Combination of Drugs and Drug-Resistant Reverse Transcriptase Results in a Multiplicative Increase of Human Immunodeficiency Virus Type 1 Mutant Frequencies Louis M. Mansky, Dennis K. Pearl, and Lisa C. Gajary Presented by Manjari Dani Presented by Manjari Dani

Background information HIV-1 is a retrovirus that has RNA as its nucleic acid or genome. It reproduces inside the host cell by reverse transcription. Reverse transcription is the process of copying information from RNA into DNA (complementary DNA). RNADNARNADNA

OVERVIEW OF HIV-1 infection 1. Attachment – Getting into the host cell 2. Reverse transcription – converting Viral RNA into DNA. Reverse transcriptase is an enzyme which converts viral RNA into complementary DNA. Without reverse transcriptase HIV cannot reproduce.

OVERVIEW OF HIV-1 infection 3. Integration - viral DNA joins host DNA 4. Transcription- making multiple viral RNAs 5.Translation – producing viral proteins. 6. Viral Protease- cleaving viral proteins. When viral RNA is translated into protein, that protein is assembled in a long chain that includes several individual proteins (reverse transcriptase, protease, integrase). these proteins are functional only when they cut from the longer polypeptide chain. When viral RNA is translated into protein, that protein is assembled in a long chain that includes several individual proteins (reverse transcriptase, protease, integrase). these proteins are functional only when they cut from the longer polypeptide chain.

OVERVIEW OF HIV-1 infection Viral protease is an enzyme which cuts the long chain into its individual proteins 7. Assembly & budding- Getting out of the host cell.

HIV-1 treatment Treatment of HIV-1 targets its replication by using reverse transcriptase (RT) and protease inhibitors. RT inhibitor- a molecule that prevents RTs function. AZT and 3TC are RT inhibitors used as drugs against HIV-1.

HIV-1 treatment Protease inhibitor – a drug that blocks protease function to cleave the viral polypeptide into functional enzymes, thus interferes with HIV-1 infection.

Highly Active Antiretroviral Therapy HAART is the therapy, composed of multiple anti-HIV drugs, that is prescribed to many HIV-positive people, even before they develop symptoms of AIDS. The therapy usually includes one nucleoside analog (DNA chain terminator), one protease inhibitor and either a second nucleoside analog or a non-nucleoside reverse transcription inhibitor nucleoside analogue A synthetic molecule that resembles a natural nucleoside, but it can not link to an adjacent nucleotide.

Here comes the problem The problem with these therapies is that they lead to the development of drug resistance in HIV viruses. Like AZT and 3TC drugs prevents hiv infection by inhibiting RT but use of these drugs develop resistance in viruses thus they become able to carry reverse transcription even in the presence of inhibitors.

Reason of drug resistance in HIV-1 Mutation Mutation rate for HIV-1 IS Mutation rate for HIV-1 IS 4*10^-5 mutations per base pair per replication cycle i.e. about 1 mutation for 3 new genomes. Drug treatment increases the selection and accumulation of drug resistance mutations. Drug resistant mutations make viruses less susceptible to drug and able to replicate in the presence of drug which result in greater level of resistance.This result in failure of drug therapy.

Several studies Shown that drug as well as drug resistant RT effect mutation rate of HIV-1. AZT increased the HIV-1 mutation rate 7.6-fold in a single round of replication. 3TC increased the virus mutation rate 3.4-fold. AZT-resistant RTs increased the mutation rate as much as 4.3-fold, while 3TC-resistant RT had no significant effect on the mutation rate.

Objective of this paper To study the combined effect of drug and drug resistant virus on HIv-1 mutant frequency. means the effect of replication of drug resistant HIV in presence of drug means the effect of replication of drug resistant HIV in presence of drug They studied – AZT,3TC – RT inhibitor AZT,3TC – RT inhibitor HU and Thy –alter intracellular dNTP pools, used in HIV treatment HU and Thy –alter intracellular dNTP pools, used in HIV treatment Drug resistant virus= drug resistant Rt=drug resistant mutation

Experimental Protocol A.One cycle of HIV-1 replication was constituted 1.Construction of HIV vector 2.Transfection of vector into step 2cells i.e. virus producing cells. 3.Infection of provirus produced by step2 cells into step 3 cellsi.e.target cells. 4.Cocultivation of step 2 and step3 cells also result in infection of target cells note - G418 resistance is the indicator of target cells infected with the HIV-1 and also gives the relative amount of infectious virus produced from the step 2 cells.

Experimental protocol B. influence of the antiretroviral drugs on HIV-1 mutant frequencies was determined HIV-1 mutant frequencies was determined The target cells were treated with drug for 2h before infection and 24 hr after infection Infected target cells were pooled and total DNA was purified,digested with restriction enzyme. The vector was purified with lac repressor protein, ligated and introduced into E.coli. Ratio of light blue and white bacterial colonies to total was used to determine mutant frquencies.

Exp.1 Combined effects of AZT and AZT-resistant RT M41L/T215Y and M41L/D67N/K70R/T215Y –are AZT resistant mutations. AZT and AZT-resistant RT individually led to increase mutant frequencies. The combined effect of AZT resistant mutation and AZT could be multiplicative, additive, synergistic and antagonistic.

Combined effect is multiplicative Mutant frequency = no.of mutant colonies / total colonies Relative mutant frequency = mutant frequency / standard mutant frequency Wt or normal HIV-1 vector’s mutant frequency was considered as standard frequency

Exp2. Combined effects of 3TC and AZT-resistant RT 3TC with AZT resistant mutation has multiplicative effect on virus mutant frquency.

Exp3. Effect of AZT and 3TC treatment together with AZT resistant or AZT/3TC dually resistant RT AZT and 3TC along with drug resistant mutation consistent with a multiplicative model (9 *3.4 =30.6) but not with an additive model ( =12.4).

4.Effect of HU treatment of infected target cells on virus mutant frequency Infected target cells were grown in the presence of HU ranging from 0 to 3.0 mM HU treatment increased the mutant frequency in a dose-dependent manner 2 The relative amount of infectious virus produced decreased with the increase in HU conc.

HU treatment together with drug-resistant RT AZT resistant mutation AZT resistant mutation with 2.0 mM HU resulted in a 21.8-fold increase The effect either a multiplicative (35) or additive (11.7)

5.Effect of Thy treatment of cells Infected target cells were grown in the presence of Thy, ranging from 0 to 75 µM Thy treatment increased the mutant frequency of HIV-1 in a dose-dependent manner.

Thy treatment together with drug- resistant RT AZT resistant RT AZT resistant RT with o.4 mM Thy resulted in a 16.7-fold increase Consistent either with multiplicative (29) or additive (10.8)

discussion for increased virus mutant frequency AZT and 3TC both are nucleoside analog so may have similar mechanism for increased virus mutant frequency Potential mechanisms for AZT : (i) AZT alters nucleotide pools, (ii) AZT is incorporated into plus-strand DNA and may result in discontinuous DNA synthesis that integrate with subsequent error-prone repair by the host cell, and (iii) AZT may bind non catalytically to RT and cause a conformational change that influences enzyme Fidelity.

Area of further studies it has been found that AZT mechanisms doent involve alteration of nucleotide pools. Predicted is that that AZT resistant RT has higher fidelity but AZT resistant RTs were observed to have lower fidelity.

discussion Mechanisms for increased mutant frequency by HU and Thy – Both HU and Thy alters intracellular dNTP pools.So the increase in mutation rate is may be due to increase in error rate in RT

discussion An altered mutation rate is dependent on the population dynamics of HIV-1. two mathematical models has been proposed to predict the effects of mutation on population - 1Deterministic model-in which the parameters and variables are not subject to random changes, so that the system at any time is entirely defined by the initial conditions. 2Stochastic model - which considers the presence of some randomness in parameters or variables. Thus the model do not give a single point estmate but a probability distribution of possible estimates. possible

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