Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jan 6;94(2):e01183-19.
doi: 10.1128/JVI.01183-19. Print 2020 Jan 6.

Human Cytomegalovirus Alters Host Cell Mitochondrial Function during Acute Infection

Joseph A Combs  1 , Elizabeth B Norton  1 , Zubaida R Saifudeen  2 , Kerstin Honer Zu Bentrup  1 , Prasad V Katakam  3 , Cindy A Morris  1 , Leann Myers  4 , Amitinder Kaur  5 , Deborah E Sullivan  1 , Kevin J Zwezdaryk  6
Affiliations

Human Cytomegalovirus Alters Host Cell Mitochondrial Function during Acute Infection

Joseph A Combs et al. J Virol. .

Abstract

Human cytomegalovirus (HCMV) is a large DNA herpesvirus that is highly prevalent in the human population. HCMV can result in severe direct and indirect pathologies under immunosuppressed conditions and is the leading cause of birth defects related to infectious disease. Currently, the effect of HCMV infection on host cell metabolism as an increase in glycolysis during infection has been defined. We have observed that oxidative phosphorylation is also increased. We have identified morphological and functional changes to host mitochondria during HCMV infection. The mitochondrial network undergoes fission events after HCMV infection. Interestingly, the network does not undergo fusion. At the same time, mitochondrial mass and membrane potential increase. The electron transport chain (ETC) functions at an elevated rate, resulting in the release of increased reactive oxygen species. Surprisingly, despite the stress applied to the host mitochondria, the network is capable of responding to and meeting the increased bioenergetic and biosynthetic demands placed on it. When mitochondrial DNA is depleted from the cells, we observed severe impairment of viral replication. Mitochondrial DNA encodes many of the ETC components. These findings suggest that the host cell ETC is essential to HCMV replication. Our studies suggest the host cell mitochondria may be a therapeutic target.IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus present in up to 85% of some populations. Like all herpesviruses, HCMV infection is for life. No vaccine is currently available, neutralizing antibody therapies are ineffective, and current antivirals have limited long-term efficacy due to side effects and potential for viral mutation and resistance. The significance of this research is in understanding how HCMV manipulates the host mitochondria to support bioenergetic and biosynthetic requirements for replication. Despite a large genome, HCMV relies exclusively on host cells for metabolic functions. By understanding the dependency of HCMV on the mitochondria, we could exploit these requirements and develop novel antivirals.

Keywords: CMV; OXPHOS; cytomegalovirus; electron transport chain; membrane potential; mitochondria; mitochondrial biogenesis; mtDNA; oxidative stress; reactive oxygen species.

PubMed Disclaimer

Figures

FIG 1
FIG 1
HCMV-infected cells display increased glycolysis. The extracellular acidification rate was measured at 24 h (A) or 48 h (B) after HFFs were mock or HCMV infected using the Seahorse XFe24 glycolytic stress assay. Glycolysis (C and D), glycolytic capacity (E and F), glycolytic reserve (G and H), glycolytic reserve % (I and J), and nonglycolytic acidification (K and L) were derived from these measurements. All samples were normalized to protein. The data are representative of pooled data from at least three independent experiments. Error bars indicate ± the standard errors of the mean (SEM) of at least triplicates. *, P < 0.05; **, P < 0.01; ns, not significant.
FIG 2
FIG 2
HCMV-infected cells display increased OXPHOS. The OCR was measured at 24 h (A) or 48 h (B) after HFFs were mock or HCMV infected using the Seahorse XFe24 MitoStress kit. Basal OCR (C and D), maximal OCR (E and F), spare respiratory capacity (G and H), nonmitochondrial oxygen consumption (I and J), ATP production (K and L), and proton leak (M) were derived from these measurements. All samples were normalized to protein. (N) Flow cytometry viability assays were used to measure cell death in mock- and HCMV-infected cells. Data pooled from at least three independent experiments. Error bars indicate ± the SEM of at least triplicates. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant.
FIG 3
FIG 3
Mitochondrial respiration and membrane potential are increased in HCMV-infected fibroblasts. HFFs infected with HCMV were loaded with MitoTracker CMTMRos dye (A) or DiOC6 dye (B) at the indicated times and analyzed by flow cytometry. Samples were gated on live cells only. Graphs represent pooled data from three independent experiments. Mean ± the SEM of at least triplicates. **, P < 0.01; **, P < 0.001; ****, P < 0.0001; ns, not significant.
FIG 4
FIG 4
HCMV upregulates ETC complex proteins and increases reactive oxygen species levels. (A) Whole-cell extracts were prepared from mock- and HCMV-infected cells. ETC complexes I to V and GAPDH protein levels were determined by Western blotting. Total ROS (detected by CellROX Deep Red) (B) and superoxide (detected using MitoSOX Red) (C) levels were obtained by flow cytometric analysis. The data of three independent experiments were pooled. Means ± the SEM are shown.
FIG 5
FIG 5
Changes in mitochondrial gene expression after HCMV infection of HFFs. (A) Schematic of mitochondrial functions controlled by genes altered in HCMV-infected HFFs. (B) Table displaying the total numbers of mitochondrion-associated genes increased, unchanged, or decreased in expression at 24 and 48 h after HCMV infection. (C) Heat map organized by mitochondrial function showing differential gene expression at 24 h postinfection (n = 3 biological replicates).
FIG 6
FIG 6
HCMV infection induces mitochondrial fission. (A) Representative microscopy images of mock- and HCMV-infected HFFs stained with an antibody to Tom20 located on the outer mitochondrial membrane. (B) The time course of fission/fusion protein expression was analyzed by Western blotting. (C) Expression of genes related to fusion (MFN2) and fission (OPA1), as well as HCMV IE, was determined by qRT-PCR. The data are shown as the means ± the SEM of three independent experiments.
FIG 7
FIG 7
Ultrastructure of mitochondria after HCMV infection. (A) Representative electron micrographs of mock-infected (left) and HCMV-infected (right) HFFs. Mock-infected cells display tubular mitochondria. HCMV-infected cells show circular mitochondria with intact cristae. (A to D) The areas (B), perimeters (C), and circularities (D) of individual mitochondria decrease with time in infected cells. The image is representative of n = 3 biological controls. Scale bar, 500 nm. Magnification, ×6,000. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.
FIG 8
FIG 8
HCMV infection induces mitochondrial biogenesis. (A) Flow cytometry analysis of mitochondrial mass (content) using MitoTracker green accumulation. (B) Citrate synthase activity in lysates from mock- and HCMV-infected HFFs. (C) qPCR analysis of fold change mtDNA copy number from mock- and HCMV-infected HFFs. (D) RNA expression of PGC1α in HCMV-infected HFFs was determined using qRT-PCR. Citrate synthase data are representative of a single experiment. All other data represent means ± the SEM from three independent experiments. *, P = 0.05; ***, P = 0.001; ****, P = 0.0001.
FIG 9
FIG 9
Host cell mtDNA integrity is required for HCMV replication. (A) RNA expression of mitochondrial gene ND1 in wild-type (WT) and ρ0 clone HFFs. (B) HFF wild-type and HFF ρ0 cells were infected, and the titers of released HCMV progeny were determined. (C) The levels of apoptosis were compared by using eFluor780 fluorescent flow cytometry detection assays. The data are representative of one experimental replicate. (D) HCMV viral protein expression from HFF wild-type (WT) or ρ0 (Rho) cell lysate was determined by using Western blot analysis. The data are representative of at least three independent experiments unless otherwise indicated. Error bars indicate ± the SEM of at least triplicates.
FIG 10
FIG 10
Model depicting changes to host cell mitochondria after HCMV infection. Changes to mitochondrial morphology are induced due to increasing glycolytic and OXPHOS demands. Mitochondrial fission occurs enabling increased mitochondrial membrane potential, ETC output, and mitochondrial biogenesis. This results in increased ROS production. HCMV prevents fusion events through unknown mechanisms.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cannon MJ, Schmid DS, Hyde TB. 2010. Review of cytomegalovirus seroprevalence and demographic characteristics associated with infection. Rev Med Virol 20:202–213. doi:10.1002/rmv.655. - DOI - PubMed
    1. Azevedo LS, Pierrotti LC, Abdala E, Costa SF, Strabelli TMV, Campos SV, Ramos JF, Latif AZA, Litvinov N, Maluf NZ, Caiaffa Filho HH, Pannuti CS, Lopes MH, Santos V. A d, Linardi C. d C G, Yasuda MAS, Marques H. H d S. 2015. Cytomegalovirus infection in transplant recipients. Clinics (Sao Paulo) 70:515–523. doi:10.6061/clinics/2015(07)09. - DOI - PMC - PubMed
    1. Fowler KB, Boppana SB. 2018. Congenital cytomegalovirus infection. Semin Perinatol 42:149–154. doi:10.1053/j.semperi.2018.02.002. - DOI - PubMed
    1. Lim Y, Lyall H. 2017. Congenital cytomegalovirus: who, when, what-with, and why to treat? J Infect 74(Suppl 1):S89–S94. doi:10.1016/S0163-4453(17)30197-4. - DOI - PubMed
    1. Pawelec G, McElhaney JE, Aiello AE, Derhovanessian E. 2012. The impact of CMV infection on survival in older humans. Curr Opin Immunol 24:507–511. doi:10.1016/j.coi.2012.04.002. - DOI - PubMed

Publication types

MeSH terms

Substances