[BMB411 READINGS IN BMB 09/01/17] 1

Paper: Takata, et al. (2017). CG dinucleotide suppression enables antiviral

defence targeting non-self RNA." Nature 550:124-127 + supplemental online material
(see also the fasta and sequence alignment files in course docs).

Please fashion preliminary answers to the following questions before our class meeting
on Friday 10/13/17 and email me your responses (rmyers@miami.edu); this will help
direct your reading of the paper. Write your answers directly into this Pre-Discussion
File following each question; do not modify the file in any other way. Save your answers
to a file named exactly like this:

Pre-Discussion BMB411 Fall 2017 Paper Four YOUR NAME

(where YOUR NAME is your namee.g. RIK MYERS)

Send this file as an attachment to rmyers@miami.edu with the following subject heading
(exactly as written below):

Pre-Discussion BMB411 Fall 2017 Paper Four

After the meeting on 10/13/17, revisit your answers and modify them to suit your
evolved understanding. Mark your altered text using yellow highlights. Return these re-
answered questions via email to rmyers@miami.edu before 5 PM on Sunday 10/15/17.
Save the second set of answers as before, except replace Pre-Discussion with Post-
Discussion. Also send your email with the subject as I said before except again replace
Pre-Discussion with Post-Discussion. I want two sets of answers from you. If your
understanding remains the same after the discussion, submit your answers again with
the appropriate change to the file name. I use this information to see how your thinking
is altered by the in-class discussion.

Q1: What is the overall hypothesis of the paper?

-CG dinucleotide suppression enables anti-viral defense targeting non-self

RNA

Q2: In general terms, what is the plan Takata et al. came up with to test
their hypothesis in Q1? For example: Did they create mutants, knock-down
expression of a key gene and examine phenotypes, look for protein-protein
interactions, etc.
-They created mutants and examined phenotypic consequences in the
mutants

Q3: What experimental techniques were used to carry out the plan in Q2?
-Dna splicing
Q4: Why did the authors choose to use human 293T (likely derived from an
embryonic adrenal precursor cell next to kidney, stably transfected by a
temperature-sensitive variant of the SV40 T antigen gene and the gene for
resistance to neomycin), HOS (human osteosarcoma), HeLa (human papilloma
virus 18 infected cervical cancer) cells and primary human lymphocytes for the
reported studies? What unique features led the investigators to use these cell
lines? What experimental limits did this choice of research material create?
Human cells were a better model system to study RNA viruses because HIV
is predominantly a human-organism threatening issues.

Q5: What were the overall conclusions from these studies?

The induced mutation was successful.

Q6: What topic or question do you think the authors will take up next based
on this paper? What are the important next questions raised?
Will this have the same effect with different cell types?

Q7: Identify three different experiments that the authors used to come to the
conclusion you stated in answer to Question 5. State what you perceive to be a
limitation to one or more of these experiments that might weaken their
conclusions.
They did not check for mycoplasma contamination.

Q8: How did transduction by a lentivirus expressing CRISPR/Cas with a

ZAP exon 1-targeting guide sequence generate ZAP-deficient HOS cells?
CRISPR/Cas9 can target and remove a gene using markers. So the
CRISPR would remove the ZAP gene.

Q9: What are the implications of the observation that 5-CG-3 sequences in
exons and 3 untranslated regions - but not introns - of HIV-1 pre-mRNA are
necessary for inhibition of HIV-1 protein synthesis by ZAP protein?
Introns are spliced out, and the exons represent coded data.

Q10: One implication of this paper is that HIV-1 quasi-species populations

(ask if you are unfamiliar with this term) have evolved resistance to ZAP. In the
context of the analogy of an arms race between adaptive offensive and
defensive strategies, what do you anticipate the next move to be by human
evolution? Likewise for HIV evolution. Consider the relative rates of evolution of
the human species and of retroviral quasi-species.
 [BMB411 READINGS IN BMB 09/01/17] 3

The next move human evolution may take would be to develop a

mechanism that suppresses CG expression etc.