1 Mukund Modak, Ph. D. Dental Biochemistry 2013 Lecture 39

2 Learning Goals for AIDS (HIV):  HIV as a causative agent for AIDS ( infection target and role of CD4)  HIV anatomy ( important structural proteins)  HIV Life Cycle ( vRNA---RNA:DNA---ds proviral DNA(integration into host DNA)  HIV Genome and major gene products ( LTR, pol gene etc)  Structural and NON-structural proteins  Chemotherapy intervention sites and treatment strategies  Commonly used inhibitor-drugs ; mode of their action  Conclusions

HIV (AIDS) HTLV Family, Retrovirus class (lentivirus subclass) Target: T4 lymphocytes (CD4 receptor)* *CD4 (Ig like structure) binds MHC2 complement on the surface of antigen presenting cell T4 is a helper T-cell (helps both T and B cell functions); HIV infection destroys T4 cell, resulting in the state of immunodeficiency and ultimate demise due to bacterial or viral infections. Viral replication occurs via retroviral lifecycle Viral RNA  proviral DNA  mRNA  Protein 3

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1.HIV attaches to T4 cell gp120 (of virus) to CD4 and CCR5 (chemokine receptor) present on the cell surface allows entry of the virus. 2.Viral RNA + viral RT + nucleotides = viral DNA RT RTIntegrase 3.V RNA RNA.DNA ds DNA into host DNA +tRNA primer ss ss its RNase H Proviral DNA 4. Proviral DNA  Cell DNA  transcription +translation  viral proteins +RNA New Virus LIFE CYCLE OF AIDS VIRUS (HIV) 6

 Diploid genome (2 RNA copies/ virus particle)  RNA physically linked as a dimer by hydrogen bonds; Harbors tRNA lys for initiating reverse transcription  RNA is single-stranded, positive sense, composed of 9749 nucleotides and has 5' cap and 3' poly-(A) tail  HIV genome encodes nine open reading frames, 15 proteins 7

8 MA (matrix) CA (capsid) NC (nucleocapsid) p6 PR (protease) RT (reverse transcriptase) IN (integrase Gp160 SU (Gp120) TM (Gp41) 5’ 3’

LTR Region contains sites for transcriptions factors 9

HIV GENE PRODUCTS AND THEIR FUNCTIONS Structural genes LTR : Binding site for host transcription factors gag :Nucleocapsid, core protein (matrix), P-6 pol:RT, RNase H, integrase, protease env:Coat proteins (CD4 specific). Gp120 & Gp41 Non-structural genes1 tat:Transcription activator ( 100 X) binds to vRNA (TAR) rev:Regulation of mRNA export vif:Promotes infectivity vpr:Nuclear import of viral DNA; G-2 cell cycle arrest. vpu:enhances release of virus; also down regulates CD4 expression nef:suppresses v-gene expression; also regulates CD4 and MHC class I expression 10

 CD4 antigen: primary receptors, binds HIV gp120  Chemokine receptors: essential co-receptors, seven- transmembrane G protein-coupled receptors, tropism -CCR5: employed by macrophage-tropic HIV strains involved in critical early stages of infection, CCR5 receptor gene was mapped to human chromosome 3p21 only 18 kb away from CCR2B receptor gene -CXCR4: ligand is a B cell stimulatory factor called fusin, promotes infection /fusion of CD4+ T cells -CCR2: recently identified co-receptor 11

-Nature of co-receptor may explain why people who are exposed repeatedly to HIV remain uninfected  CCR2: heterozygous mutation in CCR2 present in all races in the U.S. at a frequency of about 20-25%, accounts for long term survivors  CCR5: binds to the chemokines- RANTES, MIP-1 , and MIP-1  and suppress HIV’s ability to infect cells; 32 base pair deletion in CCR5 prevents its expression, two copies of defective CCR5 gene confers immunity from HIV infection and a single defective gene results in delayed AIDS progression 1 in 100 Caucasians have this double mutation and 17% have a single defective gene in contrast to only 2% African Americans with single mutation 12

Chemotherapeutic Intervention Sites in the HIV Life Cycle 13

 Nucleoside Class: competitive inhibitors, bind to the enzyme's active site, DNA chain terminators. These drugs are phosphorylated to the triphosphate form by host cell enzymes before being incorporated into the growing DNA chain and inhibiting further elongation. Eg. AZT(azidothymidine), DDI(didanosine), 3TC(Lamivudine) etc.  Non-Nucleoside Class: these drugs do not need to be phosphorylated to be active, bind elsewhere than the enzyme’s active site and function in a non- competitive fashion. These drugs work synergistically with nucleoside analogs, exhibit high therapeutic index and good bio-availablity. Eg. Nevirapine, delavirdine, efavirenz etc. 14 Reverse Transcriptase Inhibitors

Treatment strategies Pol (replication) and env (receptors, viral membrane) gene products Pol gene assembly 17 kD65 kD36 kD NH 2 Protease pol RNase H Integrase Protease : protease inhibitors Pol (Rt) : AZT/dideoxynucleosides/non-nucleoside inhibitors e.g. Nevirapine Integrase: Issentress (Merck) Entry Site (CCR-5): Maraviroc Fusion Inhibition: Enfuvirtide (EFV) Vaccines: ???? 15

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 Peptide mimicking compounds that are transition state analogs. They bind the enzyme much more tightly than the natural substrate and function as competitive enzyme inhibitors. Drugs in this category include saquinavir, indinavir etc. 18

 Combination therapy, or the simultaneous use of multiple anti-HIV drugs, is the most effective means of controlling HIV-1 infection. Typically, one combines one protease inhibitor with two reverse transcriptase inhibitors 19

 No effective HIV vaccine available  Attempts to develop 3 classes of anti-HIV vaccines  Therapeutic vaccines: designed to boost the immune system of an already-infected person  Protective/Prophylactic vaccines: to prevent HIV infection in uninfected population  Perinatal vaccines: prevent mother to child transmission 20