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

Viral Early/Intermediate/Late Protein Expression of Lu-

ABSTRACT Infection of Hela Cells with MAV-M062R/Lu-M062R M062R and MAV-M062R
Figure 3A. Western blot results for early (V5-M062, M040) and intermediate
Poxviruses are useful for studying host species barrier and host immunity,
(M038) viral protein expression.
due to their ability to successfully evade host defense. Various members of
the poxvirus family possess proteins that inhibit a mammalian protein,
called sterile alpha motif protein 9 (SAMD9), for a successful infection.
SAMD9 is important to human health, but its function is poorly
understood. We utilized myxoma virus (MYXV), a rabbit specific poxvirus,
as a tool to study SAMD9, as MYXV encodes a sole inhibitor of human
SAMD9, called M062 protein. We thus can investigate SAMD9’s function by 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.
exploiting the interactions between SAMD9 and MYXV M062 protein. Liu 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) Figure 3B. Western blot results for late viral protein expression (SERP1 and
lab generated a panel of mutant MYXVs for the study and among them one
mCherry)
mutant virus expressing a mutated M062 called MAV-M062R is chosen for
the study. As control, a wildtype MYXV called Lu-M062R is used. The
Protein Concentrations using Bradford Assay
mutation in MAV-M062R (I79T) abolished its binding to the SAMD9
protein, resulting in abnormal viral protein synthesis. In this study, we Table 1-2. Protein concentrations (ug/mL) found using Bradford Assay.
utilized Western blot to examine viral protein production that
characterized such effect. This defect in viral protein synthesis leads to Samples Mock 2 MAV 1H MAV 4H MAV 8H MAV 12H Table 1. Early/intermediate time point
samples to analyze early/intermediate
attenuated infection. We thus can understand how SAMD9 protein OD595 0.176 0.191 0.184 0.182 0.187 protein expression. The samples were
Figure 3. Western Blot Results. At the given time points during infection (Fig. 3A), early (V5 tagged M062 and
performs antiviral effect in host cells. diluted with water at a 1:10 ratio. 2 uL M040) and intermediate (M038) gene expression were compared between wildtype (Lu-M062R) and mutant
ug/mL 0.341 1.168 0.801 0.691 0.095
of sample loaded with 100 uL virus. In Fig 3B, late (Serp1 and mCherry) protein expression is compared between the two viruses. Beta-
Bradford reagent. The optical density actin served as internal control for loading. Lu-M062R and MAV-M062R showed comparable early protein
DF 10 10 10 10 10
(OD), concentrations (ug/mL) and expression. Intermediate (M028) and late (SERP1 and mCherry) protein expression was shown to be
dilution factors are indicated. attenuated in MAV-M062R when compared to Lu-M062R.

INTRODUCTION Table 2. Later time points of infection.

CONCLUSIONS/FUTURE DIRECTIONS
Samples Mock 1 Lu 1H Lu 4H Lu 8H Lu 12H
The samples were diluted with water
SAMD9 is a conserved cytoplasmic protein in mammalian cells. Its at a 1:10 ratio. 2 uL of sample loaded Infection with MAV-M062R virus showed reduced intermediate and late protein
deleterious mutation is associated with human diseases (NFTC, MIRAGE OD595 0.189 0.179 0.178 0.191 0.192
with 100 uL Bradford reagent. The synthesis, indicating translation inhibition in the host cell.
Syndrome, Myeloid Malignancies). SAMD9 presents anti-poxvirus properties ug/mL 1.095 0.562 0.470 1.168 1.223 optical density (OD), concentrations
with an unknown mechanism. In order to understand the function of this (ug/mL) and dilution factors are Biological replicates are needed to confirm or refute whether late viral protein
DF 10 10 10 10 10
protein, recombinant MYXV viruses with mutated SAMD9 inhibitor (e.g., I79T indicated. expression (SERP1 and mCherrry) of MAV-M062R is completely inhibited or just
MAV-M062R) were generated. Two recombinant MYXV, Lu-M062R (wildtype attenuated.
virus) and MAV-M062R (mutant virus) were engineered (Fig. 1).
BSA Standard Curve Analyze possible mechanisms of translational control that can be responsible for
this attenuated protein expression (i.e., phosphorylation of eIF2 alpha)

Figure 2. Bradford Assay Standard Curve.

Figure 2. The BSA standard curve was
used to accurately quantify the
concentration of our protein samples
(Table 1-2), by comparing them to
these known BSA concentrations. The
equation for the line of best fit is
Fig. 1. Lu-M062R is the wildtype MYXV. MAV-M062R is a mutant with a I>T
shown on the graph.
replacement at the 79th residue. Both recombinant viruses were engineered to
have V5 tag at the c-terminus of the corresponding M062.
This mutation of MAV-M062R is shown to have affected the secondary
structure of the M062 protein. Since MYXV M062 protein is seen to bind with
human SAMD9, we aim to analyze whether this mutation will affect the Known BSA concentrations:
1000 ug/mL, 500 ug/mL, 250 ug/mL, 125 ug/mL, 62.5
interaction between MAV-M062R and host SAMD9 protein. ug/mL, 31 ug/mL, and 0 ug/mL
ACKNOWLEDGMENTS
-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 – NIGMS (P20 GM103429) from the National Institutes of Health.
REFERENCES
1. Liu, Jia et al. “The poxvirus C7L host range factor superfamily.” Current opinion in virology vol.
2,6(2012): 758-766.
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 Report
Fall 2022.