Plant could be used as safe potent material to isolate drug that can interact with and inhibit RT of HIV.

1. HIV RT INHIBITION in
vitro and Cell
Inhibition of retroviral revertase (EC 2.7.7.49) by isolated
molecules from Plants.
TITAS MALLICK, UNIVERSITY OF CALCUTTA

2. INTRODUCTION
• 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.
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3. HIV STATISTICS
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, UNIVERSITY OF CALCUTTA

4. •HIV VIRUS
Virus classification
Group: Group VI (ssRNA-RT)
Order: Unassigned
Family: Retroviridae
Subfamily: Orthoretrovirinae
Genus: Lentivirus
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5. HIV BIOLOGY
©AIDSinfo-NIH
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6. Characters
• Diameter 120nm
• 2 +ve sense strand RNA genome
• 9 genes in total
• 19 total proteins.
(Gag, Pol, Env, Tat, Rev, Nef, Vif, Vpr, Vpu, Tev [10th gene sometime
present])
• P24 coat protein
• P17 capsid protein
TITAS MALLICK, UNIVERSITY OF CALCUTTA

7. HIV virology
• Gag, Pol, Env is important for new virus production
• Gag encodes for GP120, which also get spliced into GP41
• Tat encode p16, P14 transcriptase transactivator.
• Tar element processed to miRNA and can regulate ERCC1, IER3 :
important apoptotic gens
• Rev bind preRNA element
• Vif prevents APOBEC3G: a cellular protein that identifies poly C
and poly U in viral tract and deactivate RT activity
• Vpr induces cell cycle arrest at G2/M checkpoint
TITAS MALLICK, UNIVERSITY OF CALCUTTA

8. continue
• Nef down regulate CD4 as well as both Class I & II MHC
• Vpu helps in packaging.
• During packaging Psi elemnt must present in the 3’ end of the LTR
• SLIP element helps in Gag-Pol frameshift during transcription.
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9. PROCESSES
• GP120 interact with CD4, corecptor CCR5 also plays vital role.
(alternate of CD4-CCR5 mechanism is Mannose specific C-type lectin
mediated receptor DC-SIGV mediated infection)
• GP41 initiate initial injection of capsomeric proteins.
• 2-contact point stable attachment later established.
• GP120 interact with Integrin α4β7, initiate LFA-1 integrin mediated
cell to cell spreading
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10. HIV GENOME
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11. continue
• Viral RNA -> RT -> cDNA -> Integration -> Transcription
• Rev one among the first encoded protein have a NLS, first shuttle to
the nucleus and with the help of Tat it helps the complete vRNA +ve
sense strand to shuttle to the cytoplasm
• Gag, Env help to form the complete virus.
General structure is like-
LTR-U3-R-U5-LTR
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12. continue
• RT developed from the Gag-Pol protein by protein splicing
mechanism by the Cleavage by Viral Protease PR.
• RT have two subunits p66 and p51, developed by a p6 coding
region frameshift read through.
• P66 is a 560 aminoacid protein
• P51 is a 440 aminoacid protein.
TITAS MALLICK, UNIVERSITY OF CALCUTTA

13. HIV ENZYME SYSTEM
• Reverse Transcriptase
• Integrase
• Protease
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14. Reverse Transcriptase
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15. TITAS MALLICK, UNIVERSITY OF CALCUTTA

16. Protein Seqence
IV-1 (HXB2):
10 20 30 40 50 60 70 80 90 100
| | | | | | | | | |
PISPIETVPV KLKPGMDGPK VKQWPLTEEK IKALVEICTE MEKEGKISKI GPENPYNTPV FAIKKKDSTK WRKLVDFREL NKRTQDFWEV QLGIPHPAGL
110 120 130 140 150 160 170 180 190 200
| | | | | | | | | |
KKKKSVTVLD VGDAYFSVPL DEDFRKYTAF TIPSINNETP GIRYQYNVLP QGWKGSPAIF QSSMTKILEP FRKQNPDIVI YQYMDDLYVG SDLEIGQHRT
210 220 230 240 250 260 270 280 290 300
| | | | | | | | | |
KIEELRQHLL RWGLTTPDKK HQKEPPFLWM GYELHPDKWT VQPIVLPEKD SWTVNDIQKL VGKLNWASQI YPGIKVRQLC KLLRGTKALT EVIPLTEEAE
310 320 330 340 350 360 370 380 390 400
| | | | | | | | | |
LELAENREIL KEPVHGVYYD PSKDLIAEIQ KQGQGQWTYQ IYQEPFKNLK TGKYARMRGA HTNDVKQLTE AVQKITTESI VIWGKTPKFK LPIQKETWET
410 420 430 440
| | | |
WWTEYWQATW IPEWEFVNTP PLVKLWYQLE KEPIVGAETF
TITAS MALLICK, UNIVERSITY OF CALCUTTA

17. Reverse Transcription
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18. information
• RT is a DNA/RNA dependant DNA polymerase.
• Have a plam, finger and thumb domain.
• Active Open conformation is denoted A form.
• An intermediate form also characterized termed H form
• Kd for DNA/DNA templte-Primer efficiency of RT is 4 μM whereas
it is 14 μM for RNA/DNA
• In active conformation it bends the DNA/RNA hybrid at -40degree
• DNA/DNA bending is also reported by some workers, thus making
the RNA/DNA reverse transcription much efficient.
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19. continue
• Important stage in the procedure is host tRNAlys3 is used as primer
• Two sharp strand transfer is needed to generate both the +ve and -
ve sense strand RNA.
• One theory suggests as cDNA synthesis processes overtime the RTc
matures by conformational changes into a complex called PIC, that
later imported into the nucleus and get involved in some host gene
interfering processes.
TITAS MALLICK, UNIVERSITY OF CALCUTTA

20. Main project: identification of RT inhibitor
• Accumulation of biomass.
• Isolation and purification of RT.
• Quantification.
• Plant Crude extraction.
• Protein isolation.
• Exclusion and Screening.
• RT functional assay.
• Pico-green assay.
• In-vivo assay.
TITAS MALLICK, UNIVERSITY OF CALCUTTA

21. BEGINNING WITH CULTURE
• The HIV RT GENE is placed in a vector in it’s MCS site under an
IPTG inducible enhancer.
• The plasmid was transferred in the E. coli strain BL21(DE3)
• The first batch of culture begun with reviving the cells in liquid
media for overnight in 37 degree.
• Then the primary culture was established in 37 degree.
• From the late log phase of the primary culture the secondary
culture was seeded, when it reached a OD of 0.6 then IPTG added
for induction.
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22. TO DO
• The secondary cultures must be centrifuged for the collection of the
biomass.
• This phase should be repeated several time for collection of proper
amount of biomass.
• The induced RT then must be isolated from the biomass
• For this process we must use hexa-His tag affinity chromatography
• Then this proteins must be assayed for quantification (Barford’s),
dialysis must be performed for condensation of the proteins.
TITAS MALLICK, UNIVERSITY OF CALCUTTA

23. PLANT SCREENING
• We must begin with a rough initial screening and then proceed
from a material with previous reports of RT inhibition.
• Choice of plants-
1. Catharanthus roseus (L.) G.Don (Apocynaceae)
2. Moringa (Moringaceae)
3. Carica papaya L. (Caricaceae)
4. Ocimum basilicum L. (Lamiaceae)
TITAS MALLICK, UNIVERSITY OF CALCUTTA

24. Next Stages
• The Proteins must be isolated from the plant extract after proper
cleaning and drying of the plant leaves.
• The protein fractionation must be done by salting in.
• Then other processes is done.
• Nickel column chromatography is performed for separation of
different phases of the solution.
• Different fractions are dissolved in different grades of non-polar to
polar solvents.
• Each fractions are tested for the RT inhibition activity in vitro
possibly through Pico-green Assay.
TITAS MALLICK, UNIVERSITY OF CALCUTTA

25. Functional RT assay
• Isolated RT in vitro tested by rtPCR and gel electrophoresis if it is
functional or not.
• MLV RT used as positive control.
TITAS MALLICK, UNIVERSITY OF CALCUTTA

26. Pico-green assay
• Pico green binds with only DNA/DNA or DNA/RNA hybrid but
not with ssRNA or ssDNA.
• Only pico green shows a high pick.
• Pico green+RNA shows low pick.
• Pico green+RNA+RT shows fall in pick if RT functional.
• Pico green+RNA+RT+inhibitor tested if show fall in the pick it
indicates the inhibitor inhibiting RT.
TITAS MALLICK, UNIVERSITY OF CALCUTTA

27. Next Stages
• After a fraction showing its inhibition action further exclusion
stages are performed.
• To identify a single molecule interacting with the RT for its
inhibition we must use first molecular docking and GCMS for
proper identification of the molecule,
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28. NEXT
• After the in vitro assays we must go for some in cell assay where we
should introduce both the RT and the inhibitory protein in the cell
line and will test for its action.
• Before working in cell culture the compunds CC50 level is
measured. The compounds are tested only below the cytotoxicity
level.
TITAS MALLICK, UNIVERSITY OF CALCUTTA

29. Current status
• Primary culture had been seeded
• Secondary culture was inoculated
• Induction was done.
• The work stopped here.
TITAS MALLICK, UNIVERSITY OF CALCUTTA

30. THANK YOU.
TITAS MALLICK, UNIVERSITY OF CALCUTTA

31. reference.
• Wikipedia
• Structure and function of HIV-1 reverse transcriptase: molecular
mechanisms of polymerization and inhibition: Stefan G. Sarafianos
et al. NCBI-NLM-NIH
• HIV-1 Reverse Transcription: Wei-Shau Hu et al. NCBI-NLM-NIH
• Strand transfer events during HIV-1 reverse transcription: Basu VP et al. NCBI-NLM-
NIH.
• Jmol Proteopedia.
• http://www.bioafrica.net/proteomics
• The Mechano-Chemistry of Reverse Transcriptase: Omri Malik et al.; The CELL
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