Using Myxoma Virus to Understand the Intrinsic Immune Properties of SAMD9 Protein

Jennifer Chen1, Naiha Ahmad2*,Jia Liu2**

1University of Arkansas at Fayetteville

2Department of Microbiology and Immunology, University of Arkansas for Medical Sciences (UAMS), Little Rock, AR, USA

*Co-author

**Corresponding author

Conclusions and Future Directions

Abstract
Poxviruses are useful for studying host species barrier and host immunity, due to their ability to successfully evade host Table 1. Protein concentrations (ug/mL) found using Bradford Assay.
Results The results generated in the presented experiment differ from the results generated previously by the Liu lab. The
defense. Various members of the poxvirus family possess proteins that inhibit a mammalian protein, called sterile alpha motif apparent attenuation of early gene expression caused by MAV-M062R can not be confirmed by previous results. A
Samples Mock 1 Lu 1H Lu 4H Lu 8H Lu 12H Table 1. The protein concentrations of the samples, found using Bradford
protein 9 (SAMD9), for a successful infection. SAMD9 is important to human health, but its function is poorly understood. We OD595 0.189 0.179 0.178 0.191 0.192 biological replicate of this experiment will be needed to examine the differences in results.
Assay. The samples were diluted with water at a 1:10 ratio. 2 uL of sample
utilized myxoma virus (MYXV), a rabbit specific poxvirus, as a tool to study SAMD9, as MYXV encodes a sole inhibitor of ug/mL 1.095 0.562 0.470 1.168 1.223 -If the mutant virus infection indeed affects early viral protein expression and not just intermediate and late gene
loaded with 100 uL Bradford reagent. The optical density (OD),
DF 10 10 10 10 10 expression (as seen previously), the antiviral mechanism will have to be triggered much earlier during the infection. In
human SAMD9, called M062 protein. We thus can investigate SAMD9’s function by exploiting the interactions between
concentrations (ug/mL) and dilution factors are indicated.
this scenario, it is speculated that SAMD9 may be triggered very early during infection, if it is not effectively inhibited.
SAMD9 and MYXV M062 protein. Liu lab generated a panel of mutant MYXVs for the study and among them one mutant Samples Mock 2 MAV 1H MAV 4H MAV 8H MAV 12H
OD595 0.176 0.191 0.184 0.182 0.187 In this case, the cause of such antiviral effect administrated by SAMD9, will need to be further examined.
virus expressing a mutated M062 called MAV-M062R is chosen for the study. As control, a wildtype MYXV called Lu-
ug/mL 0.341 1.168 0.801 0.691 0.095 -If when this experiment is repeated and consistent results are observed with what others have previously reported, the
M062R is used. The mutation in MAV-M062R (I79T) abolished its binding to the SAMD9 protein, resulting in abnormal viral
DF 10 10 10 10 10 following discussion will explain the cause of the results in this experiment. Before the cell lysate was harvested for
protein synthesis. In this study, we utilized Western blot to examine viral protein production that characterized such effect. This infection at 8H or 12H post infection, the cells were not examined under a fluorescent microscope to determine the
Figure 2. Bradford Assay Standard Curve.
defect in viral protein synthesis leads to attenuated infection. We thus can understand how SAMD9 protein performs antiviral infectivity outcome of using these viruses. Both WT and mutant viruses were engineered to express mCherry protein, a

effect in host cells. bright red fluorescent protein. The percentage of red cells, in a given view, will provide information on how well the

infection occurred. This confirmation step is important and will be performed when this experiment is repeated.

Figure 2. The BSA standard curve was used to accurately quantify the concentration of
our protein samples (Table 1), by comparing them to these known BSA concentrations
(1000 ug/mL, 500 ug/mL, 250 ug/mL, 125 ug/mL, 62.5 ug/mL, 31 ug/mL, and 0 ug/mL.
Inefficient infection by the mutant virus may have caused the phenotype seen from this experiment.​
-In several probings, the expected bands did not appear, e.g., V5 tagged M062 bands (Fig 3A), an early viral proteins,
Serp-1 and mCherry, both late proteins (Fig 3B). This is likely due to insufficient primary antibody incubation,
insufficient protein loading, or insufficient substrate exposure time. These will be carefully managed in the replicate

The equation for line of best fit is shown on the graph. experiments, when this study is repeated.

-In the results shown, intermediate viral protein expression (M038 in Fig 3A and B) were attenuated in the mutant viral

infection, which is expected and consistent with results from others.

-Through this study, the aim is to determine the defect of mutant viral infection caused by antiviral translation control at

the molecular levels.

1 2 3 4 5 6 7 8 9

Introduction
SAMD9 is a conserved cytoplasmic protein in mammalian cells. Its deleterious mutation is associated with human diseases Figure 3. Western Blot to Analyze Viral Early/Intermediate/Late Protein Expression of Lu-M062R and MAV-M062R in HeLa Cells.
(NFTC, MIRAGE Syndrome, Myeloid Malignancies). SAMD9 presents anti-poxvirus properties with an unknown mechanism.

In order to understand the function of this protein, recombinant MYXV viruses with mutated SAMD9 inhibitor (e.g., I79T

MAV-M062R) were generated. Two recombinant MYXV, Lu-M062R (wildtype virus) and MAV-M062R (mutant virus) were
A B
engineered (Fig. 1).

10 11 12 13 14 15 16 17 18

Figure 3. Western Blot Results. At the given time points during infection (Fig. 3A), early (V5 tagged M062 and M040) and intermediate (M038) gene expression were compared between wildtype (Lu-M062R) and mutant virus. In

Fig 3B, intermediate (M038) and late (Serp1 and mCherry) protein expression are compared between the two viruses. Beta actin served as internal control for loading. Infection by MAV- M062R caused significant reduction in early

gene expression along with intermediate gene expression, when compared to WT Lu-M062R. This is consistent with the consequence that viral intermediate and late protein expression are also attenuated in MAV-M062R infection,

when compared to Lu-M062R. This experiment will be repeated to account for the probings that did not work.
Acknowledgements
Fig. 1. Lu-M062R is the wildtype MYXV. MAV-M062R is a mutant with a I>T replacement at the 79th residue. Both

recombinant viruses were engineered to have V5 tag at the c-terminus of the corresponding M062. -Dr. Jia Liu, PhD, mentor

-Naiha Ahmad, UAMS Graduate Student

-University of Arkansas for Medical Sciences INBRE

-Diane McKinstry , INBRE Program Director

-This project was supported by the Arkansas INBRE program, with the National Institute of General Medical Sciences –

Methodology
NIGMS (P20 GM103429) from the National Institutes of Health.

Western Blot to Analyze Viral Protein Expression:

Cell Seeding: 1.8x105 HeLa cells were seeded, per well, in a 12 well plate.

Infection: HeLa cells infected with Lu-M062R and MAV-M062R (MOI=5). Cells incubated in DMEM at time points: 1H, 4H, 8H,12H.
References
Harvesting: DMEM was aspirated out at the different time points p.i. Lysis buffer was used to release protein contents. The samples were then centrifuged (10 min, 13,500 RPM, 4º)
1. Liu, Jia et al. “The poxvirus C7L host range factor superfamily.” Current opinion in virology vol.
Bradford Assay: protein concentrations were found using samples with a 1:10 dilution with water.
2,6(2012): 758-766.
SDS-PAGE: 10%, 10 well SDS-PAGE gel was used.

Antibodies probed: V5-M062 (early/late protein expression), M040 (early), M038 (intermediate), Serp1 (late), and mCherry (late). Beta actin was also probed for and acted as internal control.
2. Liu, Jia et al. “M062 is a host range factor essential for myxoma virus pathogenesis and function
as an antagonist for host SAMD9 in human cells.” Journal of virology vol. 85,7(2011): 3270-82.

3. Ahmad, Naiha. “Understanding the function of SAMD9 using Myxoma virus as a tool.” Progress