Combination of Drugs and Drug-Resistant Combination of Drugs and Drug-Resistant Reverse TranscriptaseReverse Transcriptase

Results in a Multiplicative Increase of Human Results in a Multiplicative Increase of Human ImmunodeficiencyImmunodeficiency

Virus Type 1 Mutant FrequenciesVirus Type 1 Mutant Frequencies

Louis M. Mansky, Dennis K. Pearl, and Louis M. Mansky, Dennis K. Pearl, and Lisa C. GajaryLisa C. Gajary

Presented by Manjari DaniPresented by Manjari Dani

Background informationBackground information

HIV-1 is a retrovirus that has RNA as its HIV-1 is a retrovirus that has RNA as its nucleic acid or genome.nucleic acid or genome.

It reproduces inside the host cell by It reproduces inside the host cell by reverse transcription.reverse transcription.

Reverse transcription is the process of Reverse transcription is the process of copying information from copying information from RNARNA into into DNADNA (complementary DNA).(complementary DNA).

OVERVIEW OF HIV-1 infectionOVERVIEW OF HIV-1 infection

1. 1. AttachmenAttachment – Getting into the host cellt – Getting into the host cell

2. 2. Reverse transcriptionReverse transcription – converting Viral RNA into DNA. – converting Viral RNA into DNA. Reverse transcriptase is an enzyme which converts viral Reverse transcriptase is an enzyme which converts viral RNA into complementary DNA. Without reverse RNA into complementary DNA. Without reverse transcriptase HIV cannot reproduce.transcriptase HIV cannot reproduce.

OVERVIEW OF HIV-1 infectionOVERVIEW OF HIV-1 infection

3. 3. IntegrationIntegration - viral DNA joins host DNA - viral DNA joins host DNA

4. 4. TranscriptionTranscription- making multiple viral RNAs- making multiple viral RNAs

5.5.Translation Translation – producing viral proteins.– producing viral proteins.

6. 6. Viral Protease-Viral Protease- cleaving viral proteins. cleaving viral proteins.

When viral RNA is translated into protein, that protein When viral RNA is translated into protein, that protein is assembled in a long chain that includes several is assembled in a long chain that includes several individual proteins (reverse transcriptase, protease, individual proteins (reverse transcriptase, protease, integrase). these proteins are functional only when integrase). these proteins are functional only when they cut from the longer polypeptide chainthey cut from the longer polypeptide chain . .

OVERVIEW OF HIV-1 infectionOVERVIEW OF HIV-1 infection

Viral protease is an Viral protease is an enzyme which cuts the enzyme which cuts the long chain into its long chain into its individual proteins individual proteins

7. 7. Assembly & budding-Assembly & budding-

Getting out of the host Getting out of the host cell.cell.

HIV-1 treatmentHIV-1 treatment

Treatment of HIV-1 targets its replication by Treatment of HIV-1 targets its replication by using reverse transcriptase (RT) and protease using reverse transcriptase (RT) and protease inhibitors.inhibitors.

RT inhibitor- a molecule that prevents RTs RT inhibitor- a molecule that prevents RTs function.function.

AZT and 3TC are RT inhibitors used as drugs AZT and 3TC are RT inhibitors used as drugs against HIV-1.against HIV-1.

HIV-1 treatmentHIV-1 treatment

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

Highly Active Antiretroviral TherapyHighly Active Antiretroviral Therapy

HAARTHAART is the therapy, composed of multiple anti-HIV is the therapy, composed of multiple anti-HIV drugs, that is prescribed to many HIV-positive people, drugs, that is prescribed to many HIV-positive people, even before they develop symptoms of AIDS. The even before they develop symptoms of AIDS. The therapy usually includes one nucleoside analog (DNA therapy usually includes one nucleoside analog (DNA chain terminator), one protease inhibitor and either a chain terminator), one protease inhibitor and either a second nucleoside analog or a non-nucleoside reverse second nucleoside analog or a non-nucleoside reverse transcription inhibitortranscription inhibitor

nucleoside analoguenucleoside analogueA synthetic molecule that resembles a natural nucleoside, A synthetic molecule that resembles a natural nucleoside,

but it can not link to an adjacent nucleotide.but it can not link to an adjacent nucleotide.

Here comes the problemHere comes the problem

The problem with these therapies is that they The problem with these therapies is that they lead to the development of drug resistance in lead to the development of drug resistance in HIV viruses.HIV viruses.

Like AZT and 3TC drugs prevents hiv infection Like AZT and 3TC drugs prevents hiv infection by inhibiting RT but use of these drugs develop by inhibiting RT but use of these drugs develop resistance in viruses thus they become able to resistance in viruses thus they become able to carry reverse transcription even in the presence carry reverse transcription even in the presence of inhibitors.of inhibitors.

Reason of drug resistance in HIV-1Reason of drug resistance in HIV-1

MutationMutationMutation 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 Drug treatment increases the selection and accumulation of drug resistance mutations.accumulation of drug resistance mutations.

Drug resistant mutations make viruses less Drug resistant mutations make viruses less susceptible to drug and able to replicate in the susceptible to drug and able to replicate in the presence of drug which result in greater level of presence of drug which result in greater level of resistance .This result in failure of drug therapy.resistance .This result in failure of drug therapy.

Several studiesSeveral studies

Shown that drug as well as drug resistant RT effect Shown that drug as well as drug resistant RT effect mutation rate of HIV-1.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.

ObjectiveObjectiveof this paper of this paper

To study the combined effect of drug and drug resistant virus on HIv-1 To study the combined effect of drug and drug resistant virus on HIv-1 mutant frequency.mutant frequency.

means the effect of replication of drug resistant HIV in presence of drugmeans the effect of replication of drug resistant HIV in presence of drug

They studied –They studied – AZT,3TC – RT inhibitorAZT,3TC – RT inhibitor HU and Thy –alter intracellular dNTP pools, used in HIV HU and Thy –alter intracellular dNTP pools, used in HIV

treatment treatment

Drug resistant virus= drug resistant Rt=drug resistant mutationDrug resistant virus= drug resistant Rt=drug resistant mutation

Experimental ProtocolExperimental Protocol

A.A. One cycle of HIV-1 replication was constitutedOne cycle of HIV-1 replication was constituted1.Construction of HIV vector1.Construction of HIV vector

2.Transfection of vector into step 2cells i.e. virus producing cells.2.Transfection of vector into step 2cells i.e. virus producing cells.

3.Infection of provirus produced by step2 cells into step 3 3.Infection of provirus produced by step2 cells into step 3 cellsi.e.target cells.cellsi.e.target cells.

4.Cocultivation of step 2 and step3 cells also result in infection of 4.Cocultivation of step 2 and step3 cells also result in infection of target cellstarget cells

note - note - G418 resistance is the indicator of target cells infected with the HIV-1 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 and also gives the relative amount of infectious virus produced from the step 2 cells.step 2 cells.

Experimental protocolExperimental protocol

B. B. influence of the antiretroviral drugs oninfluence of the antiretroviral drugs on

HIV-1 mutant frequenciesHIV-1 mutant frequencies was determinedwas determined The target cells were treated with drug for 2h before infection and The target cells were treated with drug for 2h before infection and 24 hr after infection24 hr after infection

Infected target cells were pooled and total DNA was Infected target cells were pooled and total DNA was purified ,digested with restriction enzyme.purified ,digested with restriction enzyme.

The vector was purified with lac repressor protein, ligated and The vector was purified with lac repressor protein, ligated and introduced into E.coli.introduced into E.coli.

Ratio of light blue and white bacterial colonies to total was used to Ratio of light blue and white bacterial colonies to total was used to

determine mutant frquenciesdetermine mutant frquencies..

Exp.1 Exp.1 Combined effects of AZT and Combined effects of AZT and AZT-resistant RTAZT-resistant RT

M41L/T215Y and M41L/D67N/K70R/T215Y –are AZT resistant M41L/T215Y and M41L/D67N/K70R/T215Y –are AZT resistant mutations.mutations.

AZT and AZT-resistant RT individually led to increase mutant AZT and AZT-resistant RT individually led to increase mutant frequencies.frequencies.

The combined effect of AZT resistant mutation and AZT could be The combined effect of AZT resistant mutation and AZT could be multiplicative , additive , synergistic and antagonistic.multiplicative , additive , synergistic and antagonistic.

Combined effect is multiplicativeCombined effect is multiplicative

Mutant frequency = no.of mutant colonies / total coloniesMutant frequency = no.of mutant colonies / total colonies

Relative mutant frequency = mutant frequency / standard mutant Relative mutant frequency = mutant frequency / standard mutant frequencyfrequency

Wt or normal HIV-1 vector’s mutant frequency was considered as Wt or normal HIV-1 vector’s mutant frequency was considered as standard frequency standard frequency

Exp2. Combined effects of Exp2. Combined effects of 3TC 3TC and AZT-resistant RTand AZT-resistant RT

3TC with AZT resistant mutation has multiplicative 3TC with AZT resistant mutation has multiplicative effect on virus mutant frquency.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 ( 9 +3.4 =12.4).

4.Effect of HU treatment of infected target 4.Effect of HU treatment of infected target cells on virus mutant frequencycells 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-resistantRT

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 The effect either a multiplicative (35) or additive (11.7)(11.7)

5.Effect of Thy treatment of cells5.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)

discussiondiscussion

AZT and 3TC both are nucleoside analog so may have similar mechanism for increased virus mutant frequency for increased virus mutant frequency

Potential mechanisms for AZT :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.

discussiondiscussion

Mechanisms for increased mutant frequency by Mechanisms for increased mutant frequency by HU and Thy –HU and Thy –

Both HU and Thy alters intracellular dNTP Both HU and Thy alters intracellular dNTP pools.So the increase in mutation rate is may be pools.So the increase in mutation rate is may be due to increase in error rate in RTdue to increase in error rate in RT

discussiondiscussion

An altered mutation rate is dependent on the population An altered mutation rate is dependent on the population dynamics of HIV-1.dynamics of HIV-1.two mathematical models has been proposed to predict two mathematical models has been proposed to predict the effects of mutation on population -the effects of mutation on population -

11 Deterministic model-inDeterministic model-in which the parameters and which the parameters and variables are not subject to random changes, so that variables are not subject to random changes, so that the system at any time is entirely defined by the initial the system at any time is entirely defined by the initial conditions.conditions.

22 Stochastic modelStochastic model - which considers the presence of - which considers the presence of some randomness in parameters or variables. Thus some randomness in parameters or variables. Thus the model do not give a single point estmate but a the model do not give a single point estmate but a probability distribution of probability distribution of possiblepossible estimates . estimates .

That’sThat’s

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