Kim Tran
Bio 509
28 November 2010
Perez, J., Pham, A., Lorini, M., Chua, M., Steel, J., & TenOever, B. “MicroRNA-mediated species-specific attenuation of influenza A virus.” Nature Biotechnology , 27 (6), 572-576.
(31 May 2009).
Objective
“The study conducted in “MicroRNA-mediated species-specific attenuation of influenza A virus,” proposes a method to develop a complementary attenuation strategy using microRNAmediated gene silencing. Most microRNAs are evolutionarily conserved, but a few are species specific. The species specific microRNA allows for the attenuation of the influenza A virus in mammalian, but not in avian. Attenuation in mammalians is by the direct binding of microRNA to mRNA which will result in translational repression. The specificity of the microRNA only in mammalian permits a high yield of vaccines when reproduced in chicken eggs.
Experiments and Results
The first experiment tested for affects on formation, maturation, and post-transcriptional gene silencing of microRNA when a cell is infected by the influenza A virus. miR-124 (a tissue specific microRNA) and miR-93(a ubiquitous microRNA) were transfected into HEK293 cells
(Human embryonic kidney celss) using a red fluorescent protein minigene. The exogenous miR-
124 and miR-93 were processed to maturation when cells were infected by influenza A virus.
Endogenous miR-93 expression was observed in human and murine fibroblast using a Northern blot. There was no change in expression throughout the course of infection. A luciferase reporter with miR-124 target sequences was used to monitor miR-124 in HEK293 cells. Under normal condition miR-124 supressed 90% of luciferase activity and suppression was not affected when exposed to influenza A virus or NS1(protein from virus that disrupts cellular activity of host cells). This experiment concluded that influenza A virus does not affect formation, maturation, or gene silencing function of microRNA.
The second experiment is to design a virus that would attenuate in humans and mice but not in chicken eggs. First, miR-93 target sites were incorporated in influenza a virus. miR-93 was ideal because it is a species specific microRNA, only expressing in mammalian and not in avian.
Segment 5 of the viral genome, encoding nucleoproteins, had high amino acid conservation; this made it a perfect location to form target sites while also reducing the chances of mutants escaping and enable targeting of diverse sets of recombinant influenza A virus strain. Two sites on segment 5 of the viral genome were altered to form microRNA response elements(MRE) that had high affinity to miR-93. The first MRE was positioned at site 1, 93NP1and the second MRE was positioned at site 2, 93NP2. The codon usage at the two sites was not conserved in H1N1,
H3N2, and H5N1; however they did conserve the hydrophobicity, polarity and charge; so, alterations in the two sites were three amino acid substitutions that maintained the original class of amino acids. A control virus (PRNTL) was designed using strain H1N1 with the three amino acid substitutions, but also had mutations that prevented miR-93 binding. This was used to study the effects of changes in the nucleoprotein structure caused by the substitutions.

The third experiment used the PRNTL virus to test if the nucleoprotein encoded by segment 5 maintained its function after having the three base substitutions. An antisense reporter construct with RNA-dependent RNA-polymerase-driven luciferase expression was used. It showed a 20% reduction in RdRp activity, when compared to wild type, which is not a substantial effect on function of the nucleoprotein. In vitro infection of HEK293 cells with PRNTL and wild type
H1N1 showed no difference in amount of nucleoprotein present; however in vivo infection of
BALB/c mice showed that PRNTL virus was attenuated, showing fewer amounts of nucleoproteins. This may have reflected the 20% reduction of RdRp activity. Overall, there was not a substantial loss in the function of segment 5.
The fourth experiment compared MRE-containing virus 93NP1/2 (H1N1 strain with the three base substitutions) with PRNTL virus (H1N1 with mutations that inhibit binding of miR-93).
Mice were exposed to each virus strain by intranasal inoculation. The median lethal dose for
PRNTL was 1.7 x 10
3
PFU, while the lethal dose for 93NP1/2 had a greater than 2 log difference,
2.15 x 10
5
PFU.
The fifth experiment tested the species specificity of miR-93. 10 day old chicken eggs were inoculated with 100PFU of wild type, PRNTL, 93NP1 (only site 1 altered) , 93NP2 (only site 2 altered), and 93NP1/2. Two days after infection, allantoic fluid was harvested and each strain showed approximately 10
8
PFU/ml. This meant that there was no attenuation in chicken eggs with any of the strains. H5N1 reassortment strain containing MRE-containing nucleoprotein segment 5 from H1N1 was also inoculated into chicken eggs. Titers of 10
8
PFU/ml were observed, revealing that H5N1 strain with H1N1 MRE-containing segment 5 is not attenuated in ovo .
The sixth experiment was to make sure the attenuation was due to microRNA-mediated gene silencing and second, to make sure it was specifically due to miR-93. Wild type murine fibroblast and Dicer
-/-
murine fibroblast (cannot process miRNA) were infected with the four diff strains of virus. When infected with PRNTL, wild type murine fibroblast produced hemagglutinin. With 93NP1 and 93NP2, wild type murine fibroblast showed reduction in production of hemagglutinin. With 93NP1/2, complete attenuation was observed and no hemagglutinin was produced. Dicer
-/-
murine fibroblast showed high levels of hemagglutinin in all four strains because dicer was not present to process microRNA to attenuate the viral strain.
This proved that microRNA was responsible for attenuation. To test that attenuation was miR-93 specific, HEK293 cells were treated with Mercury LNA anti-miR-93 or scrambled RNA
(control). Then cells were infected with PRNTL, 93NP1, or 93NP1/2. Cells treated with antimiR-93 and infected with 93NP1/2 had double the levels of nucleoprotein than cells treated with scrambled RNA and infected with 93NP1/2. This was because cells with anti-miR-93 bounded to miR-93 allowed segment 5 in the virus to produce nucleoprotein. Scramble DNA was not miR-
93 specific, so miR-93 directly bounded to segment 5, attenuating production of nucleoprotein.
Further test of the attenuation mechanism in HEK293 cells infected with 93NP1/2 revealed high levels of nucleoprotein mRNA, but low levels of protein. This is due to nucleoprotein mRNA present but attenuated by miR-93; therefore translation was repressed leading to low levels of protein.

The seventh experiment tested the effectiveness of H1N1 with MRE as a live attenuated influenza vaccine (LAIV). Mice were infected with PRNTL H1N1 or 93NP1/2 recombinant
H1N1. PRNTL mice had greater weight loss then 93NP1/2 mice. A lethal dose of H1N1 was given 21 days after the mice were infected with PRNTL or 93NP1/2 and resulted in 100% survival. Serum from mice also showed high level of antibodies.
The final experiment demonstrated the versatility of a MRE-based vaccine. H5N1 with MRE strain and H5N1 with PRNTL strain were used to infect mice. PRNTL caused 50% mortality and
20% loss in body weight. H5N1 containing MRE had 100% survival and only a small loss in body weight. 21 days after infection mice were given a lethal dose of H5N1. Mice that were previously infected with H5N1 containing MRE had 100% survival and high levels of antibodies.
Conclusion
The MRE base strategy has been proven to attenuate influenza A virus. It maintained its species specificity and succeeded in attenuating production of nucleoprotein which plays a role in the production of viral RNA. The H1N1 containing MRE strain that was used in the experiments does not provide protection against any current circulating strains; however versatility of the
MRE base method has been proven with viral strain H5N1. When MRE was incorporated into
H5N1, the strain was species specific and was able to attenuate the virus. The benefit of the
MRE base strategy is that it can be customized for how many MRE sites and can also be used with tissue specific microRNA. This method can be used with current attenuating methods to increase safety of vaccines.