Regulation Is Essential for Retrovirus and Retroelement Expression RNA export via: LTR gag pol env LTR Viral Rev protein LTR gag prt pol env LTR Retroelements: IAP LTR gag prt pol RTE LTR Cellular Factors musd element musd LTR gag prt pol LTR

32 AAA AAA AAA AAA Rem/RmRE (MMTV) CRM1 nuclear Export pathway NXF nuclear export pathway EXPORT

hnrnp G 50 Contains 3 conserved RNA-binding domains (RRM) at N-terminus

33 Identification of the Cellular Factors Binding to RTE RTE binding factors: RNA helicase A RTE CTE 160 KDa RNA Binding Motif protein 15 (RBM15) hnrnp G 50 Contains 3 conserved RNA-binding domains (RRM) at N-terminus U1A Nuclear protein HA-RBM15 35 Lindtner et al J Biol Chem 281: , 2006

34 RRMs RNA Recognition Motif (3664aa) RID Nuclear receptor interaction domain (977aa) (890aa) SPOC Spen paralogue and orthologue C-terminal SID/RD SMRT interaction domain/repression domain SHARP RBM15/OTT OTT3 transcription posttranscription posttranscription Human Spen(MuMint) transcription (3576aa) (962aa) Rbm15 Mouse Rbm15b (564aa) (5554aa) SPEN transcription Drosophila (793aa) SSP (2738aa) DIN-1LA (CeF07A11.6) F29C4.7 C.elegans (520aa)

35 RBM15 Tethers RTE-RNA to the NXF1 Export Pathway NXF1 RBM15 Gag expression (ng) gag AAA 10 0 Fold Activation: RBM15 and NXF1 interact in vitro and in vivo, and act cooperatively

5 Preliminary study indicates a role of RMB15 in the expression

36 RBM15 is Essential for Mouse Development control RBM15-ko homozygous RBM15-ko homozygous Day 11.5 Preliminary study indicates a role of RMB15 in the expression of a subset of cellular mrnas Soren Warming, Nancy Jenkins, Neal Copeland Mouse Cancer Genetics Program

37 AAA AAA AAA AAA Rem/RmRE (MMTV) CRM1 nuclear Export pathway NXF nuclear export pathway EXPORT

38 Retroviruses and Retroelements Utilize Distinct RNA Export Mechanisms Virus RNA Export element Viral Export Factor Cellular Export Factor Nuclear receptor HIV-1 RRE Rev N/A CRM1 HTLV-I RXRE Rex N/A CRM1 HTDV/HERK RcRE Rec N/A CRM1 MMTV RmRe Rem N/A CRM1 SRV/D CTE no NXF1 NXF1 IAP CTE IAP no NXF1 NXF1 IAP RTE no RBM15 NXF1 MusD MusD element no RBM15 NXF1 RSV DR no?? MLV? no?? * * * * *

39 Diversity of Retroviral RNA Export Elements Rev Rex Different sequence Different RNA structure Different binding factors RTE musd element NXF1 NXF1 RBM15 Preserved NXF1 recognition sites RBM15 RBM15 binds to RTE and musd element suggests conformational recognition site(s)

40 Study Cancer 7: LUNG TUMOR MODEL Control IL-12

delivery of Optimized Murine IL-12 DNA in Mice N=10 No.

muil 12p70 (pg/ml) Spearman r 0.8105 P<0.0001 No.

Lung tumor model: Day 0: intravenous injection of 3x10 5 B16

41 Reduced No. of Lung Nodules in the B16 Melanoma Model After hydrodynamic delivery of Optimized Murine IL-12 DNA in Mice N=10 No. of Lung Nodules Log muil 12p70 (pg/ml) N=9 N=9 N=9 N=8 Log muil 12p70 (pg/ml) Spearman r P< No. of Lung Nodules 50 ng 5 ng DNA ng Sham wt opt Lung tumor model: Day 0: intravenous injection of 3x10 5 B16 Melanoma cells Day 2: Hydrodynamic delivery of IL-12 DNA (50 and 5 ng of wt and op Day 22: Sacrifice mice and count lung and liver nodules

42 Vitiligo In A Mouse Eliminating Large Vascularized B16 Melanoma After IL-12 DNA IT Injection: Suggests Immune Activation Participates In Tumor Remission

43 IL-15/IL-15 Receptor alpha Heterodimeric Complexes for Clinical Development George N Pavlakis NCI

44 Common γ c Chain Cytokines Modified from Waldmann, Nat. Rev. Immunol, (2006)

Co-production Leads To Mutual Stabilization of IL-15 and IL-15Rα Two Forms Of Bioactive IL-15: Cell Associated And Soluble IL-15/15Ra Complexes Trans-presented IL-15/IL-15Rα secretory

45 Co-production Leads To Mutual Stabilization of IL-15 and IL-15Rα Two Forms Of Bioactive IL-15: Cell Associated And Soluble IL-15/15Ra Complexes Trans-presented IL-15/IL-15Rα secretory vesicles ER 15 15Rα 15Rα 15 15Rα N Soluble Bioactive IL-15/15Rα Complex k d =10-11 M ERAD (ER Associated Degradation) Bergamaschi, J Biol Chem :4189; J Immunol :3064

46 IM Injection of IL-15/15sRa DNA Vectors Leads to Increased Plasma IL-15 Levels Plasma IL-15 in Lymphoablated humans

Single administration of IL-15 encoding DNA is sufficient for the

cells # NK cells (total spleen) Spleen NK cells p=0.

004 * NK cells x 10 6 pneumocytes Lung NK cells * p=0.

47 Single administration of IL-15 encoding DNA is sufficient for the complete recovery of NK cells in spleen and lung within Recovery of NK cells # NK cells (total spleen) Spleen NK cells p=0.008 * Control CTX+empty vector CTX+IL-15 p=0.004 * NK cells x 10 6 pneumocytes Lung NK cells * p=0.017 days CTX DNA CTX DNA vs CTX+empty vector Non parametric t test NK recovery is complete within 14 days after lymphodepleting treatment

48 Absolute NK And CD8 Counts In Macaque Plasma During 12 Daily IV Administrations IgG1Fc IL-15Rα IL-15 IL-15 IL-15sRα IL-15

Retroviruses. ---The name retrovirus comes from the enzyme, reverse transcriptase.

Retroviruses. ---The name retrovirus comes from the enzyme, reverse transcriptase. Retroviruses ---The name retrovirus comes from the enzyme, reverse transcriptase. ---Reverse transcriptase (RT) converts the RNA genome present in the virus particle into DNA. ---RT discovered in 1970.