Milken Institute School of Public Health Poster Presentations (Marvin Center & Video)

Title

Attempts to Develop a Peptide Based Inhibitor to Prevent HCMV Growth

Poster Number

31

Document Type

Poster

Publication Date

3-2016

Abstract

Background/Objective:

Human cytomegalovirus (HCMV) is a beta-herpesvirus that causes asymptomatic infections in healthy adults, but severe illnesses in neonates, transplant patients, and immunodeficient individuals. HCMV encoded antiapoptotic protein, vMIA, prevents infected cells from undergoing mitochondria-mediated apoptosis, thus allowing productive viral replication. vMIA trafficking to the mitochondria is required for its antiapoptotic function. It has been proposed that vMIA interaction with the mitochondrial morphology regulating cellular protein mitofusin 2 (Mfn2) is required for vMIA to traffic to the mitochondria. In this study we aimed to develop an Mfn2 polypeptide - vMIA binding domain (VBD; Mfn2aa262-390) as a tool to prevent vMIA localization and function at the mitochondria.


Methods:

Fluorescence microscopy was used to determine localization of Mfn2 and vMIA. Mouse embryonic fibroblasts expressing fluorophore tagged, mitochondrial outer mitochondrial membrane marker Tom20, as well as Mfn2, and vMIA were imaged by confocal microscopy. Images were analyzed and locations were quantified using MetaMorph. Statistical analyses were performed using SAS. Mfn2 VBD, was cloned, fluorophore tagged, and co-transfected with Tom20 to determine its localization.


Results:

Mfn2 was localized at the mitochondria in the presence or absence of vMIA. The presence of vMIA caused Mfn2 to redistribute similarly to vMIA - more evenly along the outer mitochondrial membrane. Mfn2 and vMIA colocalization suggests that vMIA interacts with Mfn2 and changes its distribution. The difference in Mfn2 distribution induced by vMIA was statistically significant. Mfn2 VBD did not localize to the mitochondria on its own or when vMIA was expressed in the cell indicating this Mfn2 polypeptide is unable to interact with vMIA.


Conclusion:

vMIA interacts with and alters the distribution of Mfn2. However, vMIA trafficking to mitochondria is neither affected by the proposed vMIA binding domain of Mfn2 nor does this domain localize with vMIA. Thus, VBD may not be suited for inhibiting HCMV growth.

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Presented at: GW Research Days 2016

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Attempts to Develop a Peptide Based Inhibitor to Prevent HCMV Growth

Background/Objective:

Human cytomegalovirus (HCMV) is a beta-herpesvirus that causes asymptomatic infections in healthy adults, but severe illnesses in neonates, transplant patients, and immunodeficient individuals. HCMV encoded antiapoptotic protein, vMIA, prevents infected cells from undergoing mitochondria-mediated apoptosis, thus allowing productive viral replication. vMIA trafficking to the mitochondria is required for its antiapoptotic function. It has been proposed that vMIA interaction with the mitochondrial morphology regulating cellular protein mitofusin 2 (Mfn2) is required for vMIA to traffic to the mitochondria. In this study we aimed to develop an Mfn2 polypeptide - vMIA binding domain (VBD; Mfn2aa262-390) as a tool to prevent vMIA localization and function at the mitochondria.


Methods:

Fluorescence microscopy was used to determine localization of Mfn2 and vMIA. Mouse embryonic fibroblasts expressing fluorophore tagged, mitochondrial outer mitochondrial membrane marker Tom20, as well as Mfn2, and vMIA were imaged by confocal microscopy. Images were analyzed and locations were quantified using MetaMorph. Statistical analyses were performed using SAS. Mfn2 VBD, was cloned, fluorophore tagged, and co-transfected with Tom20 to determine its localization.


Results:

Mfn2 was localized at the mitochondria in the presence or absence of vMIA. The presence of vMIA caused Mfn2 to redistribute similarly to vMIA - more evenly along the outer mitochondrial membrane. Mfn2 and vMIA colocalization suggests that vMIA interacts with Mfn2 and changes its distribution. The difference in Mfn2 distribution induced by vMIA was statistically significant. Mfn2 VBD did not localize to the mitochondria on its own or when vMIA was expressed in the cell indicating this Mfn2 polypeptide is unable to interact with vMIA.


Conclusion:

vMIA interacts with and alters the distribution of Mfn2. However, vMIA trafficking to mitochondria is neither affected by the proposed vMIA binding domain of Mfn2 nor does this domain localize with vMIA. Thus, VBD may not be suited for inhibiting HCMV growth.