Influence of wobble RNA on MxA protein folding, localization and function One of the key differences observed was the impact of the antiviral-null mutant

T103A, which although attenuated in comparison to wt wMxA, was not completely antiviral-null in the polymerase reconstitution assay using the polymerase and NP proteins of either A/Udorn/72 virus or an H5N1 virus. Interestingly a recent publication looking at the impact of G-domain mutations in MxA showed a

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Figure 4.4 The effects of MxA on A/Thailand/1(KAN-1)/04 (H5N1) virus polymerase

activity in a luciferase-based mini-genome assay. A. 293T cells were transfected with plasmids expressing the polymerase subunits and NP of A/Thailand/1(KAN-1)/04 (H5N1) virus (10 ng each), a renilla-encoding mini-genome plasmid (250 ng), a plasmid encoding firefly luciferase (10ng) and the various wMxA constructs (200 ng each). The relative luciferase activity was measured in a dual luciferase assay. Results are represented as a percentage of luciferase activity in the no MxA control and are the average of three independent experiments ± S.D. B. Immunoblot showing the level of MxA expressed following 24h expression in 293T cells. MxA was detected through an !-Rabbit MxA antibody and actin levels were detected as a loading control. (* denotes p-value = <0.05 following students T-test)

WT MxA AKAK KEKE D 478A T

103A _{R640A V}268M _L612K _I376D

G 255E F 561V _F602D _-VE Actin A B

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phenotypically different immunostaining pattern for T103A MxA than observed in section 4.1 for T103A wMxA (Dick et al. 2015a). Furthermore it was also shown that one of the antiviral-null human polymorphism mutations, G255E, showed a similar staining pattern to T103A MxA, again differing from what was observed for G255E wMxA.

Upon recognising these differences, the previously assessed mutations were then introduced to the wt MxA mRNA background and cloned into the mammalian expression vector pCAGGS to evaluate the phenotypic expression patterns of these mutations in both the wt mRNA background and the wobble mRNA background. 293T cells were transfected with each of the constructs and the distribution of MxA determined by immunofluorescence. Distribution of the wt MxA protein was similar to the wMxA constructs, as they displayed a distinct punctate phenotype associated with the over-expression of MxA with an otherwise diffuse cytoplasmic staining (Fig. 4.5). The distribution of MxA proteins containing other mutations was also similar in both mRNA backgrounds as seen for V268M, R640A, F561V and both lipid binding mutants AKAK and KEKE. Both the monomeric mutations F602D and L612K also show diffuse cytoplasmic staining with no punctate staining of MxA, indicating that the change in mRNA sequence did not impact the overall localization and functionality of these mutants.

However, there are some clear phenotypic differences in the appearance of a number of different mutants. Firstly, the antiviral-null mutant T103A showed a significantly altered expression pattern when expressed from the wt mRNA sequence when compared to the wobble mRNA sequence. MxA derived from the wt mRNA sequence

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WT MxA!

MxA

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T103A MxA! R640A MxA!

wMxA

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F561V MxA F602D MxA L612K MxA

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I376D MxA AKAK MxA KEKE MxA

Figure 4.5 Immunofluorescence comparison of MxA derived from wt MxA mRNA- and wMxA mRNA-expressing constructs. 293T cells were transfected with 500ng plasmid to express MxA from either the endogenous mRNA background or the wobble mRNA background. 24 hours post-transfection cells were fixed and probed with an !-MxA polyclonal rabbit antibody followed by an Alexa-488 conjugated goat !-rabbit antibody. Nuclei were stained using DAPI. Green; !-MxA, Blue; DAPI-stained nuclei.

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showed an aggregation pattern within the cytoplasm as described previously by Dick et al. (2015) whereas the MxA expression pattern from the wobble mRNA background shows a distinctly wt-like phenotype. This suggests that the mRNA sequence may play an important role in the overall protein expression pattern as these two proteins share the exact same amino acid sequence. A similar difference was observed between MxA proteins expressed from the wt-mRNA and wobble mRNA sequences contianing the human polymorphism G255E, with MxA derived from the wt mRNA sequence showing a clear punctate pattern, whereas that from the wobble mRNA demonstrated a wt-like expression pattern.

Two other mutants showed differing phenotypes when expressed from the different mRNA backgrounds. Both I376D and D478A present with a wt-like punctate over- expression phenotype from the wobble mRNA background. However, from the wt mRNA sequence these two mutants offer very different staining patterns. I376D was previously described to disrupt tetramer formation resulting in a predominantly dimeric form of MxA (S. Gao et al. 2010). However no immunofluorescence or antiviral functionality data was presented. The introduction of I376D into the wt mRNA background produced a striking phenotype suggestive of long oligomeric structures in the perinuclear region, which appears to be dependent on the level of MxA protein available as surrounding cells with less MxA expression appear to only have the beginnings of this structure whilst having a diffuse cytoplasmic background staining. D478A is a stalk mutant that has been shown to behave similarly to wild type in vitro and this mutant was shown to localise with LACV Nucleoprotein (N) (S. Gao et al. 2011). The expression phenotype seen in the wt mRNA background matches the previously published phenotype, however, this is not in the context of

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LACV N, suggesting that this staining pattern is indicative of MxA localizing with another cellular organelle in the perinuclear region.

Following the identification of these differences in phenotype it was necessary to ascertain the impact of these mutations on the antiviral activity of MxA against influenza A virus post primary transcription. Figure 4.6 shows the comparison between the A/Thailand/1(KAN-1)/04 virus polymerase activity in the presence of the MxA mutants expressed from both the wt mRNA and wobble mRNA backgrounds. Both T103A and G255E show complete attenuation in the ability to produce an antiviral effect against influenza A virus when expressed from wt mRNA. This matches the data published by Dick et al. (2015) for both T103A and G255E, and is not surprising considering the aggregated phenotypes displayed by both of these mutants. Interestingly, the two other mutants that appear to display a non-wt like expression pattern after expression from wt MxA mRNA (D478A and I376D) do not appear to differ in antiviral activity regardless of the mRNA background, displaying inhibition at around the 50% and 60% mark for D478A and I376D respectively, which matches the antiviral activity previously stated for the D478A mutation (S. Gao et al. 2011). Although the change in mRNA background appears to have accounted for the majority of discrepancies between the data presented here and previously published data, R640A demonstrated a decent level of antiviral activity in both mRNA backgrounds, despite being previously described as antiviral null (S. Gao et al. 2011).

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Figure 4.6 Comparison between wMxA and MxA constructs in A/Thailand/1(Kan- 1)/04 viral polymerase activity in a luciferase-based mini-genome assay. 293T cells were transfected with plasmids expressing the polymerase subunits and NP of A/Thailand/1(KAN-1)/04 (H5N1) virus (10 ng each), a renilla-encoding mini-genome plasmid (250 ng), a plasmid encoding firefly luciferase (10ng) and the various MxA and wMxA constructs (200 ng each). The relative luciferase activity was measured in a dual luciferase assay. Results are represented as a percentage of luciferase activity in the no MxA control and are the average of three independent experiments ± S.D.

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