1. HIV 1981 objeven 1983 izol. retrovir - Pasteur institute 1984 izol. retrovir - NIH 1986 nzev HIVdo 2002 20 mil. mrtvch 20025 mil. nov infikovanch 200242 mil. infikovanch 1 ze 100 mezi 15 a 49 r.Subsaharsk Afrika 2,4 mil. mrtvch 25-30% osob infikovno 1/3 dt mladch 15 let ztratilo rodie

2. Od HIV+ k AIDS 10% infikovanch lid do 2-3 let AIDS 80% do 10 let progrese viru z nich polovina AIDS 10-17% AIDS free po vce ne 20 let HIV II mnohem pomalej navc Vpx, Vt nebezpe kdy aktivovan imun systm vce aktiv. T-lymf. + aktivn Nf-kappaB Delece v Nef, MHC I alely, chemokinov receptory 3. lnky pro pt tden Trafficking HIV Sherer NM, Lehmann MJ, Jimenez-Soto LF, Ingmundson A, Horner SM, Cicchetti G, Allen PG, Pypaert M, Cunningham JM, Mothes W.Related Articles, Links Visualization of retroviral replication in living cells reveals budding into multivesicular bodies. Traffic. 2003 Nov;4(11):785-801. DC-SIGN DC jako Trojsk k Exp Med. 2004 Nov 15;200(10):DC-SIGN-mediated infectious synapse formation enhances X4 HIV-1 transmission from dendritic cells to T cells. Arrighi JF, Pion M, Garcia E, Escola JM, van Kooyk Y, Geijtenbeek TB, Piguet V. Pirozen antiviremika TRIM5 Stremlau, M. et al. Nature 427:848-852, 2004, APOBEC3G Gu Y & Sundquist WI. Naure 424:21-22, 2003. Evolun atenuace viru HIV Arien KK, Troyer RM, Gali Y, Colebunders RL, Arts EJ, Vanham G.Related Articles, Links Replicative fitness of historical and recent HIV-1 isolates suggests HIV-1 attenuation over time. AIDS. 2005 Oct 14;19(15):1555-1564. 4. 1981 HIV I - minimln 3 nezvisl penosy z impanze1986 HIV II - tm identick s mangabejm SIV podobnouchorobu dostane i makak 5. Regionln rozloen subtyp 6. Prevalence 7. Osoby nov infikovan v roce 2003 Eastern EuropeWestern Europe & Central Asia 30 000 40 000 180 000 280 000 North America36 000 54 000 East Asia & PacificNorth Africa & Middle East 150 000 270 000 Caribbean43 000 67 000South45 000 80 000& South-East AsiaSub-Saharan Africa 610 000 1.1 millionLatin America 3.0 3.4 million Australia 120 000 180 000 & New Zealand700 1 000 celkem: 4.2 5.8 milion 8. Projected life expectancy at birth Selected sub-Saharan countries 65 years 60 55 Kenya50 Botswana Zimbabwe45ZambiaUganda40Malawi35 1955 1960196519701975 1980198519901995 2000Source: World Population Prospects: The 1996 revision, United Nations Population Division, 1996 10 -GE-98001/10 1 December 1997UNAIDS/WHO 9. U.S. HIV/AIDS epidemic: New infections 2001Men 70% Women 30% 10. AIDS Incidence* for Women and Percentage of AIDS Cases, January 1986 - June 2000, United States Number of Cases Percent of Cases1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000Half-Year of Diagnosis*Adjusted for reporting delaydelays 11. HAART 12. HIV structure: 13. Struktura virionu 14. HIV Lifecycle Rambaut A. et al. Nat. Rev. Genet. 5:52-61, 2004 15. Glykoproteiny virovho obalu Synthesis. Initial product is gp160; processed by a cellular protease into gp41 (transmembrane) and gp120 (surface). Gp41 and gp120 are linked by a non-covalent interaction. Gp120. Oligomeric and heavily glycosylated. Includes highly variable surface domains (such as V3) which influence virus host cell range and antigenicity. Gp120 interacts with CD4 and also with the chemokine receptors (CCR5, CXCR4). Gp41. Oligomeric. Contains a hydrophobic N- terminus fusion domain, that mediates fusion of the viral envelope with the host cell plasma membrane. 16. Structure of HIV-1 gp120/CD4 Complexgp120CD4 Blue: glycosylation sites 17. Vazba HIV a vstup do buky CD4 binding.Gp120 binds CD4, via conserved conformational domains in gp120. Gp120 conformational change. CD4 binding exposes the coreceptor binding domain in gp120. Coreceptor binding. Gp120 binds to the chemokine receptors which act as viral coreceptors (principally, CCR5 and CXCR4). Gp41 conformational change. The combined effect of CD4 binding and coreceptor binding is that a coiled- coil structure is induced in gp41, thereby exposing the hydrophobic fusion domain in gp41. This allows fusion to occur. 18. Vstup viru HIV vstup mukznmi povrchy epiteliln buky infikovateln M buky a Langerhansovy buky DC-SIGN dendritic cell specific Icam-grabbing nonitregrin -vysokoafinn interakce s HIV -v krvi jako prvn monocyty a aktivovan T-lymfocyty 19. R5 and X4 VirusesR5 strains of HIV: Use CC-chemokine receptor 5 (CCR5). CCR5 is found on dendritic cells, macrophages and T cells. R5 viruses predominate in early infection and are believed to be the major species involved in mucosal (sexual) transmission. X4 strains of HIV: Use CXCR4 as a coreceptor. X4 viruses can infect T cells only, and come to predominate over time. These strains are associated with the elimination of TH cells. 20. Other ReceptorsAdditional chemokine receptors: CCR2b, CCR3, CCR8, APJ, Bonzo (STRL33), BOB (GPR15). None of these is as important as CCR5 or CXCR4, though CCR3 may be important in microglia.Dendritic cells: Can capture virus via DC-SIGN (DC-specific ICAM3-grabbing non-integrin, which is a lectin-like receptor). DC-SIGN can bind and store HIV in a stable infectious form, and transmit it subsequently to T cells during the process of T cell activation. 21. Tropismus viru HIVMutace v ligandu pro CXCr4 Mutace v CCr5 rezistence k HIV rezistence blokuje vazu HIV nakoreceptor 22. HIV-1 Fusion and EntryFlint. Fig. 5.5 23. CCR5 Binding Site on gp120Yellow: CD4 Green: gp120Gp120 domains: Blue: Glycosylations Purple: CD4-binding Orange: Coreceptor-binding PDB (http://www.rcsb.org/pdb/): 1GC1 24. Genetics of HIV SusceptibilityCCR5: CCR5D32. 32 bp deletion in gene leads to translational frameshift and protein truncation; no CCR5 is made. Heterozygotes (10% of caucasians) have delayed progression; homozygotes (1% of caucasians) resist infection with R5 strains. CCL3L1 gene dosage. The number of copies of a segmental duplication encompassing the gene for CC chemokine ligand 3- like protein (CCL3L1), a ligand for CCR5. A low CCL3L1 copy number is associated with an increased susceptibility to AIDS progression. Gonzalez S et al. Science 307:1434, 2005. Major histocompatibility complex: human HLA genes. Influence immune responsiveness. Full heterozygosity for class I HLA results in delayed progression, presumably because it allows the host to respond to a greater diversity of viral antigenic epitopes. 25. Figure 1: Generalised molecular interactions taking place between cells in the immunological synapse (top) and the virological synapse (bottom). Some of the interactions are probably shared between the two types of synapse, such as LFA-1-ICAM-1 and LFA-1-talin-actin, whereas others are specific for each type of synapse. 26. Schematic diagram of the virological synapse formed between a retrovirally infected (effector) and an uninfected (target) T cell. The cell cell contact zone contains tight junctions and, in the case of the HIV-1 VS, a synaptic cleft into which virions are released from the effector cell. The HTLV-1 VS may differ from the HIV-1 VS: there is no evidence for HTLV-1 virion budding or release or of a synaptic cleft (C. Bangham, unpublished results). The MTOC is polarised towards the site of cell cell contact and viral Gag may be associated with, and be transported along, microtubules. Figure based on results presented in (20 22). 27. DC-SIGN DC-SIGN is present in the infectious synapse between DCs and CD4+ T cells. Mature DC- SIGN+ DCs were loaded with HIV- GFP for 2 h at 37C and incubated with highly purified resting CD4+ T cells for 30 min at 37C, allowing infectious synapse formation. Two representative examples are shown (ac and df). DC-SIGN was readily detected at the infectious synapse by confocal microscopy, appearing sometimes enriched (f), but not consistently (c). (green) HIV-GFP; (red) DC-SIGN. 28. HUT 78 infikovan HIV + epiteliln buka 29. Struktura genomu 30. Cis-Acting Regulator: LTRStructural Genes: Gag, Pol, Env Essential Trans-Acting Genes: Tat, Rev Accessory Genes: Vif, Vpr, Vpu, Nef 31. Genetick diverzita viru HIV v jednominfikovanm lovku je vt ne diverzita chipkovch vir celosvtov populace pi epidemii!!! Umouje efektivn nik novch variant, protiltkynesthajNejvyy titr aktuln protiltky asto proti historickvariantProtiltky interferuj, ale i pomhaj viru do buky Fc receptory 32. Strategie niku imunitn odpovdiStealth: Infection of cells in immune-privileged sites (CNS, resting T cells) & establishment of latency Downregulation of MHC class I by Nef. HLA-A, B are downregulated (needed for target recognition by CD8+ cells) but HLA-C, -E are not (transduce inhibitory signals to NK cells) Glycosylation and conformation of Env hides epitopes 33. Strategie niku imunitn odpovdi Counter-attack: HIV selectivelyinfects HIV-specific CD4+ memory T cells HIV targets DC-SIGN and infects dendritic cells (DC); it then uses DC to infect T cells HIV interferes with bothT cell and DC function/maturation Vif counteracts the endogenous antiviral activity of APOBEC3G (CEM15) Nef induces FasL, which leads to death of Fas+ T cells 34. Dynamika viru HIV 90% partikul v krvi produkovno CD4-T lymfocyty s dlkou ivota okolo 1 dne Zbytek dleijcmi T-lymfocyty s odlinm fenotypem Lymfocyty v krvi pouze 1/50 vech T-bunk zbytek periferie hlavn uzliny zde mnohem vt procento infikovanch bunk ne v krvi 35. 1/3 pacient s AIDS neurologickproblmy!!! virus do NS vstupuje brzy po infekci makrofgotropn varianty infikovnyastrocyty a mikroglie 36. Virus HIV a ndoryobecn incidence a nebezpenost nkterch typ stouphlavn ty spojen s infekc onkogennmi viry Kaposhiho sarkom endothelilnho pvodu, 20% homosexulnch mu, kauzalita s human herpes virus 8 HHV-8 B-bunn lymfomy dsledek infekce EBV a HHV-8 Anogenitln karcinomy 3x vy incid. - papilomaviry 37. Patologie imunitnho systmu pi AIDS CD 4 T-buky kad rok bytek o cca 60/ml, target CTL, sm virus mn jejich fyziologii sniuje CD4, MHC I (A,B), CD28, produkci IL-2, IL-2 receptor CTLn aopak vce, hlavn na zatku, aktivace targety, een disbalance v potu T-lymfocyt Monocyty a makrofgy defekty v chemotaxi, funkci Fc receptor B-buky zpotku overprodukce IgG, IgA, IgD, hlavn u HIV 1 kles mnostv mlo helper, mno se in vitro bez aktivace asto infikovny EBV a cytomegalovirem NK buky aktivita se sniuje IL-2 dependentn Autoimunita obvykl dramatick disbalance imun. syst. 38. HIV-1 Env glycoproteinsSurfac e glycoprotein (SU)Transmembrane glycoprotein (TM)gp120gp41 Variable domain s (gp120)1100200300400 500 600 70| |||| | | | signaltransmembrane peptideanchor V3 loopGRCD4 binding Fus ionP A domaindomain G Fproteolytic cleavageC C (c ellular enzyme) important for ch emok ine re ceptor binding 39. Genetics of HIV Susceptibility Major histocompatibility complex: human HLA genes. Influenceimmune responsiveness.Full heterozygosity for class I HLA results in delayed progression, presumably because it allows the host to respond to a greater diversity of viral antigenic epitopes. 40. Nuclear Import & IntegrationNuclear import. The viral preintegration complex (the partially uncoated core particle, including RT and RNA) enters the nucleus via an active uptake process that depends on (multiple) viral nuclear localization signals. The ability to traverse the nuclear pore allows HIV to infect non-dividing cells, unlike oncoretroviruses. Integration.This is non-random and occurs preferentially in transcriptionally active genes. 41. Viral TranscriptionThe viral long terminal repeat (LTR) contains the major promoter which drives viral RNA transcription. Elements include: Core promoter. Recruits cellular RNA pol II. Enhancer elements. Bind NFAT, NFkB; allow virus to respond to cellular activation. Modulatory elements. Additional regulatory domains. TAR (trans-activation response element). Located in R region of LTR; allows HIV LTR to undergo Tat- mediated activation 42. Trans cription elements within the HIV-1 LTRAdapted from Luciw, Chap ter 60 , Fields Virolog y, 3rd Edn. U3R U5modulatory elements enhancerTAR coremRNATAT AAgag gene Various fac torsNF- kB Sp1LBP1Cellular stimuliHIV LTR - Transcriptional Regulation 43. Viral Transactivators Multiply spliced mRNAs.Simple retroviruses encode only unspliced or singly spliced mRNAs.Tat: trans-activator of transcription Regulatesviral transcription (enhanceselongation) Binds RNA (TAR: Tat-responsive element) Acts in concert with cellular proteins (P-TEFb)which bind Tat & TAR (P-TEFb = CYCT1 + CDK9)Rev: regulator of viral expression Regulates splicing (overcomes restriction on nuclear export of unspliced transcripts) Binds RNA (RRE: Rev-response element) Acts in concert with cellular proteins which bind 44. HIV-1 Transactivators: SchematicHIV-1 transactivators Ref.: Cullen. J. Virol. 6 5:105 3, 1 991 DNA provirus +RNA trans cription(regulated by Tat)RNA nuclear exportRNA s plicing Early(regulated by Rev) Late regulatory mRNAs struc tural mRNAs (doubly splic ed) (unspliced, s ingly spliced) - +Gag-pol (uns pliced mRNA) Env (singly spliced mRNA)Tatalso: Vpr, Vpu, Vif encoded byRevsingly spliced mRNAsalso: Nef encoded bydoubly spliced mRNA 45. Action of TatTat interacts cooperatively with the TAR RNA sequence and with the cellular protein complex, positive transcription elongation factor b (P-TEFb). P-TEFb includes cyclin T1 (CYCT1) and cyclin- dependent kinase 9 (CDK9). CDK9, once recruited to nascent HIV RNA, phosphorylates the C-terminus of RNA polymeraseII (RNAPII)and negative transcription elongation factor (N-TEF), thereby enhancing elongation. 46. Action of HIV-1 TatRef .: Cullen Cell 93:685, 1998 CDK9 Tat inte racts cooperative lywith TAR & with the cyclinT1 compone nt of pos itiveCyclintranscription elon gation loopT1 factor-b (P-TEFb). Thisallows the CDK9 compon ent No TatTAR + Tatof P-TEFb to phos phorylate bulgeTAT polII .Pre mature te rmination (short transcripts) Greatly e nhanced elongation RNA pol II transcript ionCDK9 ph osphoryl ate scomplexpolII P HIV DNATAT AAHIV DNATAT AAAction of HIV-1 Tat 47. Additional Actions ofTat Translocated across the plasma membrane. A basic domain within Tat (RKKRRQRRR) allows it to enter intact cells; this is being exploited for gene therapy and immunization. Tat is thus the prototype of so- called protein transduction domains or PTDs.Causes T cell activation. May prime virus-negative T cells for HIV-1 infection and replication.Induces apoptosis. Occurs in T cells and in neurons; may contribute to HIV neuropathogenesis and immune evasion 48. Effect of RevNuclear export of late HIV-1 mRNAs. Incompletely spliced (singly spliced or unspliced) mRNAs contain the RRE and fail to exit the nucleus in the absence of Rev. These late mRNAs encode the viral structural proteins (Gag, Pol & Env). Inhibits splicing of late HIV-1 mRNAs. Rev binding to the RRE may prevent the formation of a spliceosome on the late HIV-1 mRNAs. 49. Effect of HIV-1 Rev on ViralRNAs Flint. Fig. 10.15 50. Action of Rev1. Nuclear import of Rev: The Rev NLS binds importinb (IMPb), and the complex enters the nucleus2. Rev binds to the RRE, and multimerizes3. Nuclear export of Rev and its cargo: The Rev NESis bound by CRM1, and the ATP-dependent RNAhelicase DDX3 (a nucleo-cytoplasmic shuttlingprotein) then binds CRM1 and localizes to thenuclear pore (and subsequently to the cytosol). 51. Tat & Rev: Common Themes Bind specific RNA sequences Require cellular cofactors Nuclear acting Made up of modular domains Act in concert to regulate HIVtranscription 52. Virion Proteins: Core &Enzymes Core proteins. Gag gene products (MA, CA, NC). Initial product is Pr55Gag; proteolytically cleaved by viral protease. A molecular chaperone (Cyclophilin A) interacts with CA and is required for virus replication. Viral enzymes. Pol products (IN, PR, RT); made by read-through of Gag stop codon (translational frameshift).Initial product is Pr160Gag-Pol; processed by viral protease. PR (protease) is a major antiviral target, as is RT (reverse transcriptase), which also has a high error 53. Virion Proteins:Enzymes Viral enzymes. Pol products; made by read-through of Gag stop codon (translational frameshift). Initial product is Pr160Gag-Pol; processed by viral protease. PR (protease). Cleaves Gag, Gag-Pol precursors. Involved in virion maturation. Antiviral target. RT (reverse transcriptase). An RNA-dependent DNA-polymerase; lacks proof-reading capacity (high error rate). Antiviral target. IN (integrase). Catalyzes insertion of viral DNA into the host chromosome. 54. Viral Accessory ProteinsVif (virion infectivity factor). Important for virion maturation and infectivity; counteracts an innate antiviral protein (APOBEC3G/CEM15). Vpu (viral protein, unknown). Downregulates CD4; enhances virus release. Absent in HIV-2. Vpr (viral protein, regulatory). Causes G2 cell cycle arrest; facilitates infection of macrophages; induces apoptosis. 55. Nef Initially described as a dispensible negative factor; later realized to be critical for pathogenicity. Functions: CD4 and MHC class I downregulation. Binds the cytoplasmic domains of these molecules & targets themtotheintracellular sorting/degradation pathways. Enhances virion infectivity. Increases viral replication in primary cells by increasing virion infectivity. Apoptosis. May upregulate FasL expression, leading to killing of virus-reactive cytotoxic T cells. Modulation of host cell; kinase binding. A central SH3-binding domain recruits cellular protein tyrosine 56. Attenuated SIV (Nef-delete)Attenuated SIVRef.: Kestter et al., Cell 65:651, 1991 gag vifre vnef LTRpolvpxtat LTR vprenvVirus Clone Deaths (5 mo)-----------------------------Mac2393/7M a c 2 3DN e f9 0/6-----------------------------Deletion(Mac239DNef) 57. SIVDnefViral attenuation: Deletion of Nef from SIVmac239 rendered the virus much less pathogenic for macaques, and dramatically reduced in vivo virus replication. Protection by attenuated virus: Animals infected with SIVDNef were found to be protected against infection with other strains of SIV. Attenuation is not complete: SIVDNef can cause disease in neonatal animals, and some adult animals infected with SIVDNef also go on to develop disease at later times 58. Attenuated HIV-1(Nef-delete)Attenuated HIV-1 Ref.: Deacon et al., S cience 270:988, 1995 gagvif re v nef LTR poltatLTRvpr vpuenvDeletions 59. Sydney Blood Bank CohortHIV+ blood donor: use of contaminated blood from this person resulted in the infection of 7 recipients. Non-progression: 1 patient died of unrelated causes soon after, but the other 7 (donor + 6 recipients) remained healthy for 10+ years. All turned out to have the same strain of HIV, with has a deletion in Nef. Incomplete attenuation: In 1999, at 14-18 years after infection with the virus, several of the members of this cohort showed evidence of a decline in their CD4 count. 60. Endogenous Antivirals:APOBEC3G Reuben S. Harris & Mark T.Liddament. NatureReviews Immunology 4,868-877 (2004) 61. Endogenous Antivirals: APOBEC3G (II)Gu Y & Sundquist WI. Naure 424:21-22, 2003. 62. Endogenous Antivirals: APOBECFamily Reuben S. Harris & Mark T.Liddament. NatureReviews Immunology 4,868-877 (2004) 63. EndogenousAntivirals: TRIM5 Greene, W.C. Nature Med. 10:778-80, 2004; Stremlau, M. et al.Nature 427:848-852, 2004. 64. Funkce protein HIVCD4 downregulation.Nef, Vpu, gp120 can downregulate CD4. Important for virus release? Nuclear import.There are nuclear localization signals in Vpr, MA and IN, which contribute to infection of non-dividing cells. T cell activation. Gp120, Tat and Nef activate T cells. Immune evasion. Vpu and Nef downregulate class I major histocompatibility complex (MHC) proteins. Nef can also upregulate FasL and trigger apoptosis of virus-reactive T cells. Rev can regulate epitope density on target cells. 65. FunkceRev 66. ivotn cyklus viru HIV 67. HIV targets for therapy CD4 cell X Integrase NRTI NNRTI inhibitorPI X Fusion inhibitor 68. CD4 cell count and opportunistic infection 69. HIV Testing Drug resistance testing- genotypic and phenotypic assays Antibody detection ELISA- color change (optical density) P24 antigen test -present early in disease and later ELISA sandwich technique Western blot- confirmatory need at least two bands (p24, gp41, gp160/120) Drug resistance testing- genotypic and phenotypic assays 70. HIV seroconversion + - 0 15 21 34 47d 71. Akutn retrovirln syndrom Occurs in up to 75% of patients Flu-like/mono-like illness- fevers, myalgias, exudative pharyngitis, rash (maculopapullar), thrush, May be the best time to start treatment Produkce velkho mnostv virion v aktivovanch lymfocytech v uzlinch 5x103 inf. virion na ml plasmy 72. asn choroba CD4 counts above 500 cells Infections Sinusitis Pneumonias Pneumococcal vaccine Herpes infections Papilloma viruses Other- Lymphoma 73. Pozdn stdium CD4