Proviral insights of glycolytic enolase in Bamboo mosaic virus replication associated with chloroplasts and mitochondria

Kuan-Yu Lin, Ying-Wen Huang, Liang-Yu Hou, Hsin-Chuan Chen, Yu Wu, I-Hsuan Chen, Ying-Ping Huang,

 Shu-Chuan Lee, Chung-Chi Hu, Ching-Hsiu Tsai, Yau-Heiu Hsu, and Na-Sheng Lin

PNAS; May 6, 2025; 122 (19) e2415089122; https://doi.org/10.1073/pnas.2415089122

Significance

Viruses are obligate parasites that often use internal membranous structures to create environments enriched with host factors, providing a protection environment for replication. Previous evidence shows that Bamboo mosaic virus (BaMV) hijacks nucleus-encoded chloroplast proteins for RNA targeting to chloroplasts and for subgenomic RNA transcription within chloroplasts. Moreover, it recruits several cytoplasmic proteins and mitochondria outer membrane protein, voltage-dependent anion channel (VDAC) into BaMV replication complex, which is essential for its replication. In this study, we identified pivotal role of the glycolytic enzyme enolase (ENO) in BaMV replication by maintaining ENO-VDAC metabolon, which depends on ENO structural integrity, and connects the chloroplasts and mitochondria. Our findings reveal that BaMV replication requires dynamic and sequential interactions between these two intracellular organelles.

Abstract

Diverse single-stranded RNA viruses employ different host cellular organelles or membrane systems to compartmentalize their replication intermediates and proviral factors, ensuring robust replication. Replication of Bamboo mosaic virus (BaMV), an Alphaflexiviridae family, is tightly associated with chloroplasts and dynamic cytosolic viral replication complex (VRC) clusters. BaMV VRC clusters comprise double-stranded viral RNA, BaMV replicase (Rep_BaMV), and mitochondrial outer membrane protein, voltage-dependent anion channel (VDAC). In this study, we demonstrate that host glycolytic enolase (ENO) binds to untranslated regions of BaMV RNA independently of ENO hydrolytic activity. However, the structural integrity of ENO is essential for its direct interaction with Rep_BaMV, and its positive regulating role in BaMV replication and the size of BaMV VRC clusters. Additionally, ENO, pyruvate kinase (PYK), and VDAC colocalize within cytosolic BaMV VRC clusters embedded in the convoluted endomembrane reticulum (ER) along with ER-targeted viral movement proteins under BaMV infection. This association suggests that the ENO-PYK-VDAC metabolon, with ENO serving as a scaffold to link chloroplasts and mitochondria, may play a pivotal role in BaMV robust replication. Collectively, our findings offer significant insights into how glycolytic ENO acts in BaMV replication.

See https://www.pnas.org/doi/10.1073/pnas.2415089122

Figure 1: Enolase (ENO) is a BaMV 5′ UTR binding protein that interacts with Rep_BaMV by EMSA analysis and coimmunoprecipitation assay. (A) RNA binding activity of different amount of recombinant ENO wild-type (WT) protein incubated with 5′ UTR of BaMV (Ba 5′ UTR), CMV (CMV 5′ UTR), and 3′ UTR of BaMV (Ba 3′ UTR) and CMV (CMV 3′ UTR) was assayed by EMSA. (B) Interaction of ENO and Rep_BaMV in satBaMV replication fraction. P30 fraction of ENO-DsRed infiltrated WT N. benthamiana leaves (WT) or ENO-DsRed and pKF4 coinfiltrated Rep_BaMV transgenic N. benthamiana leaves (O1) at 2 days-post-agroinfiltration (dpa) were purified and immunoprecipitated by ENO antiserum. After immunoprecipitation, Rep_BaMV and ENO were detected by protein blot. O1: Rep_BaMV transgenic N. benthamiana.