Dynamic interplay between the co-opted Fis1 mitochondrial fission protein and membrane contact site proteins in supporting tombusvirus replication

Abstract

Plus-stranded RNA viruses have limited coding capacity and have to co-opt numerous pro-viral host factors to support their replication. Many of the co-opted host factors support the biogenesis of the viral replication compartments and the formation of viral replicase complexes on subverted subcellular membrane surfaces. Tomato bushy stunt virus (TBSV) exploits peroxisomal membranes, whereas the closely-related carnation Italian ringspot virus (CIRV) hijacks the outer membranes of mitochondria. How these organellar membranes can be recruited into pro-viral roles is not completely understood. Here, we show that the highly conserved Fis1 mitochondrial fission protein is co-opted by both TBSV and CIRV via direct interactions with the p33/p36 replication proteins. Deletion of FIS1 in yeast or knockdown of the homologous Fis1 in plants inhibits tombusvirus replication. Instead of the canonical function in mitochondrial fission and peroxisome division, the tethering function of Fis1 is exploited by tombusviruses to facilitate the subversion of membrane contact site (MCS) proteins and peroxisomal/mitochondrial membranes for the biogenesis of the replication compartment. We propose that the dynamic interactions of Fis1 with MCS proteins, such as the ER resident VAP tethering proteins, Sac1 PI4P phosphatase and the cytosolic OSBP-like oxysterol-binding proteins, promote the formation and facilitate the stabilization of virus-induced vMCSs, which enrich sterols within the replication compartment. We show that this novel function of Fis1 is exploited by tombusviruses to build nuclease-insensitive viral replication compartment.

Subversion of selective autophagy for the biogenesis of tombusvirus replication organelles inhibits autophagy

“…Interestingly, both GFP-ATG8a and GFP-ATG8i were usurped into TBSV and CIRV VROs, forming small number of punctate structures (Figs 1C and S1A–S1C ). We also found that the minus stranded replicon RNA, which is the replication intermediate and marks the site of virus replication [ 49 , 89 ] was co-localized with p33 replication protein and ATG8a in the replication compartment in N . benthamiana replicating the closely-related cucumber necrosis (CNV) helper virus, which supplied the RdRp ( S2 Fig ).…”

“…Interestingly, both GFP-ATG8a and GFP-ATG8i were usurped into TBSV and CIRV VROs, forming small number of punctate structures (Figs 1C and S1A-S1C). We also found that the minus stranded replicon RNA, which is the replication intermediate and marks the site of virus replication [49,89] was co-localized with p33 replication protein and ATG8a in the replication compartment in N. benthamiana replicating the closelyrelated cucumber necrosis (CNV) helper virus, which supplied the RdRp (S2 Fig) . Therefore, we suggest that ATG8a is recruited to VROs that are active in viral RNA synthesis. Based on these experiments, we conclude that ATG8 autophagy-related proteins are efficiently recruited by tombusviruses to VROs in N. benthamiana.…”

“…One of the characteristic features of TBSV infection is the formation of virusinduced membrane contact sites (vMCSs) [25,[46][47][48]. vMCS forms between the hijacked subdomain in the ER and the peroxisome with the help of p33 replication protein and co-opted host proteins, such as oxystrerol-binding proteins, ER-resident Sac1 PI4P phosphatase and VAP proteins and Fis1 mitochondrial fission protein [43,48,49]. vMCS function is essential for the enrichment of sterols and phosphoinositides within VROs [48] to protect TBSV replication protein from proteasomal degradation [28,44].…”

Phytosterol metabolism in plant positive-strand RNA virus replication

“…In fact, phospholipid biosynthesis in TBSV-infected yeast and plant cells is clearly enhanced (Sharma et al, 2010;Xu and Nagy, 2017). Thus, it is reasonable to speculate that de novo sterol biosynthesis in the ER acts in close coordination with internal redistribution of pre-existing sterol pools, such as PM sterols (Lin et al, 2021) or even the esterified sterols accumulated in the cytoplasmic lipid bodies (Bouvier-Navé et al, 2010;Lara et al, 2018) (Figure 2), in order to meet the enhanced demand of these compounds triggered by virus replication. To establish the origin of these sterols is certainly an interesting subject for further studies, but either case it is clear that efficient virus replication in infected cells requires an active intracellular trafficking of these compounds.…”

Viral regulation of organelle membrane contact sites

“…As such, MCS dysregulation is a hallmark of several neurodegenerative disorders, as well as genetic and metabolic diseases [14][15][16][17][18]. Similarly, viral infections can trigger reorganization of organelle contacts to modulate organelle structure and function, and to establish a successful replication cycle [19][20][21][22][23][24][25][26][27]. Viruses are obligate parasites, indicating that they must co-opt existing cellular pathways and modulate their homeostatic functions to instead support viral replication.…”