This document summarizes the objectives and methods of a study investigating HIV reverse transcriptase (RT) inhibition. The objectives are to: 1) Extract and purify HIV RT protein, 2) Isolate and purify plant crude extracts, 3) Test RT functionality using in vitro transcription and RT-PCR, 4) Screen plant extracts and fractions for functional RT inhibitors using high-throughput assays, and 5) Test effective inhibitors for cytotoxicity and ability to inhibit HIV in cell culture. Purification of HIV RT and plant extracts, as well as in vitro and cell-based assays, are described to achieve these objectives.

1. HIV RT INHIBITION (in-vitro ,/
in cell culture)
Materials and methods
TITAS MALLICK
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 1

2. Recap of previous presentation
• Presentation 1
Basic HIV biology, AIDS, the real life scenario, global statistics and
importance of new drug development and an overview of HIV biology;
i.e. the infection and replication of the virus.
• AIDS A DEADLY DISEASE
AIDS (acquired immunodeficiency syndrome) is a syndrome
caused by a virus called HIV (human immunodeficiency virus). The
disease alters the immune system, making people much more
vulnerable to infections and diseases. This susceptibility worsens if the
syndrome progresses.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 2

3. Cont.
2017 GLOBAL HIV STATISTICS
• 36.9 million [31.1 million–43.9 million] people globally were living with HIV in
2017.
• 21.7 million [19.1 million–22.6 million] million people were accessing
antiretroviral therapy in 2017.
• 1.8 million [1.4 million–2.4 million] people became newly infected with HIV in
2017.
• 940 000 [670 000–1.3 million] people died from AIDS-related illnesses in 2017.
• 77.3 million [59.9 million–100 million] people have become infected with HIV
since the start of the epidemic.
• 35.4 million [25.0 million–49.9 million] people have died from AIDS-related
illnesses since the start of the epidemic.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 3

4. Cont.
• Presentation 2
History of HIV drug development, its drawback and importance of new
drug development.
LUC MONTAGNIER, FRANCOISE BARRE-SINOUSSI’s effort to identify the
causal organism of AIDS, previously called as GRID
And finally ROBERT GALLO’s isolation of the virus.
Early treatment by Suramin and AZT(azidothymidine) as HIV inhibitor.
Their drawback; cytotoxicity and other and introduction of modern era
of treatment.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 4

5. Cont.
• HAART, and PREP
1. 2 nucleoside reverse transcriptase inhibitors (NRTI)
2. 1 non-nucleoside reverse transcriptase inhibitor (NNRTI)
3. 2 protease inhibitor (PI)
4. 1 Integrase nuclear strand transfer inhibitors (INSTI)
• 21st century approaches like CRISPER, BONE MARROW TRANSPLANT
and VACCINATION.
Their problems, cost-effectiveness, and the main need for drug
development.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 5

6. The basic need for new drug research
• The HIV replication is a very error prone process by nature, the DNA
dependent RNA polymerase incorporates wrong bases during
replication, most of them though causes lethal mutations so the
resultant virus doesn’t mature, but some of them end up altering the
RT protein structure (or other protein) that doesn’t make the enzyme
non functional but alters the site of drug binding. So the drug doesn’t
work for long, that makes it necessary to create new drugs that can
effectively attack the new and mutated proteins of the virus.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 6

7. | Plant sources may be potent source for anti-retroviral drugs |
1. Low cytotoxicity is expected.
2. Expected to be much efficient.
3. Low cost expected.
4. Can be major component of HAART.
5. Already reported plants crude extract having anti-retroviral effect.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 7

8. Objectives of the project.
O B J E C T I V E 1
ExtractionandpurificationofHIVRT
TheHIVRTgeneisclonedinaBL21(DE3)VectorinE.colistrainPET28A fusedwith
HexaHistidinetagunderaIPTGinduciblepromoter. Secondaryculture,induction,
and nickel affinity chromatography can be used to successfully isolate and purify
HIVRT.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 8

9. • PET 28 RT 66 cultured for the RT66 isolation.
• Primary culture overnight.
• Secondary culture from the primary culture done.
• Induced by IPTG.
• The cells are harvested as pellet after centrifugation at 6K for 6’’ from
the overnight grown secondary culture.
• The pellet are washed with a pellet wash buffer. (COMP: 0.2M NaCl
and 10mM Tris)
• Sonicated with lysis buffer (Comp: Binding Buffer + 0.05% NP40 +
Lysozyme + PMSF)
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 9

10. Cont.
• Initially the IMAC column recharged with NiSo4.
• Ni-IMAC done to extract the His-tagged RT66
• First the binding done with the binding buffer (comp: 50 mM Tris PH 8.0,
500mM NaCl, 5mM Imidazole)
• Washing buffer (comp:20mM Tris PH 7.5, 500 mM NaCl, 75 mM imidazole)
used to wash off other proteins.
• Bradford test used to check the protein conc in washes.
• Elution buffer (comp:20mM Tris PH 7.5, 500 mM NaCl, 500mM imidazole)
used to elute and collect the protein.
• The protein eluted in the previous stage put in 2 step dialysis (comp Dialysis
Buffer 1- 50mM Tris, 100 mM NaCl, 30% glycerol, comp Dialysis Buffer 2-
50mM Tris, 100 mM NaCl, Glycerol 50%)
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 10

11. cont.
• As same process the PBR28ART51 cultured and protein purification
done for RT51
• I quantified the RT51 by Bradford standard curve.
• The RT51 I quantified have a conc of 3.70 ug/ml
• THE RT51 and RT66 will be mixed equimolarly to get functional RT.
• RT 66 shown functionality in in-vitro transcription itself.
• To check the proteins in SDS-PAGE Negative stating of the gel is
performed for quick visualization
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 11

12. Checking RT51 by negative stating of SDS
PAGE
BSA BSA--Protein--
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 12

13. Procedure of negative staining of SDS PAGE
• After the gel electrophoresis the gel is placed on a clear tray in miliQ
water.
• The gel is hydrated for 2 min.
• Decant the water. 0.2 M Imidazole with 0.1% SDS in miliQ added, and
the gel is placed on a rocker for 15 min.
• 0.2 M ZnSO4 addedon the gel.
• Within 2 min banding pattern appears.
• Before staining with Coomassie the gel is washed multiple times with
miliQ till it turn transparent.
• Normal staining and distaining is performed.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 13

14. Protein Quantification
Std Conc Well ReplicatesMean SD %CV
1 0.5F12 0.173 0.242 0.111 45.731
G12 0.183
H12 0.37
2 1F11 0.367 0.363 0.068 18.824
G11 0.293
H11 0.43
3 2F10 0.403 0.586 0.107 21.154
G10 0.497
H10 0.617
4 4F9 0.803 0.75 0.174 24.725
G9 0.807
H9 0.503
5 8F8 1.043 1.044 0.035 3.352
G8 1.08
H8 1.01
SO1 3.7C12 0.683 0.633 0.692
D12 0.583
SO2 5.819C11 0.913 0.85 0.876
D11 0.787
0.242
0.363
0.586
0.75
1.044
0
0.2
0.4
0.6
0.8
1
1.2
0 1 2 3 4 5 6
conc.
Std
STANDARD CURVE Mean Linear (Mean)
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 14
Prepared standard curve and quantification of purified protein.

15. O B J E C T I V E 2
Isolationandpurificationofplantcrudeextract
Choice of plant for the project is used to prepare the crude extract. Column
extraction processes are used to for the isolation of concentrated crude protein
from the plant. Protein fractionations are made in different grade of polar to no-
polarsolvents.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 15

16. Procedure of Plant crude extraction.
A B C
D
E F
A. Selection of Plant, B. Collection, C. Drying and grinding, D. Column extraction, E. Rotary
evaporation, F. Collection and drying of crude extract.© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 16

17. Isolation of plant crude extract
• Catharanthus roseus and Ocimum gigatium chosen as choice of plant.
The leaf and branches are dried and powder is made.
The Powder loaded in the column.
Three separate solvents 1. Petroleum Benzene 2. Ethyl Acetate 3.
Methanol according to their polarity used for the extraction from the
plant materials.
The extracts are then dried and collected and weighed.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 17

18. O B J E C T I V E 3
AssayofRTtocheckits’functionality.
High throughput Functional RT assay to be done to check the RT activity. For the
project we should use the DNA that is in a RELA-GFP vector for other experiment
tobeclonedunderaT7promoterinasecondvector.Theninvitrotranscription is
usedtocheckRTfunctionality
RT PCR performed to check the RT functionality. Before RT PCR an in-vitro
transcriptionisperformedtogenerateRNAs.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 18

19. In vitro transcription
• Template DNA, forward and reverse primer, ATP, CTP, GTP UTP and
10x buffer used for this process. DNA dependent RNA polymerase is
used as the enzyme to generate RNA. DEPC treated H2O is used in
this process.
• After the incubation the RNA is quantified and this RNA is used for
the RT PCR.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 19

20. RT PCR
• To check the RT activity this assay is performed.
• The enzyme purified (RT 66) is used in this procedure. The first step of
this assay include a Reverse transcription process, and the DNA
developed in this process is amplified using the PCR and checked in
the gel. Positive result indicate the RT is functional.
• The first stage of this process is cDNA synthesis, RNA from the in vitro
transcription, a primer dNTPs is used, RT 66 is used as the enzyme.
And 5x Buffer and water is used.
• After that the cDNA is amplified using PCR, dNTPs, MgCl2, 10x Buffer,
2 primers, water and enzyme is used. After the incubation the
product is checked in gel. RT66 HAS SHOWN ACTIVITY.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 20

21. O B J E C T I V E 4
IdentificationofthefunctionalRTinhibitormolecule.
Firstthecrudeplantextractsaretobeusedtochecktheireffectivenessagainst
theHIVRT.Andthenthecrudesaretobefractionatedfurtherandeachfractions
areneededtobescreenedforfunctionalRTinhibition.Thefurtherfractionations
tobedoneunlessasinglemoleculecouldbeidentifiedasHIVRTinhibitor.High
throughputRTassaysneededtobedoneforthescreeningprocess.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 21

22. High throughput pico-green assay
• Pico green assay was performed in the lab. But due to some issues
related to the instruments it failed and instead the radioactive primer
extension assay was started.
• STATUS OF PICO GREEN ASSAY: STOPPED.
• Procedure: first a optimization and a standard curve generation is
crucial. Reaction is performed with 2800, 280, 28, 2.8, 0.28, 0.028
pico mole of DNA, TE buffer and Pico green dye (1:2000).
• Dye control, DNA control and RNA control with all the sets are
prepared in duplicates.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 22

23. Cont.
• After the completion of optimization and standard curve preparation
pico green should be used in reaction with RT alongside control RT
inhibitors to check different plant crudes for their RT inhibition
activity.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 23

24. Radioactive primer extension assay to check RT activity
• Poly RA is used as the RNA with oligo DT primer. HIV RT is used as the
enzyme.
• Radio labeled 3HdTTP (Tritium T) added as nucleotides.
• First optimization to be done with different amount of enzymes, RNA.
• Once after the completion of the optimization process. Different
crudes and fractions are tested for HIV RTi activity, and the reading is
taken through a Saintillation counter.
• STATUS: NOT STARTED.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 24

25. Materials and process
• First Template Primer dimer is needed to be prepared in a ratio of 1:5. It is
then diluted 200 fold with polymerization buffe (includes Tris-Cl 120mM PH
8.1, KCl 120 mM, MgCl2 16 mM, 0.5M DTT and H2O) and incubated in 37
degree C for 1hr.
• Then Reaction sets are prepared with Reaction Buffer (includes 50mM Tris
CL pH 7.8, 5mM MgCl2, 60mM KCl, 100ug/ml BSA, 1mM DTT, 0.5 uci
3HdTTPs), Poly RA-dt, RT, Water.
• Incubated in 37 degree for 1,3,5… mins. The reaction is stopped with EDTA
and incubated on ice for 30 mins.
• Transferred on a filter paper. Washed with 1ml 5% TCA. The filter paper
washed with TCA, Water, 70% Ethanol x2, dried.
• 1 mL saintilletion fluid added and reading taken in saintillation counter.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 25

26. O B J E C T I V E 5
CheckingtheRTinhibitorincellularcondition.
The molecule showing HIV RT inhibition activity is tested for it’s cytotoxicity level
byaCC50test.Thenthemoleculeisintroducedtoacellculturesystemwithsingle
cycleHIVvirusalongwiththeRTinhibitorfortestingitincellularcondition.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 26

27. Test of HIV inhibitor activity in cell culture
• 293 T cells are co-transfected with PNL43ΔGFP (RT backbone with
Green fluroscent Protein) and VSVG coat.
• After 48 hours of incubation the produced virus are separated and it
is used to infect Huh cells. The infected cells are seen green in
fluorescent microscopy.
• After 24 hr the cells should be treated with Control HIV inhibitors and
test Crude extracts and fractions to check anti HIV activity.
• In case of positive result the healthy cell must not be green.
• Status: This is expected to be started soon.
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 27

28. • Discussion
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 28

29. •THANK YOU
© Titas Mallick . M.Sc Botany. Sem II, CU. 2019 29