Nicholas Schifano
Dr. Kahn
Nucleic Acids II
December 17, 2021
Inhibition of Flavivirus by Antisense Oligomers Specifically Suppressing Viral Translation and RNA
Replication
Flaviviruses are a family of viruses that consist of a single-stranded, enveloped virus. These viruses
can be found in primarily ticks and mosquitos and some viruses can be infectious to humans. Flavivirus
can cause sporadic and epidemic illness in humans. West Nile Virus (WNV) is one of the leading causes of
mosquito-borne disease and resides in one the family of flavivirus. Although, the severity of infection is
that only 1 in 150 people develop serious illness, the complication lies that there are no commercially
available treatments for the virus as well vaccines1,2.
Antisense Oligonucleotides are synthetic polymers in which some or all of the natural monomers
of the nucleotides are chemically altered DNA and RNA sequences. These 15-25 monomeric sequences of
DNA or RNA have the ability to target a specific sense mRNA. Due to the specificity of them, this has made
antisense compounds have become effective tool for basic molecular biology and often used for drug
discovery and validation. Antisense compounds act by blocking upstream message for receptor
substrates3. A natural analog such as puromycin provided an early example of antisense inhibition of
protein synthesis4 Originally one of the limits to this biotechnology was that only short sequences can be
ligated together, in the 1960’s it was trinucleotides and by the end of the decade oligomers 10-15 U in
length were able to be constructed. Reverse transcriptase then made it more feasible for sequencing
oligonucleotides by synthesizing the primary structure of DNA enzymatically, this would then allow the
nascent DNA that is formed can be sequenced.
More recent advancements were made with the development of phosphorodiamidate
morpholino oligomers (PMO) which are short single stranded DNA analogs with morpholine rings
connected by phosphorodiamidate linkages5. PMOs specifically bind to complementary sequences of
target mRNA by Watson-Crick base pairing and block mRNA through a steric blockade. One of the greatest
appeals to PMO antisense therapies is that the PMO is resistant to a variety of enzymes in biologic fluids,
which allows for suitable in vivo applications6.
The paper that I am discussing today uses this advancement to develop a PMO based antisense
oligomer to inhibit viral transmission and RNA replication. The idea of the paper and overall goal was to
use what is known about the genome of WNV and make an effective PMO inhibitor. The first step was
looking at the genomic sequence of RNA and which parts of it can be vital for viral replication. It was seen
that there is a 5’ untranslated region (UTR), a single open reading frame (ORF), and a 3’ UTR within the
RNA genome. Through investigation, it was shown that 5’ and 3’ UTRs may direct the process of translation
and for viral replication. It has also been understood that conservation within the genome is also vital for
the replication process of the flavivirus. Using this information, they were able come up with sequences
attached to a peptide (for delivery purposes) to inhibit the viral replication and translation process. They
looked at the at the different conserved species and or formations, for example conserved start codons
or conserved stem or loop structures as a basis for PMO (Fig. 1). They used a scrambled sequence as a
negative control. The overall important point that came from this was that it’s possible that not just the
conservation of the sequence is important but specific conservations in structure like the circularization
interactions specifically on 3’ end and pseudoknots formed (Pskt). Judging from the assay results it seems
that loops formed or stems right before the loop are vital and would be interesting to see further
experiments.
Taking a look at one of the antiviral assays that they had preformed they use a developed cell line
containing a dual-reporter replicon (RlucNeoRep) (Fig 2, in my figures). The idea is that when this reporter
is incubated with something that is able to block replication, then it will suppress the Rluc expression level.
In the paper they identify a couple significant PMOs that show the highest potency for this action. They
had identified that the 3’CSI, AUG-1, and 5’ END had the greatest efficiency at preventing the replication
process. This suggests the opposite what I have previously mentioned before about confirmations or
structural conservation is more vital to replication than the sequence itself. However, they identified these
and yet there were more effective variants relative to structural targets. I would assume that this was to
illuminate the multifaceted tool set that PMOs can be. The results are indicative of PMOs being a valid
therapeutic agent because inhibition was caused at concentrations as low as 2.5 µM of PMO. The assay
shows the ability of these PMOs to a degree however, there were other things missing. I would have liked
to see couple controls done that weren’t run with the experiment. For example, there was no positive
control, there should be something that directly inhibits the Rluc expression that should be ran along side,
something that is absolutely sure to inhibit WT WNV. This could bring other information like a delta or
changes in expression level, also expectations of what to see for inhibition (is 2.5 µM an impressively low
concentration?). These controls would also allow for a subset of graphs that represent normalized values
on what the max high and max low values could be.
The PMOs proposed in this publication proved to be a valid concept for a therapeutic potential,
this is due to the high selectivity and low cytotoxicity. Regardless of whether 2.5 µM is an impressively
low concentration for a therapeutic, the important aspect is that the PMOs retain low if any at all
cytotoxicity which is vital for the development of therapeutics. This research has shown possibilities for
the future in combating flaviviruses. In terms of specific research there are numerous areas of study that
can be done from these results. Things that I would like to see would be, PMOs that target the loop
structure after the Pskt stem, this would give a good idea on what could be more important. I also, it may
be risky, but it would be interesting to see that if mutating single nucleic acids that the PMOs would still
selectively inhibit. I figured this would be a little difficult since mutating the RNA could inherently inhibit,
however coming up with a type of experiment in that nature will give understanding in a structure activity
relationship. Also, even though they claim that the arginine rich peptides are great delivery systems for
these PMOs I think that studies on that would be great as well. Under
Figures
Figure 1
Figure 2
References
1. S. W. van Leur, T. Heunis, D. Munnur, and S. Sanyal, "Pathogenesis and virulence of flavivirus
infections," (in eng), Virulence, vol. 12, no. 1, pp. 2814-2838, Dec 2021, doi:
10.1080/21505594.2021.1996059.
2. L. A. Beltz, "Chapter 1 - Introduction to Flaviviruses," in Zika and Other Neglected and Emerging
Flaviviruses, L. A. Beltz Ed.: Elsevier, 2021, pp. 1-18.
3. Custom antisense oligonucleotide synthesis. Creative Biolabs. (n.d.). Retrieved December
18, 2021.
4. D. W. Allen and P. C. Zamecnik, "The effect of puromycin on rabbit reticulocyte ribosomes," (in
eng), Biochim Biophys Acta, vol. 55, pp. 865-74, Jun 11 1962, doi: 10.1016/0006-3002(62)908995.
5. F. Sanger and A. R. Coulson, "A rapid method for determining sequences in DNA by primed
synthesis with DNA polymerase," (in eng), J Mol Biol, vol. 94, no. 3, pp. 441-8, May 25 1975, doi:
10.1016/0022-2836(75)90213-2.
6. J. Summerton, "Morpholino antisense oligomers: the case for an RNase H-independent
structural type," (in eng), Biochim Biophys Acta, vol. 1489, no. 1, pp. 141-58, Dec 10 1999, doi:
10.1016/s0167-4781(99)00150-5.
7. Y. Nan and Y.-J. Zhang, "Antisense Phosphorodiamidate Morpholino Oligomers as Novel Antiviral
Compounds," (in eng), Front Microbiol, vol. 9, pp. 750-750, 2018, doi:
10.3389/fmicb.2018.00750.
8. Inhibition of flavivirus infections by ... - journals.asm.org. (n.d.). Retrieved December 18,
2021, from https://journals.asm.org/doi/abs/10.1128/jvi.79.8.4599-4609.2005