Combination of Drugs and Drug-Resistant Reverse Transcriptase Results in a Multiplicative Increase of Human Immunodeficiency Virus Type 1 Mutant Frequencies - PowerPoint PPT Presentation

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Combination of Drugs and Drug-Resistant Reverse Transcriptase Results in a Multiplicative Increase of Human Immunodeficiency Virus Type 1 Mutant Frequencies

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3. Integration - viral DNA joins host DNA. 4. Transcription- making multiple viral RNAs ... one nucleoside analog (DNA chain terminator), one protease inhibitor ... – PowerPoint PPT presentation

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Title: Combination of Drugs and Drug-Resistant Reverse Transcriptase Results in a Multiplicative Increase of Human Immunodeficiency Virus Type 1 Mutant Frequencies


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

2
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).

3
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.

4
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.

5
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.

6
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.

7
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.

8
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.

9
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.

10
Reason of drug resistance in HIV-1
  • Mutation
  • Mutation rate for HIV-1 IS 410-5 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.

11
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.

12
Objectiveof 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
  • They studied
  • AZT,3TC RT inhibitor
  • HU and Thy alter intracellular dNTP pools,
    used in HIV treatment
  • Drug resistant virus drug resistant Rtdrug
    resistant mutation

13
Experimental Protocol
  • 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.

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15
Experimental protocol
  • B. influence of the antiretroviral drugs on
  • 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.

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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.

18
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 vectors mutant frequency was
    considered as standard frequency

19
Exp2. Combined effects of 3TC and AZT-resistant
RT
  • 3TC with AZT resistant mutation has
    multiplicative effect on virus mutant frquency.

20
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).

21
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.

22
HU treatment together with drug-resistantRT
  • 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)

23
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.

24
Thy treatment together with drug-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)

25
discussion
  • 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.

26
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.

27
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

28
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 -
  • Deterministic 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.
  • Stochastic 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 .

29
Thats
  • it
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