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- the two cDNAs made by reverse transcriptase are not complimentary to each other, they are both complimentary to the viral RNA so how do they combine with each other?
Shouldn't reverse transcriptase use the newly made cDNA as a template to make the second strand of DNA that is complimentary to the first?(29 votes)
- I think the description in the video may be incorrect.
Using the viral RNA as a template, reverse transcriptase creates a DNA-RNA complex. Another enzyme unzips this complex to create a single strand of cDNA. That piece of cDNA is then acted on by reverse transcriptase again to create the cDNA's complementary strand. This yields double stranded DNA. I think the video is wrong in saying that reverse transcriptase uses the same viral RNA to create two separate cDNA strands. Otherwise--like you said--the cDNA strands created would be identical and not complementary.(47 votes)
- I know it might seem like I'm splitting hairs but at4:15, when the video refers to viral integration into the host (human) genome, isn't the representation of circular DNA now inaccurate?(11 votes)
- At1:52she draws reverse transcriptase binding to the 5' end of the viral RNA, but shouldn't it bind to the 3' end of the RNA if it is to synthesize the complementary DNA in the 5' to 3' direction?(11 votes)
- This seemed wrong to me as well, and when I checked other sources they all agree that reverse transcriptase binds to the 3' end of RNA instead of the 5' end as shown in the video. cDNA is synthesized 5' to 3' just as in normal transcription. This is a good image: https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/Cloning/Images/0816/reverse_transcription_process.png(7 votes)
- What does protease do? Why is the virus considered immature until the protease cleaves them? What does it cleave? Why does it need to cleave it before the protein is functional?(13 votes)
- Remember, DNA -> RNA -> Proteins. When mRNA is translated, polypeptide proteins are formed. And the role of proteases are to split the polypeptides chain at specific points (cleave) to make sure that the proteins it wants are made.
By cutting other proteins off of the polypeptide chain, the proteases basically shape the new proteins (think primary structure) and this is called maturation of the proteins.(1 vote)
- At7:03, she mentions that the protease "cleaves the other proteins to make sure that they're fully functional." How does cleaving proteins make them functional? And is this the step that makes the virus "mature"?(5 votes)
- Viral RNA is translated into a single long chain that includes reverse transcriptase, integrase, and protease which can't function because they're in a long chain. Protease cuts them from the chain into their individual enzyme components so they can be used to produce new viruses.(4 votes)
- Would the retrovirus be a + sense or a - sense strand or that would not apply here?(4 votes)
- HIV is classified as a single-stranded positive-sense enveloped RNA virus
but I don't know if this applies to all retroviruses(1 vote)
- i didnt understand the function of integrase (regarding 3' ends n sticky ends) and protease.... please explain.(2 votes)
- Silly question but why can't you just target/silence the viral protease to make HIV impotent, kind of? Wouldn't it look different than the proteases we need for human cells because they wouldn't be involved in cleaving a reverse transcriptase and integrase enzyme bond?(2 votes)
- Does integrase act as both a restriction enzyme and DNA ligase while in the nucleus?(1 vote)
- No, it acts as a restriction enzyme and inserts the viral DNA through a process called transesterification. DNA ligase acts by connecting bases together, but since we're already inserting dsDNA into the host genome, transesterification is used. This is a process by which new ester bonds are formed (phosphodiester bonds) on the phosphate-sugar backbone of DNA.(2 votes)
- So you might have an understanding of viral replication, but there's one special case that doesn't quite fit neatly into the box of lytic or lysogenic. And that's what we're going to talk about. So that special case is called a retrovirus. So first let's zoom in and take a look at some unique things about the retrovirus that make it different from other viruses. So first of all, it is an enveloped, single-stranded RNA virus. And inside of this envelope it also carries three special proteins. And right now just be aware that they are three special proteins. I'll talk about them more when we get to each step where they're important. So as you know, enveloped viruses can enter in one of two ways, either through tricking receptors, receptor-mediated endocytosis, or through direct fusion. And it just so happens that in our example, and we're talking here about the retrovirus HIV, this retrovirus will enter the cell with direct fusion. So now that this nucleocapsid is inside the cell, it actually has to undergo a step called uncoating where this purple capsid is dissolved. Oh, and we forgot about the protein, so let me redraw those in right now. So these are the proteins that were originally inside of the capsid. So everything inside of that coat is released. And this is where the first special step occurs. So we're going to say that this red protein is reverse transcriptase. So reverse transcriptase will hop on to the RNA, and it reverse transcribes the RNA, which means that so it reads from five prime to three prime end. And you will form complementary DNA shown here in pink. And the reason it's called reverse transcription is usually you take DNA to make RNA. But in this case you take RNA to make DNA. And because this is the complementary DNA strand, we call this cDNA, complementary. And then reverse transcriptase will work again on this same RNA to make another cDNA strand. Because it's the same exact code, it can recombine with the other cDNA strand to make a double-stranded DNA. And so now what happens is you have integrase coming along. And let's make integrase blue. So integrase comes along, clips off each of the three prime ends. So now these are slightly shorter on each end. And sorry this is a bit hard to see because this strand's three prime end is over here. And while the first one is actually clearly labeled as this is the three prime end. And by clipping off those three prime sections, they form these sticky ends because unpaired DNA wants to be paired. And integrase has suddenly removed that part. And you might be wondering what happens to this RNA. And what happens is that it actually gets degraded by normal ribonuclease. So that's no longer there. And integrase does exactly what it says. It will follow this path and integrate this HIV DNA into the host's DNA. And one thing I would just want to very quickly mention is that if I had drawn this to be super accurate, this would need to have a nucleus around it because the HIV retrovirus infects human eukaryotic cells which have a nucleus. So it actually will travel through the nuclear membrane to get to the genome. And here integrase helps the viral DNA integrate with the host, like its name, integrase, integrate. So just imagine this is all double-stranded, but just for simple drawing sake, this will just be one line. So this is viral DNA. And this is called the provirus stage. So you can see that this is similar to the lysogenic cycle that we'd talked about before. But unlike the regular lysogenic cycle, it's not dormant or latent. It actually does not have that repressor gene that typical lysogenic viruses have. So it is actively transcribed whenever the host's DNA is transcribed. So since the host's cell thinks this is normal DNA, it will make RNA. And I just wanted to call this viral mRNA so you have an idea that the cell cannot tell that this mRNA shouldn't have happened. So this mRNA exits the nucleus. And these viral RNAs are now in the cytosol. Again, once this viral mRNA exists the nucleus and it goes into the cytoplasm, it's just like any other RNA. And some of these will be translated into proteins like the capsid proteins. And of course the three proteins that we begin with which are the reverse transcriptase, the integrase, and actually the last one we haven't yet mentioned, is the protease. The green here is protease. And we're going to hold off a little bit on what protease does. But here it's formed. And you can see that you now have all of the parts that can self-assemble into new viruses. So again, all of these viruses that are formed will have the RNA, the reverse transcriptase, the integrase, and the protease. So you'll notice that these are actually missing one thing. They're missing their envelope. And so they're called immature viruses. And unlike the typical lytic cycle, it doesn't just break open the membrane. In fact, it takes advantage of the membrane. And so these viruses will come along, and they will bud off. So this will want in here and this will want to enter here. Oops, and that's missing a border, I just realized, so there you go. And they will bud off, and that will be their envelope. And sorry, they're missing the proteins. And I'll just draw them in again. So again, these are still immature, right. And before they go on to infect other cells, they have to mature somehow. So what happens is that protease inside of here will cleave those other proteins to make sure that they're fully functional before the virus enters another cell and starts this cycle all over again. And so retroviruses replicating are a bit more complicated than traditional replication. So it's not just lysogenic or lytic. It actually has elements of both.