Co-opting the fermentation pathway for tombusvirus replication: Compartmentalization of cellular metabolic pathways for rapid ATP generation

Abstract

The viral replication proteins of plus-stranded RNA viruses orchestrate the biogenesis of the large viral replication compartments, including the numerous viral replicase complexes, which represent the sites of viral RNA replication. The formation and operation of these virus-driven structures require subversion of numerous cellular proteins, membrane deformation, membrane proliferation, changes in lipid composition of the hijacked cellular membranes and intensive viral RNA synthesis. These virus-driven processes require plentiful ATP and molecular building blocks produced at the sites of replication or delivered there. To obtain the necessary resources from the infected cells, tomato bushy stunt virus (TBSV) rewires cellular metabolic pathways by co-opting aerobic glycolytic enzymes to produce ATP molecules within the replication compartment and enhance virus production. However, aerobic glycolysis requires the replenishing of the NAD+ pool. In this paper, we demonstrate the efficient recruitment of pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) fermentation enzymes into the viral replication compartment. Depletion of Pdc1 in combination with deletion of the homologous PDC5 in yeast or knockdown of Pdc1 and Adh1 in plants reduced the efficiency of tombusvirus replication. Complementation approach revealed that the enzymatically functional Pdc1 is required to support tombusvirus replication. Measurements with an ATP biosensor revealed that both Pdc1 and Adh1 enzymes are required for efficient generation of ATP within the viral replication compartment. In vitro reconstitution experiments with the viral replicase show the pro-viral function of Pdc1 during the assembly of the viral replicase and the activation of the viral p92 RdRp, both of which require the co-opted ATP-driven Hsp70 protein chaperone. We propose that compartmentalization of the co-opted fermentation pathway in the tombusviral replication compartment benefits the virus by allowing for the rapid production of ATP locally, including replenishing of the regulatory NAD+ pool by the fermentation pathway. The compartmentalized production of NAD+ and ATP facilitates their efficient use by the co-opted ATP-dependent host factors to support robust tombusvirus replication. We propose that compartmentalization of the fermentation pathway gives an evolutionary advantage for tombusviruses to replicate rapidly to speed ahead of antiviral responses of the hosts and to outcompete other pathogenic viruses. We also show the dependence of turnip crinkle virus, bamboo mosaic virus, tobacco mosaic virus and the insect-infecting Flock House virus on the fermentation pathway, suggesting that a broad range of viruses might induce this pathway to support rapid replication.

ROS/RNS balancing, aerobic fermentation regulation and cell cycle control – a complex early trait (‘CoV-MAC-TED’) for combating SARS-CoV-2-induced cell reprogramming

“…Several metabolic changes have been reported in virus-infected animal and/or plant host cells that were related to central carbon metabolism: e.g. (a) increased rate of glycolysis linked to pools of nucleotides and amino acids essential for replication (31,32), (b) differential mTOR (mammalian target of rapamycin) pathway regulation and intracellular calcium signaling linked to enhanced Krebs cycle, (c) down-regulation of glycolysis that severely affected viral infection (33)(34)(35), (d) encoding mitochondria-related proteins that disturbed normal functioning of mitochondria (36), (e) increased production of lactic acid from glucose pumped out of cell (37), (f) knock-down of ADH (Alcohol dehydrogenase) and pyruvate decarboxylase that diminished virus replication rates (38), and, (g) interplay between glycolysis and fermentation that is suggested to serve metabolome channeling for virus replication highlighted as paradigm change (39). Bojkova et al (40) observed that blocking of glycolysis resulted in prevention of SARS-CoV-2 replication in infected cells.…”

The Mechanism of Citrus Host Defense Response Repression at Early Stages of Infection by Feeding of Diaphorina citri Transmitting Candidatus Liberibacter asiaticus

“…Very interestingly, a recent study on several viruses found that the viruses hijacked the cellular glycolytic and fermentation pathways to rapidly produce ATP locally for its replication. They also found that the knockdown of Pdc1 pyruvate decarboxylase and Adh1 alcohol dehydrogenase fermentation enzymes in plants greatly reduced the efficiency of tombusvirus replication, and enzymatically functional Pdc1 is required to support tombusvirus replication (Lin et al, 2019 ; Nagy and Lin, 2020 ). Surprisingly, both the Pdc1 and Adh1 were detected in our present study ( Supplementary Material 1 , Supplementary Table 21 ).…”

Creating Contacts Between Replication and Movement at Plasmodesmata – A Role for Membrane Contact Sites in Plant Virus Infections?

“…In the process of establishing their membrane-bound replication complexes, they extensively modify the membrane architecture into novel structures like invaginated spherules or tubules, or stacked membranes, known as viral replication complexes (VRCs), viroplasms or virus factories ( Miller and Krijnse-Locker, 2008 ; den Boon et al, 2010 ; Xu and Nagy, 2014 ). These structures may provide a variety of functions in infection: (1) hide viral replication intermediates such as double-stranded RNA from cellular defense surveillance ( Överby et al, 2010 ), (2) provide a scaffold for replication complexes ( Gouttenoire et al, 2014 ) and activate replication enzymes ( Xu and Nagy, 2017 ), (3) compartmentalize metabolic energy delivery ( Lin et al, 2019 ), translation ( Bamunusinghe et al, 2009 ; Mäkinen and Hafren, 2014 ), and virion assembly ( Annamalai and Rao, 2006 ), and (4) provide access to cellular membrane trafficking routes. VRC formation often involves de novo lipid synthesis, and shuttling of suitable lipids to the replication site.…”