Title: Combination of Drugs and Drug-Resistant Reverse Transcriptase Results in a Multiplicative Increase of Human Immunodeficiency Virus Type 1 Mutant Frequencies
1Combination 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
2Background 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).
3OVERVIEW 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.
4OVERVIEW 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.
5OVERVIEW 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.
6HIV-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.
7HIV-1 treatment
- Protease inhibitor a drug that blocks protease
function to cleave the viral polypeptide into
functional enzymes, thus interferes with HIV-1
infection.
8Highly 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.
9Here 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.
10Reason 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.
11Several 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.
12Objectiveof 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
13Experimental 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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15Experimental 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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17Exp.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.
18Combined 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
19Exp2. Combined effects of 3TC and AZT-resistant
RT
- 3TC with AZT resistant mutation has
multiplicative effect on virus mutant frquency.
20Exp3. 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).
214.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.
22HU 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)
235.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.
24Thy 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)
25discussion
- 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.
26Area 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.
27discussion
- 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
28discussion
- 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 .
29Thats