Infectious Molecular Clones

Astute ERV readers have noticed a couple of odd things about my research.

1- How the hell am I cutting and pasting bits of a retroviral (RNA) genome together?
2- How the hell do I have a (seemingly) endless supply of HIV-1 for my experiments?

The answer to both questions is, infectious molecular clones!

Short explanation-- Infectious molecular clones are just viral genomes (as DNA) inserted into plasmids. Even though plasmids are only found in bacteria (our cells have no idea what to do with the things), you can then use some chemistry tricks to get a mammalian cell line to read the plasmid, and YAAAY! They make lots and lots of virus to use in other experiments!

I can also manipulate these plasmids by using restriction enzymes to cut/paste like a kindergartener on Red Bull!

Long explanation-- Step one for making an infectious molecular clone is to turn HIV-1s RNA into DNA. Happily, HIV-1 already does that for us! Whether you isolate infected cells from a patient, OR use reverse transcription to generate cDNA ex vivo, your end goal is a provirus-- A retrovirus in its DNA state.

Next, you have to get your DNA into a plasmid. There are a billion different kinds of plasmids, or 'cloning vectors' available commercially, but a basic vector contains an antibiotic resistance gene (gotta give the bacteria a reason to keep this selfish DNA!), an origin of replication (gotta be able to make lots of plasmids!), and a 'multicloning site' chock full of targets for those restriction enzymes (gotta have a place to cut open the circular plasmid to paste in your provirus!)

Okay, so you have your provirus, you have your plasmid cut open with restriction enzymes, now you can paste all the bits together with a DNA ligase (extracted from T4 phages!).

Once your provirus is in the plasmid, the possibilities are endless. You can introduce more restriction enzyme targets with site directed mutagenesis to cut out bits you arent interested in (or if you dont want your plasmid to code for, like, an actual deadly virus, but still want to watch a precursor protein get processed). You can make an HIV-1 virus half Subtype B, half Subtype C. If you can dream it, you can make it! To see what your homemade virus does, you just have to transfect a mammalian cell line with your homemade plasmid, and then they make viruses for you! As long as you dont run out of plasmids to transfect (its easy to make more), you have an endless supply of your virus!



Its fun.

And its possible because of infectious molecular clones.

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Very interesting (and diabolical). I wonder if the biohazard level for HIV plasmid...hosts(?) is the same as that for dealing with unattenuated virus. Since it's a retrovirus, and I'm sure I'm displaying ignorance here, is there a possibility a plasmid could infect a host with HIV? Or does the nucleus provide a sufficient barrier to keep the raw genetic material isolated?

I know, I know I'm making a lot of assumptions that may not be true at all but that's what happens when you max out your knowledge of Biology at Intro to Cell and Molecular.

This doesn't really answer the question of where it comes from. Did you ever go back to HIV infected cells and isolate out cDNA type provirus? Or are all the plasmids just sitting happily in your lab, who got it from somebody else, ect.?
Or do you synthesize genes from scratch (oligo overlap extension/PCR fun timez?)?

Thanks for answering my questions! The way you make it sound, it's almost as if you have veritable cell factories for your virus, and I love it how you make it sound so simple. :) I have to admit, I LOL'ed when I read the part about the viruses being in a steel-cage death match.

BTW, how long does it take to make your viruses, and about how many of the little buggers do you get when you finish the entire process?

Thanks for posting on this, I really love learning this new stuff, and I appreciate the links that you provided too. :)

A witch! A witch!

Nope - HIV plasmids are all BSL-1. That nice layer of keratin that you have + plasma membrane + nuclear membrane + your cells are mostly quiescent (not making much protein) is enough to protect you. We have enough fights in our lab over replication-incompetent virus - if people had to miniprep plasmids in the BSL-3, I think there'd be a riot.

Making HIV is E.Z. Almost as easy as herpes. If you want a challenge, try making HepC.

By perisylph (not verified) on 29 Jul 2008 #permalink

She turned me into a NEWT!
I got better. (Sort of. But the regrowing of limbs bit is nifty.)

So you could conceivably make a tiny virus shrine, complete with a little mound of HIV-1! This begs the question, what color are your viruses, when there are enough to see with the naked eye?

Alex M said:

So you could conceivably make a tiny virus shrine, complete with a little mound of HIV-1! This begs the question, what color are your viruses, when there are enough to see with the naked eye?

I'm assuming that you would need at least a mono-layer of the virus on something like a petri-dish for it to be visible to the naked eye.(Might do with less but I was curious to do what I did next. :p)

Anyway, HIV is a spherical virus with a diameter of 120nm. I found a site that was selling petri dishes that were 101mm in diameter, so using that as my baseline I set about to see just how much virus you would need to cover the petri dish in a mono-layer of HIV. Doing some math that I will spare you, it came out to be 708,402,777,777 viruses that you would need, shoved next to each other in order to have a mono-layer (Actually, given how spherical objects behave when you start arranging them next to each other, this number should be less, but I didn't want to spend too much time on it :p).

Anyway, that's a lot of viruses to have in one spot to be able to see them with the naked eye! I honestly didn't realize just how small this virus happens to be, and when you realize what it can do to the human immune system, it almost defies reason. I'm just glad we have people like Abbie working on beating the crap out of it. :)

Thank you, Lledowyn for taking the time to do your estimate and also for inspiring me to do something interesting at work! That colossal number made me wonder what's the bare minimum that we could see.

The Internet claims that the critical factor in determining what humans can see is arc length. The Internet claims that the limit of human vision is about 15-20 arc seconds for a perfect eye, which is about .0056 degrees.

So to barely see 120nm, you would need to be about 20 microns away. That sucks!
For a comfortable distance of about 1.5ft you'd need a virus arc with length about 2.7mm. That's ~22,500 viruses just for something on the bleeding edge of visibility.

Any idea how many viruses Abbie can generate in the span of a week?

I wish my job had cage matches of any sort. Virology seems to get more interesting with every post.

For shame. I expected a "Clone Wars" reference.

My lab is FINALLY about to switch away from live virus stocks to using a plasmid for our infections. I'm endlessly thankful because if it's not the cells refusing to be infected, it's the viral stock concentration being miscalculated. I'd like to eliminate half of that problem!
We've been having so much trouble getting infections to work in our lab lately that we can't decide if we've somehow cured HIV or if the deniers are right and HIV doesn't even exist.

By Katherine F (not verified) on 30 Jul 2008 #permalink

... using chemistry tricks to make a mammalian cell read a plasmid!!??

Aaaah... now i understand why some creationists say those horrible things about "darwinism"... Someone said diabolic?? Indeed ;)

cut/paste like a kindergartener on Red Bull!

rofl! Molecular biology is fun. :D

By liveparadox (not verified) on 30 Jul 2008 #permalink