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ZINC FINGER PROTEINS FOR RETROVIRAL TREATMENT (AIDS-NO MORE A DREADFUL DIESEASE) KEYWORDS: 1) Zinc fing er p rotei ns (ZFP) 2) Zinc fin ger DNA binding protein nucleases (ZFN) 3) CCR5 co-re ceptor 4) AIDS (Acquired immuno deficiency syndrome) 5) HIV (Human Immuno Viru s) ABSTRACT: HIV-human  immune  virus  a  retro  virus  wh ose  infection  results  in  AIDS (Acquired immuno deficiency syndrome) the number of people affected by this epidemic disease is overwhelmingly increasing day by day. At the present scenario there is no cure for this disease the treatment is available only to treat the symptoms caused due to opportunistic infections and increases the life span of the infected subject.  Existing therapy for HIV treatment causes inactivation of HIV after its entry in to human cell but HIV developed resistance to this drugs. To overcome this resistance a new technology is being developed which involves zinc proteins. The target cell may be HIV or the human CCRF co-receptor present on CD4 cell which is a hu man T-cell. Zinc finger proteins are long chains of amino acids. The three dimensional structures of some proteins is maintained through a zinc atom as a magnet that attracts certain types of amino acids there fore called zinc finger proteins. Zinc finger protein present in HIV plays various roles in cell metabolism. It is important for cell modulation and gene expression. Hence when this protein is inhibited in HIV it gets inactivated. When the CCRF gene in human T-cells is mutated it can no longer function as a co-receptor for HIV. Hence entry of HIV into T-cells is inhibited and hence it becomes inactivated. For this purpose zinc finger DNA binding protein nucleases (ZFN’s) are used. Hence we can hope for the development of HIV resistant individuals by using: ZINC FINGER PROTEIN INHIBITORS ZINC FINGER DNA BINDING PROTEIN NUCLEASES INTRODUCTION: HIV is a member of retrovirus family. A retrovirus is a  virus with an RNA genome that replicates by using a viral reverse transcripta se enzyme to  transcribe its RNA into DNA in the host cell. The DNA is then incorporated into the host's genome by an integrase enzyme which is then called as provirus that can pass in to progeny. It can lead to AIDS, the condition in humans in which the immune system fails leading to life threatening opportunistic infections. Because reverse transcription lacks the usual proofreading of DNA replication, a retrovirus  mutates very often. This enables the virus to grow resistant to antiviral pharmaceuticals quickly, and impedes

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the development of effective vaccines and  inhibitors for the retrovirus. Retroviruses areenveloped viruses that belong to the viral family Retroviridae.

STRUCTURE OF HIV:

HIV is different in structure from other retroviruses. It is roughly spherical. It is

composed of two copies of positive single-stranded RNA that codes for the virus's nine genes enclosed by a conical capsid composed of viral protein the single-stranded RNA is tightly boundto nucleocapsid proteins. Enzymes needed for the development of the virion such as reverse transcriptase, proteases, ribonuclease and integrase. A matrix composed of the viral protein p17surrounds the capsid ensuring the integrity of the virion particle. This is, in turn, surrounded bythe viral envelope which is composed of two layers of fatty molecules called phospholipids takenfrom the membrane of a human cell when a newly formed virus particle buds from the cell.Embedded in the viral envelope are proteins from the host cell and about 70 copies of a complexHIV protein that protrudes through the surface of the virus particle. This protein, known as Env,consists of a cap made of three molecules called glycoprotein (gp) 120 and a stem consisting of three gp41 molecules that anchor the structure into the viral envelope. This glycoprotein complex

enables the virus to attach to and fuse with target cells to initiate the infectious cycle. Allretroviral nucleocapsid (NC) proteins contain one or two copies of an invariant 3Cys-1His array(CCHC = C-X2-C-X4-H-X4-C; C = Cys, H = His, X = variable amino acid) that are essential for RNA genome packaging and infectivity and have been proposed to function as zinc-bindingdomains which are called as ZINC FINGER DNA-BINDING PROTEIN TRANSCRIPTIONFACTORS (ZFP TFs) , where they bind to DNA to turn genes on or off.

The RNA genome consists of nine genes (gag , pol , and env, tat , rev, nef , vif , vpr , vpu, and tev)encoding 19 proteins. Three of these genes, gag , pol , and env, contain information needed tomake the structural proteins for new virus particles. The six remaining genes, tat , rev, nef , vif ,vpr , and vpu are regulatory genes for proteins that control the ability of HIV to infect cells,

produce new copies of virus (replicate), or cause disease.HIV can infect a variety of immune cells such as CD4 + T cells ,  macrophages, and microglial cells. HIV entry to macrophages and CD4+ T cells is mediated through interaction of the virionenvelope glycoproteins (gp120) with the CD4 molecule on the target cells and also withchemokine co receptors.

CCR5 is the chemokine receptor that HIV uses as a co receptor to gain entry into immune cells.CCR5 is perhaps the most important of the known co receptors for HIV, since the mostcommonly transmitted strains of HIV are strains that bind to CCR5 -- so-called "R5" strains.

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LIFE CYCLE OF HIV

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CURRENT TREATMENT OF HIV:

Using drugs for HIV or AIDS is not a cure, but it can stop people from becoming ill for manyyears. The treatment consists of drugs that have to be taken every day for the rest of a person’slife. The aim of antiretroviral treatment is to keep the amount of HIV in the body at a low level.This stops any weakening of the immune system and allows it to recover from any damage that

HIV might have caused already.

Antiretroviral drug class Abbreviations Drugs used     How they attack HIV

Nucleoside/Nucleotide ReverseTranscriptase Inhibitors

NRTIs,nucleosideanalogues,nukes

Zidovudine,Didanosine

NRTIs interfere with the action of an HIVprotein called reverse transcriptase, whichthe virus needs to make new copies of itself.

Non-Nucleoside ReverseTranscriptase Inhibitors

NNRTIs,non-nucleosides,non-nukes

Lamuvidine,stavudine

NNRTIs also stop HIV from replicatingwithin cells by inhibiting the reversetranscriptase protein.

Protease Inhibitors PIsSanquinavir,ritonavir 

PIs inhibit protease, which is another protein involved in the HIV replicationprocess.

Fusion or Entry Inhibitors Amantadine,rimantadine Fusion or entry inhibitors prevent HIV frombinding to or entering human immune cells

Disadvantages of drug treatment:

Resistance developed to drugs

Many side effects like Neurotoxicity, Neutropenia, Thrombocytopenia Myelosuppression, Thyroid dysfunction Large doses of drugs are to be taken Liver toxicity 

 

ZINC FINGER DNA- BINDING PROTEIN NUCLEASES: 

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The human immune system cells (CD4 T-cell) can be made resistant to HIV infection bytreatment with Zinc finger DNA binding protein nucleases (ZFN). The data suggest that the ZFNapproach, which results in permanent modification of the CCR5 gene encoding an importantreceptor for HIV infection, is a promising strategy for the treatment of HIV/AIDS.

A ZFN approach represents the next generation of HIV entry blocking agents and a potentially

promising class of anti HIV compounds. Permanent knock out of the CCR5 gene is importantand clinically relevant for long-term resistance for HIV infection.

ZFN’s are designed to permanently modify the DNA sequence encoding CCR5, a co receptor that enables HIV to enter and infect cells of immune system. Individuals carrying a naturallyoccurring mutation of their CCR5 gene, a variant known as CCR5-delta 32, have been shown tobe resistant to HIV infection.

Several major pharma companies have initiated programs to develop small molecules or monoclonal antibody approaches to block the binding of HIV to CCR5. however a smallmolecules  or  antibody  approach  requires  the  constant  presence  of  a  suffi

concentration of these drugs or antibody to block therapeutically relevant numbers of a CCR5protein, which is present in 1000’s of copies on the surface of each T-cell and other tissues in thebody

Zinc  Finger Nuclease  technology represents  a means  of potentially  circumventing  limitations or risks by specifically modifying only CD4 T-cells, the principle target of HIVpathology, in a one time exposure of the cells to ZFN’s. This results in permanent modificationof CCR5 protein such that HIV cannot enter and infect the cells

ZFN’s could potentially provide HIV infected individuals with a reservoir of healthy anduninfected T-cells that would be available to fight both opportunistic infections and HIV itself.

ZFN’s are designed to target the CCR5 gene and create a double strand break at pre determinedsequences. Natural DNA repair pathways, including non homologous end joining, subsequentlyrepair the double strand break. This repair is error prone and thus results in permanent disruptionof target gene.

This data support the investigation of ZFN’s as a possible methods for the therapeuticmodification of isolated patient cells to generate HIV resistant cell populations.

ZINC FINGERS CAN TURN GENES ON OR OFF:

The human body is composed of specialized cells that perform different functions, organized intotissues and organs. All cells contain the same set of genes. However, only fraction of these genesis turn on or expressed in an individual human cell at any given time. Genes are activated or repressed in response to a wide variety of stimuli and developmental signals. Distinct set of genes are expressed in different cell types and organs. Importantly, the aberrant expressions of certain genes lead to disease.

Zinc finger DNA binding protein transcription factors occur naturally inside the nucleus of allorganisms, where they bind to DNA to turn genes on or off. They are composed of a short chain

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of amino acids that are folded around and coordinated by a zinc atom. Each finger binds to aspecific short run DNA bases. By using different combinations of amino acids, Zinc finger proteins can be designed to recognize and latch onto and a specific place on the DNA. When agene is turned on it makes proteins. When turned off, it stops expressing those proteins thatwould otherwise function in the cell. If successful Zinc finger protein technology could preventand treat debilitating fatal viral diseases including AIDS.

 ZINC FINGER PROTEIN INHIBITORS:

The three dimensional structure of some proteins sections is maintained through a Zinc atomacting as a magnet that attracts certain types of amino acids therefore called Zinc finger proteins.

All the cells contain zinc finger proteins. Hence to be useful a retroviral zinc finger inhibitor should be selective. The retroviral zinc finger proteins are structurally different from the cellular zinc finger proteins.

Agents that target the two highly conserved Zinc fingers of the Human Immuno Deficiency

Virus nucleocapsid protein- Zinc finger inhibitors. These agents covalently modify Zinccoordinating cysteine thiolates of the fingers, causing zinc ejection, loss of native proteinstructure and nucleic acid binding capacity and disruption of the virus replication.

The current antiviral drugs become ineffective against HIV largely due to the emergence of drugresistant viral mutants and intolerance to side effects. HIV-1, HIV-2 and retroviruses containproteins known as CCHC Zinc Fingers in their nucleocapsids. These Zinc fingers are highlyconserved through out nearly all retroviruses including viruses responsible for leukemia andparesthesia in human being.

The zinc fingers contain sequences of 14 amino acids with four invariant residues, Cys-Cys-His-

Cys, essential in viral infectivity. Mutations in the CCHC zinc fingers render retroviruses non-infections.

Azodicarbonamide (ADA) is the only known Zinc Finger inhibitor currently involved in clinicaltrials.

ADVANTAGES OF ADA: 

• New mode of action• Absence of viral resistance• Viral load reduction•

CD4-T lymphocytes increase• Minor side effects

CONCLUSION:

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Anti Retro viral drugs are useful in prolonging life and postponing complications of AIDS, but

do not cure the infection. The complications of HIV are complex, prolonged, needs expertise,strong motivation and commitment of the patient resources and are very expensive. The

combination therapy of drugs called Highly active anti retroviral therapy (HAART) has beenemployed.

However, no specific combination can be considered optimal initial regimen for all patients.Durability of these regimens depends mainly on adherence of the patient to it. Therapy should

not be discontinued and hence life long treatment should be taken, which is expensive and may

not be compliable to patient.

But the present therapy of ZINC FINGER TECHNOLOGY can overcome all the above saidproblems of chemotherapy of AIDS. The ZPN’s provide a promising strategy to develop a HIV

resistant individual and ZINC PROTEIN INHIBITOR TECHNOLOGY inactivates the HIV such

that it cannot enter the human T-cells. These strategies provide a lot of scope for the cure of 

AIDS and to produce HIV resistant subjects. These technologies are useful in treating various

disorders like Cancer, Congestive heart failure, Coronary Heart Diseases, Auto Immunedisorders like AIDS. Thus within no time AIDS can become

REFERENCES:

Å. Pavletich et al. (1991), 

Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1

Science, 252, 809-817

Sangamo BioSciences, Inc.

 http://www.sangamo.com/ 

Shi, Yigong et al

DNA unwinding induced by Zinc finger protein binding 

Biochemistry, 1996, 35, 3845-3848http://biophysics.med.jhu.edu/berglab/pdf_files/yg96.pdf 

Web References

http://us.expasy.org/cgi-bin/get-entries?KW=Zinc-finger 

http://life.nthu.edu.tw/~lablpc/old/ccc/zinc.html

http://mcdb.colorado.edu/courses/3280/chime/tf3/right-b.htm

http://www.md.huji.ac.il/courses/biomol/Course_2002/exercise10.3.html

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Document BySANTOSH BHARADWAJ REDDY Email: [email protected]

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