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TWiV 95 Letters

Jim writes:

Some listeners might benefit from reading "The Treatment; why is it so difficult to develop drugs for cancer" in the May 17, 2010 issue of The New Yorker, a nine page article (pp 68-77).

This link goes to the digital edition ( http://archives.newyorker.com/?i=2010-05-17#folio=076 ), but you have to be a subscriber ($) to access it.

One part of the article describes the mass screening of chemicals for potential compounds and points out that although industrial research labs like Kodak and Fujifilm have millions of chemicals in their vaults, Pfizer was screening only 600,000 and Merck about 500,000. One researcher obtained a grant and purchased 22,000 chemicals from Russia and Ukraine at $10 each [AD: I have an aside about these Russian libraries], then screened them ninety-six at a time and a hundred batches a day with automatic equipment to find some suitable candidates for clinical tests that eventually produced elesclomol. They never explained why Kodak and Fujifilm were not approached. Has anyone made an effort to locate where such chemicals are assembled and how they might be accessed?

Another interesting point was the importance of the Kaplan-Meier curve that is the turning point of any clinical trial in determining its success. Interesting insight.

Jim
VA

Ricardo writes:

Hello TWIV Professors,

Please read the answers or at least the winning answer of the DROBO Contest part 2 (Influenza replication in bacteria) so all listeners can learn with the winner of the contest.

Thank you,

Ricardo
Brazil

This is the winning answer (from Jeff):

Would it be possible to genetically engineer influenza virus so it could replicate in bacteria? Why or Why not?

First, this depends on the definition of "replication". But there are lots of reasons why flu cannot infect and replicate in E. coli.

1. E. coli doesn't have the required receptor proteins to bind flu.

2. E. coli doesn't have a mechanism to internalize flu through it's cell wall.

3. E. coli doesn't have capped mRNAs which are needed to create flu mRNAs through the "cap-stealing mechanism".

4. E. coli doesn't have a mechanism to splice flu mRNAs to produce the full complement of flu proteins; this normally takes place in the nucleus of eukaryotic cells.

5. E. coli doesn't have an environment equivalent to the acidic endosome needed to allow escape of the nucleocapsid.

6. E. coli doesn't have ER/Golgi (or enzymes) to produce N-linked glycosylated proteins.

7. Flu HA and NA proteins would not be directed to inner membrane of E. coli; signal sequences wouldn't work; therefore virus would not assemble on inner membrane.

8. E. coli doesn't have protease to activate flu HA to make the protein competent to trigger fusion.

9. Even if flu mRNAs were made (with or without CAPs) they couldn't be translated in E. coli (no ribosome binding sites).

10. A different codon-usage bias may interfere with protein translation.

11. Formyl-methionine would be placed at the N-term of flu proteins, may interfere with function.

12. Proteins may not fold properly in E. coli and they may interact (bind) to E. coli proteins inappropriately, reducing their effective concentration. For example basic domains used as Nuclear Localization Signals in mammalian cells may cause problems.

However, perhaps we can use a less stringent definition of "replication".

Perhaps it's possible to try and engineer a system to replicate the viral RNAs in E. coli. This would be similar in some aspects to the reverse genetics systems used for flu in mammalian cells.

Instead of trying to express all the proteins of flu, we could limit ourselves to the proteins required to replicate a flu genome segment. The flu proteins would need to be under E. coli transcription/translation control. For replication would need PB1, PB2, PA and NP proteins. It should also be possible to use plasmids with two promoters (opposite orientation) to get 1 or more of the flu genome strands (opposite of the mRNA) made to act as template for the replicase.

Maintaining everything in E. coli, is a little tricky; plasmids with different compatibility groups (plasmid maintenance in E. coli) and antibiotic resistance for selection would be needed. This problem would go away if an in vitro transcription/translation (still prokaryotic) were used.

One further obstacle to this system is generating the proteins in correct amounts; would need a lot more NP than replication enzymes.

 

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