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. 2016 Mar 29;7(2):e00029.
doi: 10.1128/mBio.00029-16.

Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries

S Karniely  1 , M P Weekes  2 , R Antrobus  2 , J Rorbach  3 , L van Haute  3 , Y Umrania  2 , D L Smith  4 , R J Stanton  5 , M Minczuk  3 , P J Lehner  2 , J H Sinclair  6
Affiliations

Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries

S Karniely et al. mBio. .

Abstract

Infection with human cytomegalovirus (HCMV) profoundly affects cellular metabolism. Like in tumor cells, HCMV infection increases glycolysis, and glucose carbon is shifted from the mitochondrial tricarboxylic acid cycle to the biosynthesis of fatty acids. However, unlike in many tumor cells, where aerobic glycolysis is accompanied by suppression of mitochondrial oxidative phosphorylation, HCMV induces mitochondrial biogenesis and respiration. Here, we affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We found that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth under bioenergetically restricting conditions. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication.

Importance: Human cytomegalovirus (HCMV), a betaherpesvirus, is a leading cause of morbidity and mortality during congenital infection and among immunosuppressed individuals. HCMV infection significantly changes cellular metabolism. Akin to tumor cells, in HCMV-infected cells, glycolysis is increased and glucose carbon is shifted from the tricarboxylic acid cycle to fatty acid biosynthesis. However, unlike in tumor cells, HCMV induces mitochondrial biogenesis even under aerobic glycolysis. Here, we have affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We find that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication.

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Figures

FIG 1
FIG 1
SILAC-MS analysis of the changes in the mitochondrial proteome following HCMV infection. (A) Subcellular localization annotation of 1,171 human proteins identified in isolated mitochondria with at least 2 unique peptides represented as a percentage of the total. Gene Ontology (GO) information was imported using the UniProt database. (B) Scatter plot representing fold change in “heavy” (mock)/“light” (HCMV 48 hpi) protein abundance in isolated mitochondria. A total of 12.3% (144) of proteins were increased over 2-fold following HCMV infection (red), 8.6% (101) were downregulated (blue), and 79.1% (926) were not significantly changed (gray).
FIG 2
FIG 2
Upregulation of mitochondrial translation and transcription factors in HCMV-infected cells. U373 and human fibroblast (HFFF2) cells were either mock infected or infected with HCMV (Merlin) at an MOI of 5 for 48 h. WB analysis of total cell extracts using the indicated antibodies is shown. The fold change in protein abundance measured by WB is presented in the column charts.
FIG 3
FIG 3
Upregulation of mitochondrial expression factors occurs during the early phase of HCMV replication. (A) Time course analysis. U373 and HFFF2 cells were either mock infected or infected with HCMV (Merlin) at an MOI of 5 for the times indicated. Lysates were processed and analyzed as described in the legend to Fig. 2. Membranes were labeled with the indicated antibodies. The upregulation of mitochondrial expression factors was first observed at 24 hpi concomitant with the expression of the HCMV UL44 early protein. m., mock infected cells. (B) Induction of mitochondrial expression factors is dependent on the expression of immediate early/early but not late HCMV genes. HFFF2 cells were either mock infected or infected with an intact or a UV-inactivated HCMV (Merlin) at an MOI of 5 for 48 h.
FIG 4
FIG 4
HCMV β2.7 and UL37x1 are not required for the induction of mitochondrial expression factors. HFFF2 cells were either mock infected or were infected for 48 h with wild-type (WT) HCMV (Merlin-BAC in panel A or Towne-BAC in panel B) or mutant viruses with the β2.7 (A) or UL37x1 (B) genes deleted.
FIG 5
FIG 5
HCMV induces the synthesis and stabilization of mitochondrially encoded proteins. (A) Assembled mitoribosomes are induced by HCMV. Equal amounts of proteins of total cell lysates from mock-infected or HCMV-infected HFFF2 cells at 48 hpi were separated on a linear sucrose gradient (10 to 30% [wt/vol]) and analyzed by WB. Free nonassembled ribosomal subunits (SSU, small subunit; LSU, large subunit) migrate to the lower fractions, while assembled mitoribosomes appear in the higher-density fractions. (B) Induction of mitochondrially encoded proteins by HCMV. HFFF2 cells were either mock infected or were infected with HCMV at an MOI of 5. After 2 h, the inoculum was washed, and cells were refreshed with untreated medium (UT) or with medium containing ethidium bromide (EtBr) to block mitochondrial transcription. Cells were harvested at 48 hpi, and lysates were processed and analyzed as described in the legend to Fig. 2. The chart shows the fold change in protein abundance. HCMV induces mitochondrial translation. (C to D) Mock-infected and HCMV-infected cells were radiolabeled with [35S]methionine at 24 hpi in the presence of emetine (which blocks cytosolic translation) to determine mitochondrial translation or in the absence of emetine to determine cellular translation. Total cell lysates were separated on a 4 to 12% Bis-Tris Plus PAGE. Equal loading of the gels was confirmed by staining the gel with Coomassie brilliant blue G-250. Dried gels were exposed to a phosphorimager screen and visualized using a phosphorimager scanner (C). Radioactive counts in cell lysates were measured directly or after TCA precipitation and used to calculate cellular and mitochondrial translation efficiencies, as described in the text. The chart shows the fold change in translation after infection (D).
FIG 6
FIG 6
Inhibition of mitochondrial translation reduces virus titers. (A) HFFF2 cells were infected with HCMV at an MOI of 5 for 1 h and then washed and refreshed with DMEM supplemented with 10% dialyzed FBS, 2 mM glutamine, and antibiotics (DMEM-10dFBS) and 5 mM glucose. At 24 hpi, cells were washed and refreshed with DMEM-10dFBS containing either 5 mM glucose or 5 mM galactose with or without the addition of 0.2 mM uridine. Each medium was either left nontreated (nt) or was treated with chloramphenicol (50 µg/ml) to block mitochondrial translation. At 5 dpi, media were collected from cells, and released virus titers in supernatants were quantified using the 50% tissue culture infective dose (TCID50). Error bars represent the standard error of the mean (SEM) from two experiments with three replications each. (B and C) HFFF2 cells were transfected with a control siRNA or an siRNA targeting MRM3. After 22 h of transfection, cells were infected with HCMV (Merlin) at an MOI of 3, and at 24 hpi, cells were washed and refreshed with DMEM-10dFBS containing 5 mM galactose. At 5 dpi, media were collected from infected cells, and virus titers were quantified using the TCID50 (C). Cells were lysed and analyzed by WB; the results of two biological repeats are shown (B). The chart shows the fold change in protein abundance compared to control siRNA-treated cells. Error bars in panel C represent the standard errors from two biological repeats, each performed with three transfection replicates. *, P < 0.05; **, P < 0.001 (unpaired t test with Welch’s correction).
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