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!)
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!
Thats how I get to do my experiments… Frankenstein viruses… glowing viruses… ARMIES OF CLONE VIRUSES, IDENTICAL EXCEPT FOR TEENY-TINY DIFFERENCES, THROWN TOGETHER IN STEEL-CAGE DEATH MATCHES!!!! THERE CAN BE ONLY ONE!!! BWAHAHAHAHAHA!!!!!!
And its possible because of infectious molecular clones.