!
!
!
!!!"#$%&'()*+,($-)"%*./ / / / /
!!!/0)1*2+*3/45/6789/
//////////////////////////////////:+.)/8 /%;/</
Educator Materials!
Hands-on Activity
HIV Reverse Transcription and AZT!
=&>/?@>@?A@/B?CDAE?&:B&FD/CDG/CHB/
F>@?>&@I/
This!hands-on!activity!is!part!of!a!s e rie s!of!activities!and!demonstrations!focusing!on!various!as pe cts!o f!
the!human!immunodeficiency!virus!(HIV )!life!cycle.!In!th is !a ct ivity ,!st u d e n ts !w ill!model!h ow!the !a n ti-HIV!
drug!AZT!(azidothymidine)!interferes!with!the!process!of!viral!replication!and!reduces!the!amount!of!
virus!in!the!body.!!
Students!will!first!model!reverse!transcription,!the!process!that!results!in!the!production!of!a!double-
stranded!DNA!copy!of!the!HIV!J$'.K)LJ(*+'M) M/?DC /.)'%N ) .!(See!introduction!in!the!student!document!
for!a!review!of!the !pro ces s .)!U sin g!an !actual!HIV!RNA!sequence!as!a!template,!stud ents!w ill!model!the!
synthesis!of!a!complementary!strand!of!DNA!by!attaching!nucleotides!to!one!another.!Then,!students!
will!demonstrate!AZT’s!effect!on!this!process.!The y !w ill!su b stitu te !AZ T !in!p la ce !o f!th ymidine.!A ZT !lac ks !a!
hydroxyl!(OH)!group!that!is!necessary!for!subse qu en t!nucleotides!to!attach.!When!AZT !is!incorporated!in!
a!growing!DNA!sequence!it!prevents!further!nucleotides!from!being!added,!thereby!blocking!the!
production!of!HIV!DNA.!
Following!this!activity,!it!may!be!helpful!for!students!to!complete!the!HIV!integration!activity.!!
/
O@C?D&DP/F#Q@EB&> @A/
Students!will!be!able!to:!/
• Code!DNA!from!RNA.!
• Demonstrate!the!process!of!reverse!transcription.!
• Understand!the!role!of!the!enzym e!rev erse !transc riptase !in!the !form atio n!o f!viral!DNA .!!
• Review!the!chemical!structures!of!nucleic!acids!and!identify!structural!similarities!and!differences!
between!AZT!and!thymidine.!
• Model!how!AZT!inhibits!DNA!synthesis.!
/
ER??&ERORS/EFDD@EB&FDA/
E2**$,2K2N/
A(+'M+*MJ/
NGSS!(2013)!
HS-LS1-1;!HS!LS3-1;!HS-LS3-2!
AP!Biology!(2013)!
3.A.1;!3.C.3!
IB!Biology!
6.3!
/
T@U/B@?SA/
HIV,!RNA,!DNA,!reverse!transcription,!reverse!transcriptase,!bases!A,!G,!C,!T,!U!(adenine,!guanine,!
cytosine,!thymine,!and!uracil),!replication,!nucleotide,!nucleoside!
!
!
!
!!!"#$%&'()*+,($-)"%*./ / / / /
!!!/0)1*2+*3/45/6789/
//////////////////////////////////:+.)/6 /%;/</
Educator Materials!
Hands-on Activity
HIV Reverse Transcription and AZT!
B&S@/?@VR&?@S@DBA!
This!lesson!is!designed!to!be!completed!in!a!50-minute!class!period.!
/
ARPP@AB@G/CRG &@DE@/
This!lesson!is!appropriate!for!high!school!Honors,!AP,!and!IB,!and!Intro d u c to r y!Biology !c o ll eg e !s tu dents. /
/
:?&F?/TDFIO@GP@!
Students!should!have!studied!DNA!replication!and!transcription.!They!should!know!that!DNA!and!RNA!
are!made!of!nucleotides!and!have!some!familiarity!with!the!chemical!structures!of!nucleotides.!
Knowledge!of!the!atomic!structure!of!each!nucleoside!and!their!connection!via!phosphodiester!bonds!
between!the!5’-phosphate!and!3’-hydroxyl!functional!groups!is!not!required!but!will!be!helpful!to!more!
deeply!understand!how!AZT!interferes!with!DNA!synthesis.!The!introduction!to!the!student!document!
contains!information!about!the!structure!of!nucleic!acids!and!the!difference!between!nucleosides!and!
nucleotides.!
!
SCB@?&COA/
• Copies!of!the!RNA!template,!which!can!be!laminated!to!preserve!them!for!future!use.!(The!RNA!
template!is!availab le!as!a!se pa rate!d oc um e nt!on !Bio Intera ctive.)!
• Copies!of!the!DNA!nucleotides!and!AZT,!which!can!be!laminated!to!preserve!them!for!future!
use.!(The!nucleotides!are!av ailab le!as !a!sep ara te!do cu m e nt!o n!Bio Inte rac tive.) !
• Optional!Velcro!tabs!0.9!cm!(3/8!in.)!size!or!slightly!larger!
!
For!each!group!of!two!or!three!stud ents,!you!will!need!1!copy!of!the!RNA!template,!7!cytosines,!6!
guanines,!3!adenines,!2!thymines,!and!1!AZT.!
!
B@CE=&DP/B&:A//
#+,W.*%2'M/&';%*N+($%'/
• An!animation!of!the!HIV!life!cycle!is!available!at!http://www.hhmi.org/biointeractive/hiv-life-
cycle.!The!first!19!se c onds!of!this!resource!describes!the!structure!of!HIV,!including!the!RNA!
genome!and!reverse!transcriptase!enzyme.!It!then!shows!how!HIV!infects!helper!T!cells!(00:20!
seconds!through!1:25!minutes)!and!demo nstra tes !reve rse!tran sc ription ,!integ ratio n,!viral!
protein!synthesis,!reprod uctio n,!an d!b ud ding !as!step s!in!the !HIV !life!cycle.!
• Azidothymidine!(AZT)!was!the!first!FDA-approved!drug!to!treat!HIV!infection .!AZT !is!a!re ve rs e!
transcriptase!inhib itor!tha t!is!structu rally!similar!to !the!n ucleo side !thymidine.!The!3’!hydroxyl!
group!of!thymidine!is!replaced!with!an!N
3
!azide!group,!preventing!bonding!between!nucleotides!
!
!
!
!!!"#$%&'()*+,($-)"%*./ / / / /
!!!/0)1*2+*3/45/6789/
//////////////////////////////////:+.)/4 /%;/</
Educator Materials!
Hands-on Activity
HIV Reverse Transcription and AZT!
during!the!process!of!reverse!transcription.!The!structure!of!AZT!causes!it!to!block!further!DNA!
synthesis!when!it!is!incorporated!in!a!DNA!strand.!!
• The!introd uction!to!the!stud en t!do cu m en t!con tain s!inform a tion !abo ut!th e!struc ture !of!nu cleic!
acids.!Spend!some!time!reviewing!this!informa tio n .!Y o u !co u ld !a ss ign !it !as !a !re ad in g !b efo re !d o in g !
the!assignment!in!class !an d !t he n !sp e n d !so me!time !re v iewing!the !co n c ep t.!
:*)X+*$'./(Y)/?DC/B)NXK+()/+'M/D2,K)%($M)J/
• Before!class,!prepare!both!the!RNA!template!and!the!DNA!nucleotides!for!this!activity.!
o The!RNA!template!is!a!single!strand!of!nucleotides!(GGGCCUGUUGCACCAGGC).!This!
sequence!is!an!actual!HIV!sequence.!
o The!DNA!nucleotide!bases!are!individually!cut.!You!may!wish!to!laminate!them!and!
attach!either!Velcroâ!or!some!type!of!adhesive!to!the!tabs.!!
o If!you!us e !Velcroâ,!the!“h oo k”!o f!the!V elcroâ!is!a lw a ys !o n!t h e!t o p !righ t !gr ay !in se t!t ab !o f!
the!nucleotide !bas e!(facin g!up );! th is!rep rese nts!th e!3’!hydroxyl!group.!The!“loop”!of!the!
Velcroâ!is!alway s!o n !th e !to p !le ft!t ab !(fa c in g!d o w n !o n !th e !u n d erside!of!the!tab).!This!
represents!the !5’!pho sp ha te;!see!0$.2*)/8 .!!
o The!tabs!should!attach!when!the!nucleotide!bases!bond!together.!The!Velcroâ!or!
adhesives!model!the!phosphate!backbone!of!a!nucleotide!sequence.!The!edges!of!the!
nucleotides!should!touch!and!the!bases!align!with!those!of!the!HIV!RNA!sequence.!
/
0$.2*)/8"/A()XJ/$'/X*) X + *$' ./(Y ) /' 2, K) %( $M) J"/
!
Step!1:!Cut!out!the!laminated !nucleotides!and!attach!the!Velcro!to!the!tabs.!For!the!black!tabs,!the!
Velcro!goes!on!the!underside!of!the!tab!and!for!the!gray!tabs!the!Velcro!goes!on!top.!
!
Step!2:!Following!the!RNA!template,!attach!the!appropriate!nucleotides!to!one!another!with!the!black!
tabs!going!on!to p!of!th e!gra y!tab s.!
!
!
!
!!!"#$%&'()*+,($-)"%*./ / / / /
!!!/0)1*2+*3/45/6789/
//////////////////////////////////:+.)/Z /%;/</
Educator Materials!
Hands-on Activity
HIV Reverse Transcription and AZT!
!
Step!3:!Be!sure!that!the!edges!of!the!nucleotides!are!touching.!Each!nucleotide!should!align!with!the!one!
it!is!pairing !w ith !o n !th e !R N A !te mplate.!
!
E%'M2,($'./(Y)/C,($-$(3/
• Depending!on!the!size!of!the!class,!divide!students!into!groups!of!2,!3,!o r!more.!Give!eac h !gro u p !
the!RNA!tem p late!an d!th e!D NA !nu cleo tide!b ase s.!Tell!stude nts !they !will!mo de l!how !th e!en zym e !
reverse!transcripta se!b uild s!a!DN A !seq ue nce !from !th e!viral!RN A !tem p late.!!
• Explain!that!in!this!act ivit y! th ey !are!b uilding !a!single-stranded !DNA!sequence.!In!reality,!the!
reverse!transcripta se!will!also!synthesize!a!complementary!DNA!strand!and!the!double-stranded!
DNA!will!integrat e!in to !the!host!DN A!ge no m e.!
• After!students!build!the!DNA!sequence!complementary!to!the!viral!RNA,!ask!them!to!take!their!
sequences!apart.!Then,!hand!each!group!an!AZT!cutout!(without!a!top -right!Velcro!tab)!and!take!
away!one!of!their!T!nucleotides.!
• Ask!students!to!build!a!DNA!sequence!from!the!viral!RNA,!but!this!time!have!the!enzyme!reverse!
transcriptase!introd u ce!A ZT!into !the!DNA!strand.!They!should !su b stit ut e!A ZT !a t!th e!one!of!the!T!
location s!and!see!that!they!cannot!attach!more!nucleotides,!meaning!HIV!replication!is!halted.!
• The!AZT!can!be!incorporated!anywhere!in!the!sequence!where!a!T!would!normally!go.!It!does!
not!have!to!go!in!the!first!position.!!!!
!
I*+XX$'./2X!
• Once!students!complete!the!activity,!you!may!want!to!show!them!an!animation!of!how!AZT!
blocks!reverse!transcriptase!(http://www.hhmi.org/biointeractive/azt-blocks-reverse-
transcriptase).!
• The!last!part!of!the!animation!above!deals!with!drug!resistance.!To!learn!more!about!drug!
resistance!and !wh y!res earc he rs!give!m u ltiple !drug s,! watch!a!3-minute!clip!from!Lecture!3,!
“Drugs!and!HIV!Evolution,”!Chapters!10!and!11.!Open!the!lect ur e!a t!
https://www.hhmi.org/biointeractive/drugs-and-hiv-evolution!and!then!click!on!Chapter!10!and!
watch!from!time!11:41!to!15:00.!!
• AZT!is!a!nucleoside!reverse!transcriptase!inhibitor!(NRTI).!A!number!of!drugs!called!non-
nucleoside!reverse!transcriptase!inhibitors!(NNRTIs)!block!the!same!step!in!the!HIV!life!cycle!as!
!
!
!
!!!"#$%&'()*+,($-)"%*./ / / / /
!!!/0)1*2+*3/45/6789/
//////////////////////////////////:+.)/[ /%;/</
Educator Materials!
Hands-on Activity
HIV Reverse Transcription and AZT!
NRTIs!(reverse!transcription)!but!by!a!completely!different!mechanism.!Rather!than!being!
incorpo ra te d!in t o !th e!v ira l!D N A !lik e !N R T Is,!N N R T Is !b ind !d ire c tly!t o !th e !H IV !re ve rs e!t ra n sc rip ta se !
enzyme,!inhibiting!its!activity.!
• As!time!allows,!show!students!a!short!(1:50!sec)!interview!with!an!HIV-positive!woman!able!to!
lead!a!normal!life!thanks!to!antiviral!therapy.!It!is!available!at!
http://www.hhmi.org/biointeractive/benefits-antiretroviral-regimen!!!
/
CDAI@?/T@U/
1.!Draw!a!sequence!of!10!RNA!nucleotides!to!represent!a!segment!of!the!HIV!genome.!You!can!just!use!
the!shorthan d!ve rsion !with !a!string!o f!letters.!Make!sure!you!include !each!of!the!four!RNA!nucleotides!at!
least!onc e . !
A)\2)',)J/!$KK/%;/,%2*J)/-+*3"/BY)/W)3/$J/(Y+(/J(2M)'(J/JY%2KM/JY%!/+/J$'.K)/J)\2)',)/(Y+(/Y+J/(Y)/
;%2*/'2,K)%($M)J/C R PE /12(/' %(/B"/
@]+NXK)^/LRLELELELRLRLELCLPLCL!
2.!The!first!step!in!reverse!transcription!is!the!synthesis!of!a!complementary!DNA!strand.!Draw!a!
diagram!of!the!RNA!segment!from!step!1!paired!with!DNA!nucleotide!bases.!
A(2M)'(J/JY%2KM/M*+!/(Y)/J+N)/J)\2)',)/+J/$'/(Y)/+'J!)*/+1%-)/+'M/+/J),%'M/,%NXK)N)'(+*3/
J)\2)',)"/:+$*$'./JY%2KM/JY%!/R_C /`R/$'/(Y)/?DC /J(*+'M/+'M/C/$'/(Y)/GDC/,%NXK)N)'(a5/P_E5/E_P5/
C_B"//
@]+NXK)^/LRLELELELRLRLELCLPLCL//////////////////
//////////////////LCLPLPLPLCLCLP LBLELBL/
3.!The!second!step!is!the!synthesis!of!a!DNA!strand!complementary!to!the!first!to!produce!double-
stranded!viral!DNA.!Draw!a!diagram !of!a!double-stranded!DNA!mo lecule.!!
D%!/J(2M)'(J/JY%2KM/M*+!/(Y)/J+N)/J)\2)',)/+J/(Y)/GDC/J)\2)',)/$'/+'J!)*/6/+'M/+/
,%NXK)N)'(+*3/GDC/J)\2)' ,)"/:+$*$'.J/JY%2KM/+K!+3J/1)/C_B/+'M/P_E"/
@]+NXK)^/LCLPLP LPLCLCLPLBLELBL/
//////////////////LBLELELEL BLBLELCLP LCL/
4.!Compare!your!drawing!of!the!RNA/DNA!nucleotide!base!pairs!(in!Question!2)!to!the!DNA!nucleotide!
base!pairs!(in!Question!3),!and!in!each!drawing,!circle!the!nucleotide!base!pairs!that!are!different.!
A(2M)'(J/JY%2KM/,$*,K)/(Y)/R_C/X+$*$'.J/$'/V2)J($%'/6/+'M/(Y)/B_C/X+$*$'.J/$'/V2)J($%'/4"/
5.!The!double-stranded!DNA!is!incorporated!in!the!host!cell!DNA.!What!is!the!function!of!DNA!in!cells?!
A(2M)'(J/JY%2KM/J(+()/(Y+(/GDC/,%'(+$'J/(Y)/.)')($,/$';%*N+($%'/%;/(Y)/,)KK/+'M/,+**$)J/$'J(*2,($%'J/
;%*/N+W$'./X*% ()$'J "/
6.!Record'the'sequence'of'the'newly'synthesized'DNA'strand'below'and'then'take'apart'your'DNA'
sequence.'
!
!
!
!!!"#$%&'()*+,($-)"%*./ / / / /
!!!/0)1*2+*3/45/6789/
//////////////////////////////////:+.)/< /%;/</
Educator Materials!
Hands-on Activity
HIV Reverse Transcription and AZT!
EEEPPCECCEPBPPBEEP//
7.!Now'replace'one'of'your'T'nucleotides'with'AZT.'AZT'is'similar'in'molecular'structure'to'thymidine'but'
with'one'important'difference.'Examine'Figure'3'showing'the'chemical'structures'of'thymidine'and'of'
AZT.'What'are'the'differences'between'the'two'structures?!
CHBbJ/J(*2,(2*)/$J/-)*3/J$N$K+*/(%/(Y+(/%;/(Y3N$M$') /)] ,)X (/(Y+ (/$(/Y+J/D
4
/$'J()+ M /% ;/F=/%'/( Y ) /*$1 % J ) "/ /
8.!In!cells!where!AZT!is!present,!AZT!can!be!incorporated!in!a!growing!DNA!chain!in!place!of!the!
thymidine!base.!Predict!wh at !m ig ht !ha p p en !if!A ZT !is!in co rp o ra te d.!
A(2M)'(J/JY%2KM/X*)M$, (/( Y + (/* ) - )* J ) /(* + 'J , *$X ( $% ' /!$KK/J(%X/1 ) ,+ 2 J ) /(Y ) /D
4
/.*%2X/,+''%(/;%*N/+/1%'M/
!$(Y/(Y)/XY%JXY+()/%;/+'%(Y)*/'2,K)%($M)"/
9.!Repe a t!ste p !6!above!in!a!cell!where!AZT!is!present.!Write!down!the!sequence!of!the!DNA!you!
constructed.!!
EEEPPCECCEP`CHBa/
EEEPPCECCEPBPP`CH Ba/
10.!How!is!this!sequence!different!from!the!sequence!in!step!1?!
A(2M)'(J/JY%2KM/J+3/$(/$J/JY%*()*"/
11.!Using!the!information!above,!explain!ho w!and!why!A ZT !a f fe c ts !the!proc e s s!of!rever se !t ra n s c rip t io n .!!
CHBbJ/J(*2,(2*)/$J/-)*3/J$N$K+*/(%/(Y+(/%;/(Y3N$M$')/)],)X(/(Y +(/$(/Y+J/D
4
/$'J()+ M /% ;/F="/BY)/+1J)',)/%;/
(Y)/F=/.*% 2X/X*)-)'(J/(Y)/;%*N+($%'/%;/+/1%'M/1)(!))'/CHB/+'M/(Y)/')](/'2,K)%($M)/$'/(Y)/J)\2)',)5/
(Y)*)13/Y+ K($'./G DC /J3' (Y) J$J/+' M/= &>/*) X*% M2 ,($%' "/
/
CRB=F?A/
Written'by'Patricia'Nolan'Bertino,'Scotia-Glenville'High'School,'and'Anthony'Bertino,'Canandaigua'Academy.'Jam e s'Blankenship ,'
PhD,'Cornell'University,'advised'on'the'project.'Additional'writing'by'Mary'Colvard.'Illustrations'in'stud e nt'd o c ument'by'Chris'
Vargas.'
Edited'by'Leah'M.'Cataldo,'PhD,'Buckingham'Browne'&'Nichols'School;'Karen'Gulliver,'consultant;'and'Laura'Bonetta,'PhD,'
HHMI.'Reviewed'by'Melissa'Csikari,'HHMI.''
Scientific'review'by'Allen'Bateman,'PhD,'Debby'Walser-Kuntz,'PhD,'Carleton'College,'Munir'Syed,'PhD,'Hartwick'College.'
