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HIV Reverse Transcriptase Enzyme

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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HIV Reverse Transcriptase Enzyme

Postby MSpinelli » Tue Jul 17, 2007 2:35 pm

Why is it that the enzyme reverse transcriptase starts doing work when inside the cell of a host and not anywhere else?

I am just curious as to what triggers the function of the enzyme to encode the one strand of RNA to two strands of DNA.

Could it be something pertaining to positive feedback?
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Postby blcr11 » Tue Jul 17, 2007 5:31 pm

Inside the viral capsid, there isn't very much room to move, and there is no pool of dNTP, so even if it wanted to transcribe, it couldn't. The enzyme is made as an inactive polyprotein, gp160, that is packaged into an immature virion along with viral genomic RNA; the process of maturation cleaves gp160 into the mature capsid gag proteins and the two components of RT. So the active enzyme only forms in the mature capsids, where it is primed and ready to go, but can't do its thing until it is released from the virion on infection. RT is kind of promiscuous, but I think it prefers heteroduplex templates--why it evolved this crazy mechanism of making strong-stop DNA and then jumping to the 3' end of the genome to complete the strand formation--good question. It does generate convenient recombination points for incorporation into the host genome--maybe that has something to do with it; the viral genomic transcription product is reminiscent of transposons and phages--but only similar, not identical.
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interesting

Postby MSpinelli » Thu Jul 19, 2007 6:29 pm

Sorry if I sound like a newby but...

So what you're saying is the enzyme gets thrown out of the virion after it is inside the host cell because of the double duplex attraction that RT has?

Does the virion always mature inside the host cell and how exactly does it mature?

If these questions are for beginners I apologize and you do not have to answer them.
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Postby blcr11 » Thu Jul 19, 2007 10:18 pm

Here is a flash-animation describing the HIV life cycle. I’m not wild about it—there are no convenient gaps in host genomic DNA for HIV to apparently ligate itself into, for example, and the intravirion maturation process is pretty much ignored—but if you like to see things moving across your screen, you could give it a shot.

http://hopkins-aids.edu/hiv_lifecycle/hivcycle_txt.html

A better, non-animated version that at least describes in some detail the steps of infection as well as virus production and maturation is given by:

http://aids.about.com/od/newlydiagnosed ... cle_10.htm


HIV is an enveloped virus. The external surface is really just a part of pinched off cell membrane that has patches of viral envelope glycoprotein (env or gp120 for short) protruding from the surface, and an intracellular/intravirion cytoplasmic tail. Inside of the envelope is the nucleocapsid core composed of gag proteins which covers and protects the packaged viral RNA and reverse transcriptase (RT or Pol whichever you prefer to use). When the virion infects, it binds to the host-cell surface via the host’s CD4 protein, which acts as the receptor for HIV. The envelope/lipid bilayer of the virion then fuses with the cell membrane in much the same way that two lipid droplets might fuse. In the process, the nucleocapsid core is popped into the cytoplasm. The capsid contains genomic RNA, two copies per capsid; they are independent ssRNA molecules, not complementary strands; not duplex in any way, except for a tiny bit of primer-duplex set for transcription by the associated reverse transcriptase molecule. Once inside the cell, proteases weaken the capsid, which disintegrates, releasing the viral RNA-RT complex into the cytoplasm where it is now exposed to cellular pools of dNTP, thus activating RT. The action of RT is to produce a dsDNA copy of the viral RNA with a few “extras” on each end. This dsDNA genome is moved into the nucleus of the cell where the viral gene, integrase (Int for short), incorporated the DNA into the host’s genome, similar to the way lamda phage is lysogenized in E coli.

OK, that was the infection part of the cycle. Once incorportated into the host’s genome, a number of things can happen (including “silence” for some period of time). The incorporated HIV genome can be transcribed by the cell’s own RNA polymerase to make mRNAs that encode viral proteins—all of which are expressed as polyprotein precursors that need to be processed in some way in order to produce the final, active viral protein; the integrated viral genome also needs to make ss-viral genomic RNA for packaging in the soon-to-be-made virions. As gp120 gets expressed at the cell surface, the other viral components (gag-Pol precursor, gag-HIV protease precursor, and viral genomic RNA) start accumulating near the cell membrane. The immature virus eventually buds from the surface of the cell. At this point, it is little more than a “bag” of lipid bilayer with Env protein protruding. The protease begins to act once the immature virus buds, producing gag proteins that spontaneously assemble into the capsid structure, as well as active RT which packages along with the viral genome inside the capsid. So what I’m calling “maturation” doesn’t really happen inside the host (although it might start there), but really happens inside the immature, budded virus particle.
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