HIV REVERSE TRANSCRIPTION AND AZT

OVERVIEW HIV REVERSE TRANSCRIPTION AND AZT This hands-n activity is part f a series f activities and demnstratins fcusing n varius aspects f the human immundeficiency virus (HIV) life cycle. In this activity, students will mdel hw the anti-hiv drug AZT (azidthymidine) interferes with the prcess f viral replicatin and reduces the amunt f virus in the bdy. Students will first mdel reverse transcriptin, the prcess that results in the prductin f a dublestranded DNA cpy f the HIV single-stranded RNA genme. (See intrductin in the student dcument fr a review f the prcess.) Using an actual HIV RNA sequence as a template, students will mdel the synthesis f a cmplementary strand f DNA by attaching nucletides t ne anther. Then, students will demnstrate AZT s effect n this prcess. They will substitute AZT in place f thymidine. AZT lacks a hydrxyl (OH) grup that is necessary fr subsequent nucletides t attach. When AZT is incrprated in a grwing DNA sequence it prevents further nucletides frm being added, thereby blcking the prductin f HIV DNA. Fllwing this activity, it may be helpful fr students t cmplete the HIV integratin activity. LEARNING OBJECTIVES Students will be able t: Cde DNA frm RNA. Demnstrate the prcess f reverse transcriptin. Understand the rle f the enzyme reverse transcriptase in the frmatin f viral DNA. Review the chemical structures f nucleic acids and identify structural similarities and differences between AZT and thymidine. Mdel hw AZT inhibits DNA synthesis. CURRICULUM CONNECTIONS Curriculum NGSS (2013) AP Bilgy (2013) Standards HS-LS1-1; HS LS3-1; HS-LS3-2 3.A.1; 3.C.3 IB Bilgy 6.3 KEY TERMS HIV, RNA, DNA, reverse transcriptin, reverse transcriptase, bases A, G, C, T, U (adenine, guanine, cytsine, thymine, and uracil), replicatin, nucletide, nucleside Page 1 f 6

TIME REQUIREMENTS This lessn is designed t be cmpleted in a 50-minute class perid. SUGGESTED AUDIENCE This lessn is apprpriate fr high schl Hnrs, AP, and IB, and Intrductry Bilgy cllege students. PRIOR KNOWLEDGE Students shuld have studied DNA replicatin and transcriptin. They shuld knw that DNA and RNA are made f nucletides and have sme familiarity with the chemical structures f nucletides. Knwledge f the atmic structure f each nucleside and their cnnectin via phsphdiester bnds between the 5 -phsphate and 3 -hydrxyl functinal grups is nt required but will be helpful t mre deeply understand hw AZT interferes with DNA synthesis. The intrductin t the student dcument cntains infrmatin abut the structure f nucleic acids and the difference between nuclesides and nucletides. MATERIALS Cpies f the RNA template, which can be laminated t preserve them fr future use. (The RNA template is available as a separate dcument n BiInteractive.) Cpies f the DNA nucletides and AZT, which can be laminated t preserve them fr future use. (The nucletides are available as a separate dcument n BiInteractive.) Optinal Velcr tabs 0.9 cm (3/8 in.) size r slightly larger Fr each grup f tw r three students, yu will need 1 cpy f the RNA template, 7 cytsines, 6 guanines, 3 adenines, 2 thymines, and 1 AZT. TEACHING TIPS Backgrund Infrmatin An animatin f the HIV life cycle is available at http://www.hhmi.rg/biinteractive/hiv-lifecycle. The first 19 secnds f this resurce describes the structure f HIV, including the RNA genme and reverse transcriptase enzyme. It then shws hw HIV infects helper T cells (00:20 secnds thrugh 1:25 minutes) and demnstrates reverse transcriptin, integratin, viral prtein synthesis, reprductin, and budding as steps in the HIV life cycle. Azidthymidine (AZT) was the first FDA-apprved drug t treat HIV infectin. AZT is a reverse transcriptase inhibitr that is structurally similar t the nucleside thymidine. The 3 hydrxyl grup f thymidine is replaced with an N 3 azide grup, preventing bnding between nucletides Page 2 f 6

during the prcess f reverse transcriptin. The structure f AZT causes it t blck further DNA synthesis when it is incrprated in a DNA strand. The intrductin t the student dcument cntains infrmatin abut the structure f nucleic acids. Spend sme time reviewing this infrmatin. Yu culd assign it as a reading befre ding the assignment in class and then spend sme time reviewing the cncept. Preparing the RNA Template and Nucletides Befre class, prepare bth the RNA template and the DNA nucletides fr this activity. The RNA template is a single strand f nucletides (GGGCCUGUUGCACCAGGC). This sequence is an actual HIV sequence. The DNA nucletide bases are individually cut. Yu may wish t laminate them and attach either Velcrâ r sme type f adhesive t the tabs. If yu use Velcrâ, the hk f the Velcrâ is always n the tp right gray inset tab f the nucletide base (facing up); this represents the 3 hydrxyl grup. The lp f the Velcrâ is always n the tp left tab (facing dwn n the underside f the tab). This represents the 5 phsphate; see Figure 1. The tabs shuld attach when the nucletide bases bnd tgether. The Velcrâ r adhesives mdel the phsphate backbne f a nucletide sequence. The edges f the nucletides shuld tuch and the bases align with thse f the HIV RNA sequence. Figure 1. Steps in preparing the nucletides. Step 1: Cut ut the laminated nucletides and attach the Velcr t the tabs. Fr the black tabs, the Velcr ges n the underside f the tab and fr the gray tabs the Velcr ges n tp. Step 2: Fllwing the RNA template, attach the apprpriate nucletides t ne anther with the black tabs ging n tp f the gray tabs. Page 3 f 6

Step 3: Be sure that the edges f the nucletides are tuching. Each nucletide shuld align with the ne it is pairing with n the RNA template. Cnducting the Activity Depending n the size f the class, divide students int grups f 2, 3, r mre. Give each grup the RNA template and the DNA nucletide bases. Tell students they will mdel hw the enzyme reverse transcriptase builds a DNA sequence frm the viral RNA template. Explain that in this activity they are building a single-stranded DNA sequence. In reality, the reverse transcriptase will als synthesize a cmplementary DNA strand and the duble-stranded DNA will integrate int the hst DNA genme. After students build the DNA sequence cmplementary t the viral RNA, ask them t take their sequences apart. Then, hand each grup an AZT cutut (withut a tp-right Velcr tab) and take away ne f their T nucletides. Ask students t build a DNA sequence frm the viral RNA, but this time have the enzyme reverse transcriptase intrduce AZT int the DNA strand. They shuld substitute AZT at the ne f the T lcatins and see that they cannt attach mre nucletides, meaning HIV replicatin is halted. The AZT can be incrprated anywhere in the sequence where a T wuld nrmally g. It des nt have t g in the first psitin. Wrapping up Once students cmplete the activity, yu may want t shw them an animatin f hw AZT blcks reverse transcriptase (http://www.hhmi.rg/biinteractive/azt-blcks-reversetranscriptase). The last part f the animatin abve deals with drug resistance. T learn mre abut drug resistance and why researchers give multiple drugs, watch a 3-minute clip frm Lecture 3, Drugs and HIV Evlutin, Chapters 10 and 11. Open the lecture at https://www.hhmi.rg/biinteractive/drugs-and-hiv-evlutin and then click n Chapter 10 and watch frm time 11:41 t 15:00. AZT is a nucleside reverse transcriptase inhibitr (NRTI). A number f drugs called nnnucleside reverse transcriptase inhibitrs (NNRTIs) blck the same step in the HIV life cycle as Page 4 f 6

NRTIs (reverse transcriptin) but by a cmpletely different mechanism. Rather than being incrprated int the viral DNA like NRTIs, NNRTIs bind directly t the HIV reverse transcriptase enzyme, inhibiting its activity. As time allws, shw students a shrt (1:50 sec) interview with an HIV-psitive wman able t lead a nrmal life thanks t antiviral therapy. It is available at http://www.hhmi.rg/biinteractive/benefits-antiretrviral-regimen ANSWER KEY 1. Draw a sequence f 10 RNA nucletides t represent a segment f the HIV genme. Yu can just use the shrthand versin with a string f letters. Make sure yu include each f the fur RNA nucletides at least nce. Sequences will f curse vary. The key is that students shuld shw a single sequence that has the fur nucletides AUGC but nt T. Example: -U-C-C-C-U-U-C-A-G-A- 2. The first step in reverse transcriptin is the synthesis f a cmplementary DNA strand. Draw a diagram f the RNA segment frm step 1 paired with DNA nucletide bases. Students shuld draw the same sequence as in the answer abve and a secnd cmplementary sequence. Pairing shuld shw U/A (U in the RNA strand and A in the DNA cmplement), G/C, C/G, A/T. Example: -U-C-C-C-U-U-C-A-G-A- -A-G-G-G-A-A-G-T-C-T- 3. The secnd step is the synthesis f a DNA strand cmplementary t the first t prduce dublestranded viral DNA. Draw a diagram f a duble-stranded DNA mlecule. Nw students shuld draw the same sequence as the DNA sequence in answer 2 and a cmplementary DNA sequence. Pairings shuld always be A/T and G/C. Example: -A-G-G-G-A-A-G-T-C-T- -T-C-C-C-T-T-C-A-G-A- 4. Cmpare yur drawing f the RNA/DNA nucletide base pairs (in Questin 2) t the DNA nucletide base pairs (in Questin 3), and in each drawing, circle the nucletide base pairs that are different. Students shuld circle the U/A pairings in Questin 2 and the T/A pairings in Questin 3. 5. The duble-stranded DNA is incrprated in the hst cell DNA. What is the functin f DNA in cells? Students shuld state that DNA cntains the genetic infrmatin f the cell and carries instructins fr making prteins. 6. Recrd the sequence f the newly synthesized DNA strand belw and then take apart yur DNA sequence. Page 5 f 6

CCCGGACAACGTGGTCCG 7. Nw replace ne f yur T nucletides with AZT. AZT is similar in mlecular structure t thymidine but with ne imprtant difference. Examine Figure 3 shwing the chemical structures f thymidine and f AZT. What are the differences between the tw structures? AZT s structure is very similar t that f thymidine except that it has N 3 instead f OH n the ribse. 8. In cells where AZT is present, AZT can be incrprated in a grwing DNA chain in place f the thymidine base. Predict what might happen if AZT is incrprated. Students shuld predict that reverse transcriptin will stp because the N 3 grup cannt frm a bnd with the phsphate f anther nucletide. 9. Repeat step 6 abve in a cell where AZT is present. Write dwn the sequence f the DNA yu cnstructed. CCCGGACAACG(AZT) CCCGGACAACGTGG(AZT) 10. Hw is this sequence different frm the sequence in step 1? Students shuld say it is shrter. 11. Using the infrmatin abve, explain hw and why AZT affects the prcess f reverse transcriptin. AZT s structure is very similar t that f thymidine except that it has N 3 instead f OH. The absence f the OH grup prevents the frmatin f a bnd between AZT and the next nucletide in the sequence, thereby halting DNA synthesis and HIV reprductin. AUTHORS Written by Patricia Nlan Bertin, Sctia-Glenville High Schl, and Anthny Bertin, Canandaigua Academy. James Blankenship, PhD, Crnell University, advised n the prject. Additinal writing by Mary Clvard. Illustratins in student dcument by Chris Vargas. Edited by Leah M. Catald, PhD, Buckingham Brwne & Nichls Schl; Karen Gulliver, cnsultant; and Laura Bnetta, PhD, HHMI. Reviewed by Melissa Csikari, HHMI. Scientific review by Allen Bateman, PhD, Debby Walser-Kuntz, PhD, Carletn Cllege, Munir Syed, PhD, Hartwick Cllege. Page 6 f 6