Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries.

UNLABELLED: 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.

Mitochondrial and cytosolic isoforms of yeast fumarase are derivatives of a single translation product and have identical amino termini.

We have previously proposed that a single translation product of the FUM1 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae and that all fumarase translation products are targeted and processed in mitochondria before distribution. Alternative models for fumarase distribution have been proposed that require more than one translation product. In the current work (i) we show by using sequential Edman degradation and mass spectrometry that fumarase cytosolic and mitochondrial isoenzymes have an identical amino terminus that is formed by cleavage by the mitochondrial processing peptidase, (ii) we have generated fumarase mutants in which the second potential translation initiation codon (Met-24) has been substituted, yet the protein is processed efficiently and retains its ability to be distributed between the cytosol and mitochondria, and (iii) we show that although a signal peptide is required for fumarase targeting to mitochondria the specific fumarase signal peptide and the sequence immediately downstream to the cleavage site are not required for the dual distribution phenomenon. Our results are discussed in light of our model of fumarase targeting and distribution that suggests rapid folding into an import-incompetent state and retrograde movement of the processed protein back to the cytosol through the translocation pore.