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Mukund Modak, Ph. D.

Dental Biochemistry 2013 Lecture 39

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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

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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 RT Integrase3. 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)

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Diploid genome (2 RNA copies/ virus particle)

RNA physically linked as a dimer by hydrogen bonds; Harbors tRNAlys 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

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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

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HIV GENE PRODUCTS AND THEIR FUNCTIONS

Structural genes

LTR : Binding site for host transcription factorsgag : Nucleocapsid, core protein (matrix), P-6pol : RT, RNase H, integrase, proteaseenv : Coat proteins (CD4 specific). Gp120 & Gp41

Non-structural genes1tat : Transcription activator ( 100 X) binds to vRNA (TAR)rev : Regulation of mRNA exportvif : Promotes infectivityvpr : Nuclear import of viral DNA; G-2 cell cycle arrest.vpu : enhances release of virus; also down regulates CD4

expressionnef : suppresses v-gene expression; also regulates CD4

and MHC class I expression

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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

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-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-1and 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

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Chemotherapeutic Intervention Sites in the HIV Life Cycle

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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.

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Reverse Transcriptase Inhibitors

Treatment strategies

Pol (replication) and env (receptors, viral membrane) gene products

• Pol gene assembly

17 kD 65 kD 36 kD

NH2

Protease pol RNase H Integrase

Protease : protease inhibitorsPol (Rt) : AZT/dideoxynucleosides/non-nucleoside inhibitors

e.g. Nevirapine

Integrase: Issentress (Merck)Entry Site (CCR-5): MaravirocFusion Inhibition: Enfuvirtide (EFV)Vaccines: ????

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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.

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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

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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

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