1 00:00:15,395 --> 00:00:16,770 BARBARA IMPERIALI: What I'm going 2 00:00:16,770 --> 00:00:21,390 to do first of all for today, the bulk of today's lecture 3 00:00:21,390 --> 00:00:24,600 will be on HIV and Ebola viruses, 4 00:00:24,600 --> 00:00:28,380 with more time spent on HIV because it's potentially 5 00:00:28,380 --> 00:00:33,870 one of the mechanistically best understood of the retroviruses. 6 00:00:33,870 --> 00:00:37,260 And it also has offered numerous opportunities 7 00:00:37,260 --> 00:00:39,390 for therapeutic intervention. 8 00:00:39,390 --> 00:00:41,310 And there are a lot of themes and terms 9 00:00:41,310 --> 00:00:45,930 that I can bring up as I talk to you about the HIV virus, 10 00:00:45,930 --> 00:00:52,250 because it's just a great exemplar of the viruses. 11 00:00:52,250 --> 00:00:52,750 OK. 12 00:00:52,750 --> 00:00:56,382 So HIV is what is known as a retrovirus. 13 00:01:04,129 --> 00:01:07,370 So that sort of designates that there's something working 14 00:01:07,370 --> 00:01:09,710 backwards in this retrovirus. 15 00:01:09,710 --> 00:01:12,680 Based on its number in the Baltimore Classification, 16 00:01:12,680 --> 00:01:16,220 it is the most recently identified mechanism 17 00:01:16,220 --> 00:01:17,660 of viruses. 18 00:01:17,660 --> 00:01:23,990 HIV stands for Human Immunodeficiency Virus. 19 00:01:34,470 --> 00:01:36,950 And when you hear the term HIV, you'll 20 00:01:36,950 --> 00:01:44,370 often hear HIV/AIDS where the AIDS part stands for Acquired 21 00:01:44,370 --> 00:01:45,915 Immunodeficiency Syndrome. 22 00:01:54,990 --> 00:01:59,110 So what you can start to tell about the name-- 23 00:01:59,110 --> 00:02:01,470 why am I hunching down-- 24 00:02:09,340 --> 00:02:13,410 is there's something related to the immune system with respect 25 00:02:13,410 --> 00:02:14,950 to this virus. 26 00:02:14,950 --> 00:02:18,360 And in particular, it is that this virus targets, 27 00:02:18,360 --> 00:02:22,170 very specifically, a population of the cells that 28 00:02:22,170 --> 00:02:24,540 are critical in immunity. 29 00:02:24,540 --> 00:02:27,510 And when the virus attacks those cells, 30 00:02:27,510 --> 00:02:32,100 the person infected with the virus becomes immunodeficit. 31 00:02:32,100 --> 00:02:34,180 So I've talked to you about different viruses. 32 00:02:34,180 --> 00:02:35,940 Some go to the liver. 33 00:02:35,940 --> 00:02:37,380 Some go for other organs. 34 00:02:37,380 --> 00:02:42,270 So like the liver, the viruses that 35 00:02:42,270 --> 00:02:45,810 really cause a deficiency in liver 36 00:02:45,810 --> 00:02:49,410 function, but this one is very specifically deficiencies 37 00:02:49,410 --> 00:02:51,030 in immune function. 38 00:02:51,030 --> 00:02:55,560 And it has been a tremendous challenge developing vaccines 39 00:02:55,560 --> 00:03:00,670 against HIV/AIDS because of the problem with the immune system. 40 00:03:00,670 --> 00:03:04,110 The other thing that I mentioned to you in the last class 41 00:03:04,110 --> 00:03:07,560 was that HIV is often found co-infecting 42 00:03:07,560 --> 00:03:11,400 with TB patients, people with those microbial infections. 43 00:03:11,400 --> 00:03:14,610 And that's because the immunodeficiency makes you more 44 00:03:14,610 --> 00:03:17,170 susceptible to tuberculosis. 45 00:03:17,170 --> 00:03:20,370 And oftentimes there's a co-infection 46 00:03:20,370 --> 00:03:22,740 with the two diseases. 47 00:03:22,740 --> 00:03:25,470 And it makes the people with TB really even 48 00:03:25,470 --> 00:03:29,050 less able to combat the TB infection. 49 00:03:29,050 --> 00:03:33,540 So that is really what gets a lot of the TB patients. 50 00:03:33,540 --> 00:03:36,350 So that's an important aspect to know. 51 00:03:36,350 --> 00:03:38,700 And it's also an important aspect 52 00:03:38,700 --> 00:03:43,420 to be aware of when treating people for any infection 53 00:03:43,420 --> 00:03:48,420 is if there's an HIV component, the body 54 00:03:48,420 --> 00:03:52,260 is much less able to deal with mounting 55 00:03:52,260 --> 00:03:54,300 an immune response because of the fact 56 00:03:54,300 --> 00:03:56,430 that the immune cells are targeted. 57 00:03:56,430 --> 00:04:01,155 So HIV is an envelope virus. 58 00:04:05,790 --> 00:04:10,480 That means it has a membrane around it. 59 00:04:10,480 --> 00:04:15,000 It is this sort of peculiar shape 60 00:04:15,000 --> 00:04:18,920 where the outside of the membrane is coated-- 61 00:04:18,920 --> 00:04:22,560 the outside of the virus is coated with membrane. 62 00:04:22,560 --> 00:04:27,510 And then stuck in that membrane are particular proteins 63 00:04:27,510 --> 00:04:30,450 that are very critical for interaction 64 00:04:30,450 --> 00:04:32,650 with the human host cells. 65 00:04:32,650 --> 00:04:40,230 So these are often termed GP proteins. 66 00:04:40,230 --> 00:04:43,530 And the one very important one is GP140. 67 00:04:43,530 --> 00:04:51,090 And that GP stands for glycoprotein. 68 00:04:51,090 --> 00:04:56,090 Now, an important aspect, remember, of virus lifestyle 69 00:04:56,090 --> 00:04:59,030 is that they basically exploit all 70 00:04:59,030 --> 00:05:02,360 of the human cellular machinery for their benefit 71 00:05:02,360 --> 00:05:07,200 to make their proteins for all of the basic needs of a cell. 72 00:05:07,200 --> 00:05:10,910 So these glycoproteins look like the glycoproteins 73 00:05:10,910 --> 00:05:13,190 that are made by humans, because they're 74 00:05:13,190 --> 00:05:15,980 being made by the same machinery that we 75 00:05:15,980 --> 00:05:18,080 would make the glycoproteins. 76 00:05:18,080 --> 00:05:20,390 In bacteria, when we talked about them, 77 00:05:20,390 --> 00:05:22,910 it's a different machinery that glycoproteins 78 00:05:22,910 --> 00:05:25,340 and glycoconjugates look different. 79 00:05:25,340 --> 00:05:30,290 But in HIV, the glycoproteins look like our glycoproteins. 80 00:05:30,290 --> 00:05:31,970 And therefore, you could more or less 81 00:05:31,970 --> 00:05:36,950 consider the HIV virus particle to be coated with things 82 00:05:36,950 --> 00:05:39,180 that look sort of human. 83 00:05:39,180 --> 00:05:42,800 So that's a kind of decoy to the human system 84 00:05:42,800 --> 00:05:46,760 anyway with respect to recognizing a foreign entity. 85 00:05:46,760 --> 00:05:50,390 So often the glycans there serve as decoys 86 00:05:50,390 --> 00:05:52,520 to make the human body think, well, there's 87 00:05:52,520 --> 00:05:54,000 nothing wrong here. 88 00:05:54,000 --> 00:05:58,880 Now within the virus, there is RNA. 89 00:05:58,880 --> 00:06:05,240 And it's single-stranded but negative strand RNA, 90 00:06:05,240 --> 00:06:07,650 so that's what puts it into-- 91 00:06:07,650 --> 00:06:08,150 Sorry. 92 00:06:08,150 --> 00:06:10,460 Positive strand. 93 00:06:10,460 --> 00:06:11,287 Positive. 94 00:06:13,910 --> 00:06:17,000 So what puts it into a separate category 95 00:06:17,000 --> 00:06:21,440 is the fact that the genetic material in the retroviruses 96 00:06:21,440 --> 00:06:25,130 is single-stranded RNA and it's the positive strand. 97 00:06:25,130 --> 00:06:27,740 So far we've talked about two other types 98 00:06:27,740 --> 00:06:29,390 of virus mechanisms-- 99 00:06:29,390 --> 00:06:31,340 the double-stranded DNA-- remember 100 00:06:31,340 --> 00:06:33,170 that was as an example. 101 00:06:33,170 --> 00:06:35,330 That was smallpox-- and we've also 102 00:06:35,330 --> 00:06:39,290 talked about the single-stranded RNA negative strand. 103 00:06:39,290 --> 00:06:43,550 And our best example there was the influenza virus, 104 00:06:43,550 --> 00:06:46,370 which is a segmented genome. 105 00:06:46,370 --> 00:06:49,820 What you'll learn about HIV is that it 106 00:06:49,820 --> 00:06:57,575 has a non-segmented genome. 107 00:07:00,320 --> 00:07:04,070 Meaning that this strand of RNA is one continuous 108 00:07:04,070 --> 00:07:06,950 strand rather than separate pieces 109 00:07:06,950 --> 00:07:09,110 as we saw in the influenza virus, which 110 00:07:09,110 --> 00:07:12,380 was the last example at the end of the last class. 111 00:07:12,380 --> 00:07:14,300 Now, let me just take a quick look. 112 00:07:14,300 --> 00:07:21,160 So every virion, that would be a single viral particle, 113 00:07:21,160 --> 00:07:25,870 therefore will contain within its structure the RNA. 114 00:07:28,880 --> 00:07:33,980 And it will also contain two copies of a particular enzyme 115 00:07:33,980 --> 00:07:35,930 that are absolutely critical. 116 00:07:35,930 --> 00:07:38,210 When the virus infects a cell, there's 117 00:07:38,210 --> 00:07:39,920 no time for it to make anything. 118 00:07:39,920 --> 00:07:42,650 It needs a particular enzyme to get 119 00:07:42,650 --> 00:07:47,960 going with replicating that initial strand of RNA 120 00:07:47,960 --> 00:07:52,340 to start to come to make a DNA, double-stranded DNA copy of it, 121 00:07:52,340 --> 00:07:54,630 and then make a messenger RNA. 122 00:07:54,630 --> 00:07:56,600 So the enzyme that's important and there 123 00:07:56,600 --> 00:08:05,190 are two copies of an enzyme known as reverse transcriptase. 124 00:08:13,160 --> 00:08:17,210 So basically, the virus structure is fairly simple. 125 00:08:17,210 --> 00:08:20,790 There's a capsid inside with the RNA 126 00:08:20,790 --> 00:08:24,050 plus the reverse transcriptase that I'm just showing 127 00:08:24,050 --> 00:08:26,240 as a filled circle here. 128 00:08:26,240 --> 00:08:32,240 And then surrounding the virion is a membrane bilayer 129 00:08:32,240 --> 00:08:36,943 with GP proteins stuck into that membrane bilayer that 130 00:08:36,943 --> 00:08:38,360 are going to be important when one 131 00:08:38,360 --> 00:08:42,590 starts to think about the infectivity by the virus. 132 00:08:42,590 --> 00:08:43,250 All right. 133 00:08:43,250 --> 00:08:48,720 There's a second glycoprotein that's also important, 134 00:08:48,720 --> 00:08:50,930 which is known as GP41. 135 00:08:50,930 --> 00:08:55,870 And these are very specifically part of this complex, 136 00:08:55,870 --> 00:08:58,990 but associated with the GP140. 137 00:08:58,990 --> 00:08:59,730 All right. 138 00:08:59,730 --> 00:09:02,930 So now let's take a look at the virus life-- 139 00:09:02,930 --> 00:09:06,660 or details of this virus. 140 00:09:06,660 --> 00:09:10,710 But first of all, I want you to take a look at T-cells. 141 00:09:10,710 --> 00:09:13,030 You've heard a lot about T-cells. 142 00:09:13,030 --> 00:09:16,210 They're a major component of the immune system. 143 00:09:16,210 --> 00:09:18,780 So I'm going to home you right in on what 144 00:09:18,780 --> 00:09:22,980 the virus, the virion, recognizes on T-cells 145 00:09:22,980 --> 00:09:24,810 and what makes it such a disaster 146 00:09:24,810 --> 00:09:26,680 for the human immune system. 147 00:09:26,680 --> 00:09:27,180 OK. 148 00:09:27,180 --> 00:09:30,570 So here are typical T-cell receptors 149 00:09:30,570 --> 00:09:32,670 on the surface of T-cells. 150 00:09:32,670 --> 00:09:36,200 Just as a recap, I want you to remember T-cells produce 151 00:09:36,200 --> 00:09:40,560 unique antigen binding proteins that are 152 00:09:40,560 --> 00:09:43,740 put onto the T-cell surface. 153 00:09:43,740 --> 00:09:46,770 And they provide cell-mediated immunity 154 00:09:46,770 --> 00:09:50,700 by destroying antigens such as viruses. 155 00:09:50,700 --> 00:09:53,790 But what the HIV virus does is it really 156 00:09:53,790 --> 00:09:58,320 homes in on the cell-surface receptors of the T-cells, 157 00:09:58,320 --> 00:10:05,890 specifically the HIV virus recognizes the CD4 receptor, 158 00:10:05,890 --> 00:10:09,600 which is a glycoprotein on the surface of some T-cells 159 00:10:09,600 --> 00:10:13,720 that are designated as CD4 positive T-cells. 160 00:10:13,720 --> 00:10:17,070 So if you have circulating T-cells, 161 00:10:17,070 --> 00:10:22,830 that would be the first place where the HIV virus attaches 162 00:10:22,830 --> 00:10:26,130 to the T-cell to cause infectivity, 163 00:10:26,130 --> 00:10:30,060 thus debilitating the ability to mount 164 00:10:30,060 --> 00:10:33,720 the type of immune responses that T-cells are involved in. 165 00:10:33,720 --> 00:10:36,420 So it's very important to now understand 166 00:10:36,420 --> 00:10:41,910 why the physiology of this virus is to knock out 167 00:10:41,910 --> 00:10:44,280 the thing that's there to protect us 168 00:10:44,280 --> 00:10:48,700 from foreign antigens, which makes it so serious. 169 00:10:48,700 --> 00:10:54,540 Now, HIV first started to emerge in the early '80s. 170 00:10:54,540 --> 00:10:57,660 It was more or less designated as a death sentence. 171 00:10:57,660 --> 00:11:00,630 As you may be aware, HIV circulated 172 00:11:00,630 --> 00:11:04,260 around the gay community in the San Francisco area 173 00:11:04,260 --> 00:11:05,590 very rapidly. 174 00:11:05,590 --> 00:11:09,880 But it then became incredibly widespread. 175 00:11:09,880 --> 00:11:11,700 There's a possibility that the jump 176 00:11:11,700 --> 00:11:17,670 of the virus from primates to humans, the virus 177 00:11:17,670 --> 00:11:19,920 might have originated in Africa. 178 00:11:19,920 --> 00:11:23,340 There's a lot of work done on the origin of this virus, 179 00:11:23,340 --> 00:11:25,580 but it sort of came out of nowhere. 180 00:11:25,580 --> 00:11:29,100 It was something that was unexpected, unanticipated, 181 00:11:29,100 --> 00:11:32,790 and hitting people just like a cannonball. 182 00:11:32,790 --> 00:11:36,690 And it was literally a death sentence early on. 183 00:11:36,690 --> 00:11:40,710 As the mechanism of the virus became understood, 184 00:11:40,710 --> 00:11:44,010 a number of strategies could be put in place with respect 185 00:11:44,010 --> 00:11:48,810 to antiviral therapeutics to target this particular virus. 186 00:11:48,810 --> 00:11:50,910 And what we will see is the four types 187 00:11:50,910 --> 00:11:55,050 of targets that have been named as targets 188 00:11:55,050 --> 00:11:58,540 for therapeutic agents, why they work, how they work. 189 00:11:58,540 --> 00:12:01,170 And we'll see the mechanisms of those as to how 190 00:12:01,170 --> 00:12:04,680 they pertain to the HIV virus. 191 00:12:04,680 --> 00:12:07,620 And we'll talk about things like combination therapies. 192 00:12:07,620 --> 00:12:10,770 So early on, there was really nothing. 193 00:12:10,770 --> 00:12:15,310 Gradually early, therapeutic approaches started to emerge, 194 00:12:15,310 --> 00:12:18,300 but they were devastating treatments 195 00:12:18,300 --> 00:12:22,380 because the drugs were so poor against the HIV virus, 196 00:12:22,380 --> 00:12:26,460 people were taking massive handfuls of medications a day 197 00:12:26,460 --> 00:12:28,860 to try to stay ahead of the virus. 198 00:12:28,860 --> 00:12:32,370 As time has progressed, these therapeutic agents 199 00:12:32,370 --> 00:12:34,410 have been improved and improved. 200 00:12:34,410 --> 00:12:38,370 And now they're relatively simple therapeutic agents 201 00:12:38,370 --> 00:12:41,460 where you don't have to take massive doses of fairly 202 00:12:41,460 --> 00:12:44,040 non-specific therapeutic agents. 203 00:12:44,040 --> 00:12:48,210 But they're quite sort of workable. 204 00:12:48,210 --> 00:12:50,880 The problem is, and you'll see why, 205 00:12:50,880 --> 00:12:54,300 people have to take these therapeutic agents for life. 206 00:12:54,300 --> 00:12:56,970 Once cells are infected with HIV, 207 00:12:56,970 --> 00:12:59,640 there will always be a population 208 00:12:59,640 --> 00:13:03,760 of the virus genetic material in the human genome. 209 00:13:03,760 --> 00:13:06,330 So you can't just say, I feel way better, 210 00:13:06,330 --> 00:13:09,930 not taking these drugs anymore, because there is the chance 211 00:13:09,930 --> 00:13:13,170 that in some repository in some of your cells 212 00:13:13,170 --> 00:13:15,330 the virus genetic material is still there. 213 00:13:15,330 --> 00:13:19,620 And you'll see which enzymes in HIV are responsible for that. 214 00:13:19,620 --> 00:13:20,790 OK? 215 00:13:20,790 --> 00:13:24,930 So let's take a look here at the Group VI 216 00:13:24,930 --> 00:13:29,160 retrovirus named HIV/AIDS. 217 00:13:29,160 --> 00:13:36,120 In 2015, there were 37 million people infected. 218 00:13:36,120 --> 00:13:38,580 Nowhere near enough in treatment, about half 219 00:13:38,580 --> 00:13:39,900 of those in treatment. 220 00:13:39,900 --> 00:13:41,760 And about 1.2 million deaths. 221 00:13:41,760 --> 00:13:46,260 This may account also for people coinfected with TB. 222 00:13:46,260 --> 00:13:48,390 I mentioned to you when we started 223 00:13:48,390 --> 00:13:50,820 talking about infectious disease, 224 00:13:50,820 --> 00:13:54,750 I really encourage you to go to the NIAID website. 225 00:13:54,750 --> 00:13:58,110 There is tons of really interesting information, 226 00:13:58,110 --> 00:14:00,330 not just from a clinical perspective 227 00:14:00,330 --> 00:14:02,430 but from a mechanistic perspective, 228 00:14:02,430 --> 00:14:03,670 from a treatment perspective. 229 00:14:03,670 --> 00:14:05,370 There's really important things. 230 00:14:05,370 --> 00:14:07,230 So I really encourage you if you are 231 00:14:07,230 --> 00:14:10,980 interested in infectious disease, the National 232 00:14:10,980 --> 00:14:17,400 Institute of Allergies and Infectious Diseases Component 233 00:14:17,400 --> 00:14:20,490 of the NIH is the one responsible for all 234 00:14:20,490 --> 00:14:23,860 the research on infectious disease. 235 00:14:23,860 --> 00:14:26,250 And in fact, for many, many years 236 00:14:26,250 --> 00:14:28,620 during the crisis with HIV, there 237 00:14:28,620 --> 00:14:32,970 were special earmarked funds for any research 238 00:14:32,970 --> 00:14:35,880 towards the development of therapeutic agents. 239 00:14:35,880 --> 00:14:37,260 So what happens? 240 00:14:37,260 --> 00:14:39,210 How does this virus, this little particle 241 00:14:39,210 --> 00:14:41,310 that I've shown you here-- 242 00:14:41,310 --> 00:14:44,810 I should really show you this is the capsid. 243 00:14:44,810 --> 00:14:48,000 And I should really show you the membrane a little bit more 244 00:14:48,000 --> 00:14:50,490 encircling that with the capsid inside. 245 00:14:50,490 --> 00:14:55,020 So the cartoon that you see on the screen is more correct. 246 00:14:55,020 --> 00:14:57,450 So here's the virion particle. 247 00:14:57,450 --> 00:15:01,470 It has inside it the capsid that includes 248 00:15:01,470 --> 00:15:03,690 the single-stranded RNA. 249 00:15:03,690 --> 00:15:06,730 It only needs to code for very few proteins, 250 00:15:06,730 --> 00:15:09,720 remember, because the virus just exploits everything 251 00:15:09,720 --> 00:15:11,550 else from the human host. 252 00:15:11,550 --> 00:15:22,090 And GP120-- sorry for that typo, please correct that. 253 00:15:22,090 --> 00:15:26,980 And GP120 is displayed on the surface of the virion. 254 00:15:26,980 --> 00:15:30,310 And in fact, it's GP120 that interacts 255 00:15:30,310 --> 00:15:34,270 with the CD4 receptor on the surface of a target host 256 00:15:34,270 --> 00:15:36,160 cell, a T-cell. 257 00:15:36,160 --> 00:15:39,860 Now there is a second receptor that's quite important. 258 00:15:39,860 --> 00:15:43,640 It was not discovered until quite a bit later on. 259 00:15:43,640 --> 00:15:46,330 It's designated as the coreceptor. 260 00:15:46,330 --> 00:15:50,650 And the interaction of the virion with the host cell 261 00:15:50,650 --> 00:15:54,340 is only good when there's both the CD4 receptor 262 00:15:54,340 --> 00:15:56,230 plus the coreceptor. 263 00:15:56,230 --> 00:15:58,900 And this was found through careful biology that 264 00:15:58,900 --> 00:16:01,750 realized there was another critical component 265 00:16:01,750 --> 00:16:03,530 for the infectious disease. 266 00:16:03,530 --> 00:16:08,200 And that is designated as the CCR5 receptor or CXCR4 267 00:16:08,200 --> 00:16:09,070 receptor. 268 00:16:09,070 --> 00:16:13,330 So both receptors on the surface, the virus 269 00:16:13,330 --> 00:16:16,600 has evolved or selected out, to recognize 270 00:16:16,600 --> 00:16:19,300 cells that have both of those types of receptors 271 00:16:19,300 --> 00:16:23,380 on the surface which ends up making them very targeted 272 00:16:23,380 --> 00:16:25,150 towards the T-cells. 273 00:16:25,150 --> 00:16:28,840 Once that association occurs, then 274 00:16:28,840 --> 00:16:32,260 becomes the process of fusion. 275 00:16:32,260 --> 00:16:36,970 So remember, I told you that large things can get into cells 276 00:16:36,970 --> 00:16:41,440 by merging a membrane with the membrane 277 00:16:41,440 --> 00:16:43,280 on the surface of the cell. 278 00:16:43,280 --> 00:16:46,990 So what is happening as those two receptors are engaged 279 00:16:46,990 --> 00:16:53,110 is the virus membrane is coming close to the host cell membrane 280 00:16:53,110 --> 00:16:58,180 and starting to fuse through the action of GP120 281 00:16:58,180 --> 00:17:02,117 along with the CD4 receptor and coreceptor. 282 00:17:02,117 --> 00:17:03,700 And I'm going to show you a movie that 283 00:17:03,700 --> 00:17:06,490 shows that part of the fusion, because it's really 284 00:17:06,490 --> 00:17:08,619 cool to see it in action. 285 00:17:08,619 --> 00:17:13,119 Once that fusion occurs, the virus membrane 286 00:17:13,119 --> 00:17:16,750 actually just merges with the remainder of the host cell 287 00:17:16,750 --> 00:17:17,589 membrane. 288 00:17:17,589 --> 00:17:21,190 And you drop that capsid into the cell, which then becomes 289 00:17:21,190 --> 00:17:25,690 on packaged to release the single-stranded RNA that's 290 00:17:25,690 --> 00:17:27,310 the contents of that. 291 00:17:27,310 --> 00:17:31,910 And what's important to know-- whoops going backwards-- 292 00:17:31,910 --> 00:17:35,570 is that the minute the single-stranded RNA 293 00:17:35,570 --> 00:17:43,570 gets into the cell, that needs to be processed. 294 00:17:43,570 --> 00:17:47,080 But it needs to be processed with an enzyme that is 295 00:17:47,080 --> 00:17:49,550 completely unique to the virus. 296 00:17:49,550 --> 00:17:52,480 And that's the reverse transcriptase. 297 00:17:52,480 --> 00:17:55,020 Without the reverse transcriptase being there, 298 00:17:55,020 --> 00:17:59,420 it's just RNA that's just going to get chewed up in the cell. 299 00:17:59,420 --> 00:18:04,840 So what reverse transcriptase does it reverse transcribes RNA 300 00:18:04,840 --> 00:18:06,550 into DNA. 301 00:18:06,550 --> 00:18:10,360 So that gets you on the track to the infectious process. 302 00:18:10,360 --> 00:18:13,150 In the absence of RT, you don't get there. 303 00:18:13,150 --> 00:18:17,260 You're not able to make a DNA copy to then make 304 00:18:17,260 --> 00:18:19,240 a copy of the RNA and so on. 305 00:18:19,240 --> 00:18:21,460 So reverse transcriptase, that is 306 00:18:21,460 --> 00:18:26,200 why the reverse transcriptase has to be delivered along 307 00:18:26,200 --> 00:18:27,110 with the virus. 308 00:18:27,110 --> 00:18:30,020 It can't be just a virus with the genetic material. 309 00:18:30,020 --> 00:18:32,650 It's got to have this particular enzyme with it. 310 00:18:32,650 --> 00:18:38,650 So reverse transcriptase is responsible for converting RNA 311 00:18:38,650 --> 00:18:40,060 into DNA. 312 00:18:40,060 --> 00:18:44,740 And as you all know, that is not the usual direction 313 00:18:44,740 --> 00:18:46,480 with respect to the central dogma. 314 00:18:46,480 --> 00:18:49,330 We're always talking about DNA to RNA. 315 00:18:49,330 --> 00:18:51,790 But you've also heard throughout the course, 316 00:18:51,790 --> 00:18:56,440 how handy this reverse transcriptase is material is. 317 00:18:56,440 --> 00:19:00,550 When we talk about arrays and loads of biotechnology, 318 00:19:00,550 --> 00:19:03,540 using reverse transcriptase is super valuable. 319 00:19:03,540 --> 00:19:07,180 Let's say, for example, we don't want to sequence a genome, 320 00:19:07,180 --> 00:19:10,060 we want to look at the transcriptome, the part 321 00:19:10,060 --> 00:19:13,030 of the genome that's really important for being translated 322 00:19:13,030 --> 00:19:14,440 into proteins. 323 00:19:14,440 --> 00:19:17,380 What we do is we collect the transcriptome 324 00:19:17,380 --> 00:19:20,260 but reverse transcribe it into DNA, 325 00:19:20,260 --> 00:19:23,860 which is a much more stable, tractable material 326 00:19:23,860 --> 00:19:26,330 for sequencing and so on and so forth. 327 00:19:26,330 --> 00:19:29,950 So RT has become kind of a gift to biotechnology 328 00:19:29,950 --> 00:19:31,570 and biological research. 329 00:19:31,570 --> 00:19:35,860 And it was inspired from the RT in the HIV virus. 330 00:19:35,860 --> 00:19:37,820 So that's very important. 331 00:19:37,820 --> 00:19:40,180 So the reverse transcriptase, then, 332 00:19:40,180 --> 00:19:44,350 makes a DNA copy of the RNA. 333 00:19:44,350 --> 00:19:47,320 And then a second complementary copy 334 00:19:47,320 --> 00:19:51,760 of that DNA is made to make double-stranded DNA which 335 00:19:51,760 --> 00:19:54,940 represents the viral genome, but in the format 336 00:19:54,940 --> 00:19:57,070 of a double-stranded DNA. 337 00:19:57,070 --> 00:20:01,600 That DNA can be, then, taken-- 338 00:20:01,600 --> 00:20:03,160 here's the fun part. 339 00:20:03,160 --> 00:20:07,660 That DNA is actually, then, taken into the nucleus 340 00:20:07,660 --> 00:20:11,710 and it's zippered in to the host genome 341 00:20:11,710 --> 00:20:14,290 by an enzyme known as integrase. 342 00:20:14,290 --> 00:20:17,110 So it's not that you have this virus junk 343 00:20:17,110 --> 00:20:19,300 and then you send it back out again 344 00:20:19,300 --> 00:20:21,670 with the mature particles, you actually 345 00:20:21,670 --> 00:20:27,580 put this in the permanent copy of your DNA in the nucleus. 346 00:20:27,580 --> 00:20:33,820 And that is why people don't get cured from HIV/AIDS. 347 00:20:33,820 --> 00:20:36,400 They have to take treatment for the rest of their life, 348 00:20:36,400 --> 00:20:40,630 because that DNA is permanently in the copy of your genome 349 00:20:40,630 --> 00:20:44,290 that gets replicated every time your cells divide and so on. 350 00:20:44,290 --> 00:20:47,920 So you can understand the seriousness of that event. 351 00:20:47,920 --> 00:20:52,120 So that gets taken into the genome. 352 00:20:52,120 --> 00:20:56,080 But also when it's time to make new virions, 353 00:20:56,080 --> 00:21:00,520 the DNA is transcribed into messenger RNA. 354 00:21:00,520 --> 00:21:03,820 The messenger RNA leaves the nucleus, 355 00:21:03,820 --> 00:21:06,790 gets translated into proteins. 356 00:21:06,790 --> 00:21:09,070 And those proteins form the basis 357 00:21:09,070 --> 00:21:11,290 of the new virion particles. 358 00:21:11,290 --> 00:21:14,200 Now, I mentioned somewhere here that it 359 00:21:14,200 --> 00:21:16,780 is a non-segmented genome. 360 00:21:16,780 --> 00:21:24,840 So initially, the messenger RNA is a single segment 361 00:21:24,840 --> 00:21:30,640 that then gets translated into a single segment of protein, 362 00:21:30,640 --> 00:21:32,300 which is known as a polyprotein. 363 00:21:35,170 --> 00:21:40,060 So there are no stops between the various genes encoding 364 00:21:40,060 --> 00:21:42,740 frames within the messenger RNA. 365 00:21:42,740 --> 00:21:46,810 So that gets transcribed by the host machinery 366 00:21:46,810 --> 00:21:51,670 straight into a polyprotein that's all glued together 367 00:21:51,670 --> 00:21:53,660 in a single long piece. 368 00:21:53,660 --> 00:21:59,990 But then there is another important HIV enzyme, 369 00:21:59,990 --> 00:22:06,760 the protease, that chops those portions of the polyprotein 370 00:22:06,760 --> 00:22:10,300 up into useful and usable portions. 371 00:22:10,300 --> 00:22:14,020 So you also need the protease readily available 372 00:22:14,020 --> 00:22:18,910 rapidly to start dicing up the polyprotein into usable protein 373 00:22:18,910 --> 00:22:21,550 segments for the new virus assembly. 374 00:22:21,550 --> 00:22:24,970 So let's take a look here what happens. 375 00:22:24,970 --> 00:22:26,545 The messenger leaves the nucleus. 376 00:22:30,040 --> 00:22:33,970 You make a polyprotein that gets digested into protein segments. 377 00:22:33,970 --> 00:22:37,870 And then everything accumulates at the inside surface 378 00:22:37,870 --> 00:22:42,340 of the membrane and starts budding off the virion. 379 00:22:42,340 --> 00:22:45,560 When it's first budded off, it's not really ready to go. 380 00:22:45,560 --> 00:22:46,630 It's not intact. 381 00:22:46,630 --> 00:22:49,150 It has to develop a little bit further 382 00:22:49,150 --> 00:22:52,210 to become a mature virion moving forward 383 00:22:52,210 --> 00:22:54,340 to go infect another cell. 384 00:22:54,340 --> 00:22:59,530 So the initial stage, that budded stage, of the virion 385 00:22:59,530 --> 00:23:02,290 is not completely competent to go infect. 386 00:23:02,290 --> 00:23:04,810 And in fact, there has been some thoughts 387 00:23:04,810 --> 00:23:10,180 about can you ensure that the immature virion doesn't develop 388 00:23:10,180 --> 00:23:12,760 into the mature virion, because that would be one 389 00:23:12,760 --> 00:23:14,480 way of targeting the system. 390 00:23:14,480 --> 00:23:14,980 OK. 391 00:23:14,980 --> 00:23:18,010 So there are a few different pieces-- 392 00:23:18,010 --> 00:23:28,830 the reverse transcriptase, the integrase, the protease, 393 00:23:28,830 --> 00:23:29,625 and then, finally-- 394 00:23:32,610 --> 00:23:35,160 these are all critical processes that 395 00:23:35,160 --> 00:23:38,290 have been the targets of therapeutic agents. 396 00:23:38,290 --> 00:23:40,950 They are molecular targets, such as enzymes. 397 00:23:40,950 --> 00:23:47,010 And we know how to address enzymes with inhibitors. 398 00:23:47,010 --> 00:23:51,240 And one last feature of the virus life cycle 399 00:23:51,240 --> 00:23:55,500 that has been focused on respect to therapeutics 400 00:23:55,500 --> 00:23:56,565 is the viral fusion. 401 00:24:01,870 --> 00:24:06,090 So there have also been efforts at a molecular level 402 00:24:06,090 --> 00:24:12,810 to try to inhibit this process where your virus particle docks 403 00:24:12,810 --> 00:24:17,010 down on the cell and somehow the membranes fuse 404 00:24:17,010 --> 00:24:20,460 and then the virus dumps its contents into the cell. 405 00:24:20,460 --> 00:24:21,240 OK. 406 00:24:21,240 --> 00:24:26,130 This is just sort of a quick sort of more beautiful view. 407 00:24:26,130 --> 00:24:28,770 But in a minute, I'm going to show you a video. 408 00:24:28,770 --> 00:24:31,080 But I thought it was kind of nice because it really 409 00:24:31,080 --> 00:24:34,570 allows you to look at the different steps in the virus 410 00:24:34,570 --> 00:24:36,660 life cycle-- 411 00:24:36,660 --> 00:24:42,360 recognition, fusion, delivery, making the double-stranded DNA, 412 00:24:42,360 --> 00:24:45,120 integrated the DNA into the genome, 413 00:24:45,120 --> 00:24:46,920 and making new viral proteins. 414 00:24:46,920 --> 00:24:49,170 So those steps, they're pretty logical. 415 00:24:49,170 --> 00:24:52,320 It's just like step one, got to find my target. 416 00:24:52,320 --> 00:24:54,420 Step two, I've got to stick to my target. 417 00:24:54,420 --> 00:24:57,990 Step three, I've got to deliver my genomic content 418 00:24:57,990 --> 00:25:00,030 into my target and so on. 419 00:25:00,030 --> 00:25:02,040 So I don't want you to think this is 420 00:25:02,040 --> 00:25:03,570 a bunch of steps to memorize. 421 00:25:03,570 --> 00:25:07,170 They all sort of make sense in a logical progression of events 422 00:25:07,170 --> 00:25:11,250 to make the virus be able to go all the way through to budding 423 00:25:11,250 --> 00:25:15,780 off new, immature, and then mature virions. 424 00:25:15,780 --> 00:25:17,710 And so as I mentioned over there, 425 00:25:17,710 --> 00:25:19,230 these are the four targets that I'm 426 00:25:19,230 --> 00:25:20,970 going to describe to you with respect 427 00:25:20,970 --> 00:25:23,040 to therapeutic development. 428 00:25:23,040 --> 00:25:24,780 And this is where they all work-- 429 00:25:24,780 --> 00:25:28,980 the fusion early on, the reverse transcriptase 430 00:25:28,980 --> 00:25:34,830 taking that single-stranded RNA to a DNA copy, 431 00:25:34,830 --> 00:25:38,340 the integrase which zips the genomic material 432 00:25:38,340 --> 00:25:42,480 into the genome, and the protease that cleaves up 433 00:25:42,480 --> 00:25:47,400 the long polyprotein to make mature pieces of protein that 434 00:25:47,400 --> 00:25:48,375 are part of the virus. 435 00:25:52,900 --> 00:25:53,590 OK. 436 00:25:53,590 --> 00:25:56,230 So let's talk first of all about viral fusion. 437 00:25:56,230 --> 00:25:58,660 And as I mentioned, when I get to showing you-- 438 00:25:58,660 --> 00:26:03,550 it's a really beautiful video from the Howard Hughes 439 00:26:03,550 --> 00:26:06,730 Institute that really shows the fusion very well. 440 00:26:06,730 --> 00:26:09,580 But I want to describe the fusion to you 441 00:26:09,580 --> 00:26:11,080 at a molecular level. 442 00:26:11,080 --> 00:26:15,860 And then later, you'll see it a little bit more real life. 443 00:26:15,860 --> 00:26:20,860 So what I said to you is that GP120 and GP41, which 444 00:26:20,860 --> 00:26:25,870 are these proteins that are in the surface of the envelope 445 00:26:25,870 --> 00:26:32,560 then recognize the coreceptor and CD41 and form a complex. 446 00:26:32,560 --> 00:26:35,840 So it's a complex that involves what's on the virus. 447 00:26:35,840 --> 00:26:38,080 It's actually in a trimeric structure 448 00:26:38,080 --> 00:26:41,050 with the two receptors that are on the host cell. 449 00:26:41,050 --> 00:26:43,690 And they form a pretty close union. 450 00:26:43,690 --> 00:26:45,370 And then what happens is there are 451 00:26:45,370 --> 00:26:49,900 a series of events that tug the two membranes together, 452 00:26:49,900 --> 00:26:53,380 mechanically linked to conformational changes 453 00:26:53,380 --> 00:26:57,520 of the protein, and basically splosh the two membranes 454 00:26:57,520 --> 00:26:59,620 together for a fusion event. 455 00:26:59,620 --> 00:27:03,430 So it's through the confirmational dynamics 456 00:27:03,430 --> 00:27:06,460 of that large complex happening in sequence 457 00:27:06,460 --> 00:27:10,930 after the first interactions that get you to a fusion event. 458 00:27:10,930 --> 00:27:13,600 So these initially look like this, 459 00:27:13,600 --> 00:27:16,240 but then they start changing their shape, 460 00:27:16,240 --> 00:27:18,130 and making a fusion event. 461 00:27:18,130 --> 00:27:20,590 So you could look at it as, initially, you 462 00:27:20,590 --> 00:27:23,920 have the complex formed, but then, later on, there's 463 00:27:23,920 --> 00:27:25,780 been a twisting of the structures, 464 00:27:25,780 --> 00:27:27,580 the large macromolecular structures 465 00:27:27,580 --> 00:27:29,420 to fuse the membranes. 466 00:27:29,420 --> 00:27:31,720 So for quite a few years-- 467 00:27:31,720 --> 00:27:34,360 and I don't think it was the most successful 468 00:27:34,360 --> 00:27:37,210 of the approaches with respect to therapeutics-- 469 00:27:37,210 --> 00:27:41,440 people thought that maybe you could inhibit the interaction 470 00:27:41,440 --> 00:27:44,320 between GP120 and those receptors 471 00:27:44,320 --> 00:27:47,800 with small molecules that might inhibit the progress 472 00:27:47,800 --> 00:27:49,930 through the fusion process. 473 00:27:49,930 --> 00:27:52,780 So that was deemed a viable target. 474 00:27:52,780 --> 00:27:55,300 It's not the same as an enzyme target, 475 00:27:55,300 --> 00:27:59,110 but it's definitely a critical point in viral infections. 476 00:27:59,110 --> 00:28:01,930 And what was made were short peptides 477 00:28:01,930 --> 00:28:04,690 that might stabilize this complex 478 00:28:04,690 --> 00:28:09,040 and avoid the structure moving forward 479 00:28:09,040 --> 00:28:11,590 to the fusion-related complex. 480 00:28:11,590 --> 00:28:14,230 So that was one sort of series of events. 481 00:28:14,230 --> 00:28:18,070 So those peptides were designated a C-peptide. 482 00:28:18,070 --> 00:28:20,740 They would bind to this portion up here 483 00:28:20,740 --> 00:28:23,140 that's pointed to with the N. They'd stick 484 00:28:23,140 --> 00:28:25,540 and they'd basically jam the cogs. 485 00:28:25,540 --> 00:28:29,770 They'd stopped that event occurring by binding 486 00:28:29,770 --> 00:28:32,485 quite tightly to part of the fusion complex. 487 00:28:32,485 --> 00:28:34,750 OK? 488 00:28:34,750 --> 00:28:38,230 The drugs, the therapeutic agents, in that case 489 00:28:38,230 --> 00:28:40,900 were peptidic, kind of large. 490 00:28:40,900 --> 00:28:44,110 They don't diffuse into cells, but that's not a problem 491 00:28:44,110 --> 00:28:46,970 because this target is outside the cell. 492 00:28:46,970 --> 00:28:50,470 So there's no need for that to get into cells for its effect. 493 00:28:50,470 --> 00:28:53,810 All of the other therapeutic agents I'll describe to you 494 00:28:53,810 --> 00:28:56,740 are molecules that need to get into cells in order 495 00:28:56,740 --> 00:28:58,610 to do their job. 496 00:28:58,610 --> 00:29:01,960 But this particular strategy didn't involve that. 497 00:29:01,960 --> 00:29:05,350 Which was why it was fairly attractive early on, 498 00:29:05,350 --> 00:29:08,620 because it was a very different target from the others. 499 00:29:08,620 --> 00:29:11,980 So the next step I want to describe to you 500 00:29:11,980 --> 00:29:17,200 is inhibition of reserve transcriptase. 501 00:29:17,200 --> 00:29:29,690 So this is a RNA-dependent DNA polymerase. 502 00:29:29,690 --> 00:29:30,190 OK? 503 00:29:30,190 --> 00:29:33,280 So different from the other DNA polymerases. 504 00:29:33,280 --> 00:29:35,800 We have DNA-dependent DNA polymerases, 505 00:29:35,800 --> 00:29:39,190 but we don't have the RNA-dependent ones. 506 00:29:39,190 --> 00:29:42,970 And so the types of strategies that were used initially 507 00:29:42,970 --> 00:29:47,380 was to use types of analogs of the nucleosides 508 00:29:47,380 --> 00:29:50,500 that will be used in polymerization. 509 00:29:50,500 --> 00:29:56,660 So the reverse transcriptase, the first types of targets-- 510 00:29:56,660 --> 00:30:00,490 what's going on down there-- the first types of targets 511 00:30:00,490 --> 00:30:03,920 were nucleoside analogs because they 512 00:30:03,920 --> 00:30:06,040 can inhibit the polymerization. 513 00:30:06,040 --> 00:30:08,540 And you're going to realize you know quite a bit about these 514 00:30:08,540 --> 00:30:10,323 when we first look at the structure. 515 00:30:10,323 --> 00:30:11,990 So I'm going to describe to you what are 516 00:30:11,990 --> 00:30:16,130 known as nucleoside analogs. 517 00:30:20,570 --> 00:30:22,730 What does this term mean? 518 00:30:22,730 --> 00:30:27,200 Nucleoside means it's a nuclear base plus a sugar. 519 00:30:27,200 --> 00:30:28,430 There's no phosphates. 520 00:30:28,430 --> 00:30:30,970 Because, remember, there's an s there. 521 00:30:30,970 --> 00:30:34,670 And it's an analog meaning it's not one of the native ones, 522 00:30:34,670 --> 00:30:36,655 but it-- 523 00:30:36,655 --> 00:30:38,166 that looks good. 524 00:30:38,166 --> 00:30:43,753 AUDIENCE: I'm looking for [INAUDIBLE] 525 00:30:43,753 --> 00:30:44,670 BARBARA IMPERIALI: OK. 526 00:30:44,670 --> 00:30:45,360 I'll grab her. 527 00:30:45,360 --> 00:30:46,320 And you get the bag. 528 00:30:46,320 --> 00:30:49,590 [LAUGHTER] 529 00:30:49,590 --> 00:30:50,090 OK. 530 00:30:50,090 --> 00:30:52,350 So it's a nucleoside analog meaning 531 00:30:52,350 --> 00:30:55,560 it isn't the standard nucleosides 532 00:30:55,560 --> 00:30:57,540 that you put into your DNA. 533 00:30:57,540 --> 00:31:02,970 And the most critical first line actually became azidothymidine. 534 00:31:02,970 --> 00:31:07,350 So look, that's the thymide nuclear base, hits a sugar, 535 00:31:07,350 --> 00:31:10,290 but it's different from the regular ribose that's 536 00:31:10,290 --> 00:31:13,350 either in RNA or DNA. 537 00:31:13,350 --> 00:31:17,370 And it's two deoxys so it looks more like a DNA building block. 538 00:31:17,370 --> 00:31:18,270 But check it out. 539 00:31:18,270 --> 00:31:21,683 On the three prime site, there is azide. 540 00:31:21,683 --> 00:31:23,100 What do you think this thing does? 541 00:31:23,100 --> 00:31:24,310 Or have I already told you? 542 00:31:24,310 --> 00:31:24,810 Yeah. 543 00:31:24,810 --> 00:31:26,185 What do you think this thing does 544 00:31:26,185 --> 00:31:29,850 to stop the virus marching forward and turning 545 00:31:29,850 --> 00:31:31,860 its RNA into a DNA copy? 546 00:31:34,390 --> 00:31:37,440 Can it polymerase? 547 00:31:37,440 --> 00:31:38,050 No. 548 00:31:38,050 --> 00:31:39,160 So what do you think? 549 00:31:39,160 --> 00:31:40,520 So what how does it work? 550 00:31:40,520 --> 00:31:41,890 AUDIENCE: It blocks [INAUDIBLE] 551 00:31:41,890 --> 00:31:43,100 BARBARA IMPERIALI: Yeah. 552 00:31:43,100 --> 00:31:43,600 Yeah. 553 00:31:43,600 --> 00:31:46,960 It just grinds to a halt. What technology 554 00:31:46,960 --> 00:31:50,420 does this remind you of? 555 00:31:50,420 --> 00:31:50,995 Yeah? 556 00:31:50,995 --> 00:31:55,285 AUDIENCE: The sequencing with the [INAUDIBLE].. 557 00:31:55,285 --> 00:31:56,910 BARBARA IMPERIALI: So it's really cool. 558 00:31:56,910 --> 00:31:59,240 I mean, we're using something like this 559 00:31:59,240 --> 00:32:02,630 for sequencing blocking the chain growth to get pieces 560 00:32:02,630 --> 00:32:04,670 so we can tell the sequence, but this 561 00:32:04,670 --> 00:32:08,820 is a very viable drug for reverse transcriptase for kind 562 00:32:08,820 --> 00:32:13,310 of similar reasons, because it stops translation. 563 00:32:13,310 --> 00:32:15,765 It stops polymerization. 564 00:32:15,765 --> 00:32:17,390 Now, there's one important thing that I 565 00:32:17,390 --> 00:32:24,530 need to tell you about this AZT, as it's termed, azidothymidine. 566 00:32:24,530 --> 00:32:33,845 AZT is what is known in the industry as a prodrug. 567 00:32:40,350 --> 00:32:41,790 It's not the actual drug. 568 00:32:41,790 --> 00:32:42,870 Why is it a prodrug? 569 00:32:42,870 --> 00:32:45,760 What has to happen to it to be useful? 570 00:32:45,760 --> 00:32:47,820 Why is it handy to have a prodrug? 571 00:32:47,820 --> 00:32:49,630 I know there's a lot of questions there, 572 00:32:49,630 --> 00:32:51,460 but they all kind of tie together. 573 00:32:51,460 --> 00:32:53,340 So it's a prodrug. 574 00:32:53,340 --> 00:32:55,800 It can get into cells, right? 575 00:32:55,800 --> 00:33:00,120 What has to happen to it for it to be useful in inhibiting 576 00:33:00,120 --> 00:33:03,130 reverse transcriptase? 577 00:33:03,130 --> 00:33:04,720 Because it won't do it all on its own. 578 00:33:04,720 --> 00:33:05,860 Look at that structure. 579 00:33:05,860 --> 00:33:09,760 There's something missing in that structure. 580 00:33:09,760 --> 00:33:15,120 What are the building blocks in the polymerases? 581 00:33:15,120 --> 00:33:18,050 Are they nucleosides? 582 00:33:18,050 --> 00:33:19,830 No. 583 00:33:19,830 --> 00:33:21,070 Up there. 584 00:33:21,070 --> 00:33:23,220 AUDIENCE: [INAUDIBLE] 585 00:33:23,220 --> 00:33:24,870 BARBARA IMPERIALI: Yes, exactly. 586 00:33:24,870 --> 00:33:27,780 So you have to convert this neutral molecule that 587 00:33:27,780 --> 00:33:32,760 can sneak into a cell by kinases that put phosphate groups 588 00:33:32,760 --> 00:33:34,740 onto that molecule. 589 00:33:34,740 --> 00:33:38,670 So that that molecule can get engaged in polymerization, 590 00:33:38,670 --> 00:33:42,060 you can put one nucleotide onto a growing strand, 591 00:33:42,060 --> 00:33:45,750 but then it can't go any further because of that azido group 592 00:33:45,750 --> 00:33:49,020 at 3, which would be the normal place you grow. 593 00:33:49,020 --> 00:33:52,480 So prodrug strategies are very useful. 594 00:33:52,480 --> 00:33:55,620 It's a way of having drugs that aren't quite drugs yet 595 00:33:55,620 --> 00:33:59,430 but they become drugs once they are at their location. 596 00:33:59,430 --> 00:34:04,620 It's not handy to use the triphosphate of AZT as a drug, 597 00:34:04,620 --> 00:34:06,400 because it can't get in the cell. 598 00:34:06,400 --> 00:34:09,900 So we deliver a cellulary available molecule. 599 00:34:09,900 --> 00:34:12,929 We let the promiscuous enzymes in the cell 600 00:34:12,929 --> 00:34:15,000 convert it to a triphosphate. 601 00:34:15,000 --> 00:34:17,980 And then it becomes a real drug, a mature drug. 602 00:34:17,980 --> 00:34:19,800 So I think that's a very important thing 603 00:34:19,800 --> 00:34:23,870 about these types of therapeutic agents. 604 00:34:23,870 --> 00:34:27,150 And what I want you to know is that many, many viruses 605 00:34:27,150 --> 00:34:31,290 are treated with nucleoside analogs performing 606 00:34:31,290 --> 00:34:33,150 similar sorts of things. 607 00:34:33,150 --> 00:34:37,560 But in some cases, there's a lot of different tailoring goes on 608 00:34:37,560 --> 00:34:39,480 with the nucleoside analog. 609 00:34:39,480 --> 00:34:45,360 It doesn't always work to have the azide at C3 prime. 610 00:34:45,360 --> 00:34:47,230 Some of these features don't always work. 611 00:34:47,230 --> 00:34:50,250 So these have to be tailored in different ways 612 00:34:50,250 --> 00:34:52,530 to target different viruses. 613 00:34:52,530 --> 00:34:56,210 Because in this case, AZT hits reverse transcriptase. 614 00:34:56,210 --> 00:35:01,200 But there may be a different flavor of polymerase enzyme 615 00:35:01,200 --> 00:35:03,850 that you need to inhibit in other organisms. 616 00:35:03,850 --> 00:35:05,820 So it's a lot, lot, lot of options. 617 00:35:05,820 --> 00:35:06,920 Yeah. 618 00:35:06,920 --> 00:35:08,820 AUDIENCE: Does it only affect RT? 619 00:35:08,820 --> 00:35:11,670 Or could there be side effects of the cell, 620 00:35:11,670 --> 00:35:15,097 like an old using it by accident? 621 00:35:15,097 --> 00:35:16,680 BARBARA IMPERIALI: There can be, but I 622 00:35:16,680 --> 00:35:23,290 think the most important thing is that RT is very promiscuous. 623 00:35:23,290 --> 00:35:27,280 So it's much more likely to pop this building block in. 624 00:35:27,280 --> 00:35:29,460 And that doesn't have the mechanisms 625 00:35:29,460 --> 00:35:33,270 to reverse that reaction, all of the clean up that gets done 626 00:35:33,270 --> 00:35:35,320 with normal DNA polymerase. 627 00:35:35,320 --> 00:35:38,970 So our DNA polymerase is going to be a lot more tuned 628 00:35:38,970 --> 00:35:41,110 up to avoid these mistakes. 629 00:35:41,110 --> 00:35:43,320 But the viral reverse transcriptase 630 00:35:43,320 --> 00:35:45,820 just churn through in making it. 631 00:35:45,820 --> 00:35:50,700 So we're protected by our own proofreading and other types 632 00:35:50,700 --> 00:35:55,980 of mechanisms to avoid it really messing up replication. 633 00:35:55,980 --> 00:35:58,380 But that's a very good point. 634 00:35:58,380 --> 00:35:59,030 OK. 635 00:35:59,030 --> 00:36:01,710 So remember the central dogma. 636 00:36:01,710 --> 00:36:05,520 And this is why this process confounds that. 637 00:36:05,520 --> 00:36:06,930 And that's azidothymidine. 638 00:36:06,930 --> 00:36:10,960 And we've already discussed that. 639 00:36:10,960 --> 00:36:17,890 So the next enzyme that became quite an important target, 640 00:36:17,890 --> 00:36:21,790 because it was a new target, was integrase. 641 00:36:21,790 --> 00:36:25,180 So I will tell you, though, that reverse transcriptase 642 00:36:25,180 --> 00:36:29,200 inhibitors like AZT were used early on, 643 00:36:29,200 --> 00:36:34,990 but people with the virus rapidly developed resistance 644 00:36:34,990 --> 00:36:37,720 to that therapeutic agent. 645 00:36:37,720 --> 00:36:41,170 It was one enzyme that was just mutating like crazy. 646 00:36:41,170 --> 00:36:44,710 And the organism could slip through that net. 647 00:36:44,710 --> 00:36:48,040 So the development of therapeutic agents 648 00:36:48,040 --> 00:36:50,080 didn't stop with AZT. 649 00:36:50,080 --> 00:36:53,200 It was, like, we need to hit other targets for us 650 00:36:53,200 --> 00:36:57,550 to have a therapeutically viable combination of therapies 651 00:36:57,550 --> 00:37:00,740 that can really stop this virus effectively. 652 00:37:00,740 --> 00:37:05,470 So a next enzyme that was targeted is integrase. 653 00:37:05,470 --> 00:37:08,590 So this is the one that's been targeted most recently. 654 00:37:08,590 --> 00:37:13,390 And there are currently several integrase inhibitors. 655 00:37:13,390 --> 00:37:15,820 Integrase is a fascinating enzyme 656 00:37:15,820 --> 00:37:21,370 because it really is the enzyme that actually stitches 657 00:37:21,370 --> 00:37:28,370 that piece of viral DNA right into the human genome 658 00:37:28,370 --> 00:37:32,560 just so it's able to be replicated, transcribed, 659 00:37:32,560 --> 00:37:34,750 and translated at any time. 660 00:37:34,750 --> 00:37:37,340 So integrase has been targeted. 661 00:37:37,340 --> 00:37:39,370 It was a tricky target to hit. 662 00:37:39,370 --> 00:37:43,210 But as you can see with several of these drugs, 663 00:37:43,210 --> 00:37:45,970 they are successful drugs. 664 00:37:45,970 --> 00:37:47,890 One thing I want to remind you about, 665 00:37:47,890 --> 00:37:53,000 a lot of the names of the agents, 666 00:37:53,000 --> 00:38:00,850 if you see A-V-I-R within the name of a therapeutic agent, 667 00:38:00,850 --> 00:38:02,460 it means it's an antiviral. 668 00:38:02,460 --> 00:38:05,590 So that's a clue that all of these compounds 669 00:38:05,590 --> 00:38:07,630 are given names-- 670 00:38:07,630 --> 00:38:09,730 raltegravir, blah. 671 00:38:09,730 --> 00:38:12,640 I don't know who comes up with these names, but nevertheless, 672 00:38:12,640 --> 00:38:15,730 they're all names that end in A-V-I-R. 673 00:38:15,730 --> 00:38:19,450 So if you see a medicine and it ends in A-V-I-R, 674 00:38:19,450 --> 00:38:21,850 it's an antiviral agent. 675 00:38:21,850 --> 00:38:24,790 Now, the last of the really fascinating enzymes 676 00:38:24,790 --> 00:38:28,870 that was targeted is the HIV protease, 677 00:38:28,870 --> 00:38:31,420 because the HIV protease is very, very different 678 00:38:31,420 --> 00:38:34,760 from many proteases in mammalian biology. 679 00:38:34,760 --> 00:38:39,130 It's a simple dimeric structure. 680 00:38:39,130 --> 00:38:41,110 So it has quaternary structure. 681 00:38:41,110 --> 00:38:47,140 It has two monomers that are 99 amino acids each. 682 00:38:47,140 --> 00:38:52,810 And each monomer contributes an aspartic acid 683 00:38:52,810 --> 00:38:54,190 to an active site. 684 00:38:54,190 --> 00:38:56,380 And those are involved in the mechanism. 685 00:38:56,380 --> 00:39:00,130 And what generally happens when substrates or inhibitors bind 686 00:39:00,130 --> 00:39:01,990 is these two flaps-- 687 00:39:01,990 --> 00:39:04,030 one of them I've shown you in magenta-- 688 00:39:04,030 --> 00:39:07,810 close down and close up the active site. 689 00:39:07,810 --> 00:39:11,320 And ritonavir was one of the first generation protease 690 00:39:11,320 --> 00:39:15,880 inhibitors that sat in the place where 691 00:39:15,880 --> 00:39:19,270 the substrate would normally sit in the HIV protease. 692 00:39:19,270 --> 00:39:21,190 And remember that the HIV protease 693 00:39:21,190 --> 00:39:24,070 is critical for maturity of the virus 694 00:39:24,070 --> 00:39:27,040 because it chops up the polyprotein 695 00:39:27,040 --> 00:39:29,080 into the necessary enzymes. 696 00:39:29,080 --> 00:39:32,200 They can't be functional enzymes in the polyprotein. 697 00:39:32,200 --> 00:39:36,130 They have to cut up into the appropriate pieces. 698 00:39:36,130 --> 00:39:39,400 So the HIV protease had unusual specificity. 699 00:39:39,400 --> 00:39:42,760 And as soon as it was discovered and structurally characterized, 700 00:39:42,760 --> 00:39:46,420 every major pharmaceutical company jumped on-- this was 701 00:39:46,420 --> 00:39:47,830 in the late '80s-- 702 00:39:47,830 --> 00:39:50,890 to make successful protease inhibitors that 703 00:39:50,890 --> 00:39:52,750 just got better and better. 704 00:39:52,750 --> 00:39:55,730 Ritonavir is an example of first generation, 705 00:39:55,730 --> 00:39:59,140 but I think now we're on third generation-type inhibitors 706 00:39:59,140 --> 00:40:01,900 which are just a better and better and much 707 00:40:01,900 --> 00:40:05,820 more selective and specific for the HIV protease. 708 00:40:05,820 --> 00:40:08,110 And because they're much more potent, 709 00:40:08,110 --> 00:40:11,650 instead of needing grams and grams of an inhibitor, 710 00:40:11,650 --> 00:40:14,290 you can get away with milligrams of inhibitor, 711 00:40:14,290 --> 00:40:19,000 which made the therapeutic dosing for HIV much more 712 00:40:19,000 --> 00:40:21,220 palatable is sort of a good word. 713 00:40:21,220 --> 00:40:25,180 Because when you have a very promiscuous protease inhibitor, 714 00:40:25,180 --> 00:40:27,650 like the early ones that were discovered, 715 00:40:27,650 --> 00:40:30,700 they mess up your entire GI because your GI 716 00:40:30,700 --> 00:40:33,340 runs on proteases that digest your food. 717 00:40:33,340 --> 00:40:36,250 So the original, the early protease inhibitors 718 00:40:36,250 --> 00:40:37,990 had a lot of side effects. 719 00:40:37,990 --> 00:40:41,620 The most modern third generation are very viable. 720 00:40:41,620 --> 00:40:43,900 And this just shows you what other topic I 721 00:40:43,900 --> 00:40:49,470 want to hit on next is here's the green and cyan two monomer 722 00:40:49,470 --> 00:40:52,030 units of HIV protease. 723 00:40:52,030 --> 00:40:55,390 In yellow is an inhibitor that is bound. 724 00:40:55,390 --> 00:40:58,520 And just on one monomer of the two monomers, 725 00:40:58,520 --> 00:41:02,200 I've highlighted, in red, different resistance sites. 726 00:41:02,200 --> 00:41:05,470 So when any of those sites mutates, 727 00:41:05,470 --> 00:41:11,120 the protease stops being inhibited by the HIV protease 728 00:41:11,120 --> 00:41:12,210 inhibitors. 729 00:41:12,210 --> 00:41:14,030 Because you want to think about it, 730 00:41:14,030 --> 00:41:16,730 one mutation in one subunit, actually, 731 00:41:16,730 --> 00:41:21,440 will have a corresponding mutation in the partner subunit 732 00:41:21,440 --> 00:41:22,700 that is in the dimer. 733 00:41:22,700 --> 00:41:26,030 So each mutation ends up being two mutations 734 00:41:26,030 --> 00:41:31,160 in the full enzyme that has quaternary restructure. 735 00:41:31,160 --> 00:41:33,740 So these are major. 736 00:41:33,740 --> 00:41:38,550 They go all the way out to the outer parts of the enzyme. 737 00:41:38,550 --> 00:41:40,220 It's not just around the active site. 738 00:41:40,220 --> 00:41:45,290 It's just all over the enzyme you can get mutations that 739 00:41:45,290 --> 00:41:49,010 stop the typical drugs that are targeted at the protease's 740 00:41:49,010 --> 00:41:50,030 binding 741 00:41:50,030 --> 00:41:51,980 So here I just show you these are 742 00:41:51,980 --> 00:41:54,740 data that show for the protease and 743 00:41:54,740 --> 00:41:56,690 for the reverse transcriptase. 744 00:41:56,690 --> 00:41:58,550 Within the length of the protease, 745 00:41:58,550 --> 00:42:02,230 for example, there's mutations all the way along. 746 00:42:02,230 --> 00:42:03,440 I don't know why this is 101. 747 00:42:03,440 --> 00:42:06,740 The numbering system is a little different in some variants. 748 00:42:06,740 --> 00:42:11,660 But you can see which mutations will add to drug resistance. 749 00:42:11,660 --> 00:42:15,420 And there's a similar picture here for reverse transcriptase. 750 00:42:15,420 --> 00:42:16,040 OK? 751 00:42:16,040 --> 00:42:19,590 So this is a very serious issue. 752 00:42:19,590 --> 00:42:22,663 So now I'm going to give you a little break here. 753 00:42:22,663 --> 00:42:23,330 [VIDEO PLAYBACK] 754 00:42:23,330 --> 00:42:24,530 - So this is HIV. 755 00:42:24,530 --> 00:42:26,540 It's a typical retrovirus, meaning 756 00:42:26,540 --> 00:42:28,580 that it has an outer envelope. 757 00:42:28,580 --> 00:42:31,370 And in the center, it has two copies of RNA, 758 00:42:31,370 --> 00:42:33,770 as well as an enzyme here in blue 759 00:42:33,770 --> 00:42:36,800 that's reverse transcriptase, which will ultimately 760 00:42:36,800 --> 00:42:40,140 turn that RNA into DNA. 761 00:42:40,140 --> 00:42:43,130 The virus itself, with this outer envelope 762 00:42:43,130 --> 00:42:49,490 PROTEIN actually directly infects T-helper cells. 763 00:42:49,490 --> 00:42:53,330 The way that it does this is that as it comes up to the cell 764 00:42:53,330 --> 00:42:57,500 surface, it uses receptors that are on T-helper cells 765 00:42:57,500 --> 00:43:02,270 and exclusive to T-helper cells, which are CD4 molecule which 766 00:43:02,270 --> 00:43:04,100 really defines T-helper cells. 767 00:43:04,100 --> 00:43:08,450 It's a surface receptor that binds to the envelope protein. 768 00:43:08,450 --> 00:43:11,120 That causes a conformational change 769 00:43:11,120 --> 00:43:14,610 and allows a second receptor to grab hold of the envelope. 770 00:43:14,610 --> 00:43:16,550 This is the chemokine coreceptor. 771 00:43:16,550 --> 00:43:18,320 It's also called CCR5. 772 00:43:18,320 --> 00:43:20,270 And we'll talk about that more. 773 00:43:20,270 --> 00:43:25,040 What happens now is that the stock of the envelope protein 774 00:43:25,040 --> 00:43:30,440 pierces through from the virus into the host cell 775 00:43:30,440 --> 00:43:34,610 and starts to draw the cell membrane and the viral membrane 776 00:43:34,610 --> 00:43:35,630 together. 777 00:43:35,630 --> 00:43:39,110 And what ultimately happens is fusion of those two membranes 778 00:43:39,110 --> 00:43:42,980 and the viral genetic material is injected, essentially, 779 00:43:42,980 --> 00:43:44,000 into the cell. 780 00:43:44,000 --> 00:43:46,910 And the envelope protein is left at the cell surface. 781 00:43:46,910 --> 00:43:49,340 - So that's the fusion process that's so hard to explain. 782 00:43:49,340 --> 00:43:52,490 - The virus has a matrix and a capsid protein, shown here 783 00:43:52,490 --> 00:43:55,970 in green and red, that essentially are digested 784 00:43:55,970 --> 00:43:58,250 when it enters into the cell. 785 00:43:58,250 --> 00:44:02,900 That releases the viral enzymes and the viral RNA. 786 00:44:02,900 --> 00:44:04,760 And here we have reverse transcriptase, 787 00:44:04,760 --> 00:44:10,190 which takes the viral RNA, and using host nucleotides 788 00:44:10,190 --> 00:44:14,660 converts that viral RNA into a single strand of DNA. 789 00:44:14,660 --> 00:44:17,880 While it does that, it makes some random errors, 790 00:44:17,880 --> 00:44:20,420 which is characteristic of reverse transcriptase. 791 00:44:20,420 --> 00:44:24,390 It has very poor proofreading activity. 792 00:44:24,390 --> 00:44:28,800 That single-stranded DNA, now, is again reverse transcribed 793 00:44:28,800 --> 00:44:30,620 into a double-stranded DNA. 794 00:44:33,290 --> 00:44:35,660 At that point, another enzyme that 795 00:44:35,660 --> 00:44:38,150 has come in with the virus in the beginning, called 796 00:44:38,150 --> 00:44:40,670 integrase, essentially grabs hold 797 00:44:40,670 --> 00:44:43,700 of that double-stranded DNA and carries it 798 00:44:43,700 --> 00:44:48,770 through a nuclear pore into the nucleus of the cell. 799 00:44:48,770 --> 00:44:52,640 Within the nucleus of the cell, it finds the host chromosome. 800 00:44:52,640 --> 00:44:55,130 And basically, the integrase enzyme, 801 00:44:55,130 --> 00:44:59,540 makes a nick in the host's DNA and allows for HIV 802 00:44:59,540 --> 00:45:02,750 to insert itself into the host chromosome. 803 00:45:02,750 --> 00:45:08,580 And that right there is what establishes lifelong infection. 804 00:45:08,580 --> 00:45:12,140 Now, RNA polymerase comes along and makes its messenger RNA. 805 00:45:12,140 --> 00:45:15,320 - That's the host polymerase, it's just back to normal now. 806 00:45:15,320 --> 00:45:19,490 - Those messenger RNAs encode for different viral proteins. 807 00:45:19,490 --> 00:45:22,670 They end up associating with ribosomes 808 00:45:22,670 --> 00:45:25,850 at the surface of the rough endoplasmic reticulum. 809 00:45:25,850 --> 00:45:27,830 And here is a piece of mRNA that's 810 00:45:27,830 --> 00:45:31,370 making envelope protein which is directly produced 811 00:45:31,370 --> 00:45:33,830 into the endoplasmic reticulum. 812 00:45:33,830 --> 00:45:37,160 And its shuttled, then, through the endoplasmic reticulum 813 00:45:37,160 --> 00:45:41,190 and taken to the cell surface, where, at the cell surface, 814 00:45:41,190 --> 00:45:43,850 it becomes embedded in the cellular membrane. 815 00:45:43,850 --> 00:45:47,480 And at this point, coalescing with other envelope proteins 816 00:45:47,480 --> 00:45:49,430 that have been produced you have this cluster 817 00:45:49,430 --> 00:45:55,430 of envelope proteins now on the surface of this infected cell. 818 00:45:55,430 --> 00:45:59,570 At the same time, there are other messenger RNAs 819 00:45:59,570 --> 00:46:02,900 that are being produced that allow for translation 820 00:46:02,900 --> 00:46:05,370 of other viral proteins. 821 00:46:05,370 --> 00:46:08,330 So here are additional viral proteins being made 822 00:46:08,330 --> 00:46:11,270 which are going to be used to make up 823 00:46:11,270 --> 00:46:14,060 the key components that the virus, ultimately, 824 00:46:14,060 --> 00:46:16,070 is going to need. 825 00:46:16,070 --> 00:46:18,530 These are transported again to the cell surface, 826 00:46:18,530 --> 00:46:21,560 to the area where these envelope proteins are. 827 00:46:21,560 --> 00:46:25,880 And a strand of RNA, as well as some of the enzymes 828 00:46:25,880 --> 00:46:29,640 are part of that complex. 829 00:46:29,640 --> 00:46:32,730 This then buds off at the cell surface at this point, 830 00:46:32,730 --> 00:46:34,710 but it's still not a mature virion 831 00:46:34,710 --> 00:46:40,170 because the polyprotein chain needs to still be digested 832 00:46:40,170 --> 00:46:42,330 into its component parts. 833 00:46:42,330 --> 00:46:44,960 That's done by an enzyme called protease. 834 00:46:44,960 --> 00:46:48,930 Protease breaks up those polyprotein chains 835 00:46:48,930 --> 00:46:52,710 and, ultimately, allows for them to coalesce and form 836 00:46:52,710 --> 00:46:57,390 the mature structures that make up the final virion. 837 00:46:57,390 --> 00:47:00,360 And now you have a mature infectious virion 838 00:47:00,360 --> 00:47:04,840 that can go on now to infect other cells. 839 00:47:04,840 --> 00:47:09,370 Once that happens, now, the cell can produce tons of viruses. 840 00:47:09,370 --> 00:47:12,745 And this is really what, then, keeps the whole process going. 841 00:47:12,745 --> 00:47:13,360 [END PLAYBACK] 842 00:47:13,360 --> 00:47:14,290 BARBARA IMPERIALI: OK. 843 00:47:14,290 --> 00:47:14,790 All right. 844 00:47:14,790 --> 00:47:17,830 So I have gone monstrously long on HIV. 845 00:47:17,830 --> 00:47:22,390 So sadly, I cannot get you to Ebola. 846 00:47:22,390 --> 00:47:26,050 But I'll post those slides anyway. 847 00:47:26,050 --> 00:47:30,070 This question just speaks to combination therapies. 848 00:47:30,070 --> 00:47:33,980 So combination therapies are very, very important, 849 00:47:33,980 --> 00:47:36,220 particularly in cases where resistance 850 00:47:36,220 --> 00:47:41,770 to individual therapeutic agents against an individual target 851 00:47:41,770 --> 00:47:42,610 can happen. 852 00:47:42,610 --> 00:47:46,900 So HIV mutates its proteins very, very rapidly. 853 00:47:46,900 --> 00:47:51,940 But the likelihood that it will simultaneously 854 00:47:51,940 --> 00:47:55,970 mutate two targets in one cell is much lower. 855 00:47:55,970 --> 00:47:58,930 So combination therapies basically 856 00:47:58,930 --> 00:48:02,230 are there to avoid drug resistance that 857 00:48:02,230 --> 00:48:04,660 occurs, usually, because of the high mutation rate, 858 00:48:04,660 --> 00:48:08,500 not because one drug has a short half life, not 859 00:48:08,500 --> 00:48:10,990 so that multiple strains can be targeted, 860 00:48:10,990 --> 00:48:13,510 or to decrease the number of side effects. 861 00:48:13,510 --> 00:48:17,200 Obviously, combination therapies give you more side effects, 862 00:48:17,200 --> 00:48:21,560 but they are really important in mitigating the disease. 863 00:48:21,560 --> 00:48:25,240 So I was going to talk to you about vaccine development, 864 00:48:25,240 --> 00:48:27,910 but I will discuss this with Professor Martin 865 00:48:27,910 --> 00:48:32,080 to see if he wants to take that over. 866 00:48:32,080 --> 00:48:36,330 But I want to leave you with one last thing. 867 00:48:41,330 --> 00:48:43,210 OK. 868 00:48:43,210 --> 00:48:44,920 Three small points. 869 00:48:44,920 --> 00:48:49,090 On next Wednesday, this is Fred Flintstone. 870 00:48:49,090 --> 00:48:51,640 I tend to channel Fred Flintstone because he 871 00:48:51,640 --> 00:48:53,950 wears this nice blue scarf. 872 00:48:53,950 --> 00:48:57,670 Next Wednesday is the last of the classes. 873 00:48:57,670 --> 00:49:00,050 We've got some good stuff lined up for you. 874 00:49:00,050 --> 00:49:02,590 We're going to have the topoisomerase demo 875 00:49:02,590 --> 00:49:06,460 from some people who submitted a topo demo for us. 876 00:49:06,460 --> 00:49:08,770 And then we're going to do 7.016 Jeopardy. 877 00:49:08,770 --> 00:49:10,810 Now, don't think this is going to be cheesy. 878 00:49:10,810 --> 00:49:12,160 It's going to be tough. 879 00:49:12,160 --> 00:49:13,660 And it's going to be fun. 880 00:49:13,660 --> 00:49:15,610 And we're all going to be involved. 881 00:49:15,610 --> 00:49:17,590 Professor Martin and I will be the hosts. 882 00:49:17,590 --> 00:49:19,412 He's promised to dress up. 883 00:49:19,412 --> 00:49:22,130 Ha ha ha. 884 00:49:22,130 --> 00:49:24,880 Jackie will help me run the program, 885 00:49:24,880 --> 00:49:27,460 because it's in a real Jeopardy program. 886 00:49:27,460 --> 00:49:30,100 And Hannah and David will keep scores. 887 00:49:30,100 --> 00:49:34,510 And we'd like to encourage you who come to be in teams of two. 888 00:49:34,510 --> 00:49:36,730 Otherwise, there will be kind of a lot of you. 889 00:49:36,730 --> 00:49:39,980 So if when you come in, if you sit on that side of the room, 890 00:49:39,980 --> 00:49:42,850 put your two names on these boards. 891 00:49:42,850 --> 00:49:45,760 And if you're on the other side, put them on these boards 892 00:49:45,760 --> 00:49:49,810 because Hannah and David will be posted there to keep score. 893 00:49:49,810 --> 00:49:54,820 And we will muster up some small prize for the winner here. 894 00:49:54,820 --> 00:49:56,475 So, yes, question. 895 00:49:56,475 --> 00:49:58,100 AUDIENCE: Will this count as the final? 896 00:49:58,100 --> 00:50:00,520 BARBARA IMPERIALI: No. 897 00:50:00,520 --> 00:50:03,220 That would be a heck of an incentive, wouldn't it? 898 00:50:03,220 --> 00:50:05,770 What this will do for you is actually 899 00:50:05,770 --> 00:50:08,620 kind of help you refresh a lot of things, 900 00:50:08,620 --> 00:50:10,570 maybe identify blind spots. 901 00:50:10,570 --> 00:50:11,512 It was worth asking. 902 00:50:11,512 --> 00:50:12,720 It was a good question, yeah. 903 00:50:15,490 --> 00:50:17,770 So I'll post the questions afterwards as well. 904 00:50:17,770 --> 00:50:20,090 So you'll be able to kind of say, guy, 905 00:50:20,090 --> 00:50:22,570 I don't know what this question was about at all. 906 00:50:22,570 --> 00:50:25,150 And you can go and review a little of the material. 907 00:50:25,150 --> 00:50:26,180 Nice try, though. 908 00:50:28,810 --> 00:50:31,090 Oh, and there was one other thing. 909 00:50:31,090 --> 00:50:34,180 I just want to remind you that a vote is a vote. 910 00:50:34,180 --> 00:50:36,280 This is a democratic society. 911 00:50:36,280 --> 00:50:40,660 So you should please take time to fill in the evaluations 912 00:50:40,660 --> 00:50:41,570 on the course. 913 00:50:41,570 --> 00:50:43,120 OK.