Transcript wk10-ManjHIV

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
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).
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.
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 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
They studied –
AZT,3TC – RT inhibitor
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
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 ( 9 +3.4 =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 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 drugresistant 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
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
1
2
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 .
That’s
it