Molecular Basis for Mitochondrial Localization of Viral Particles during Beet Necrotic Yellow Vein Virus Infection

Abstract

During infection, Beet necrotic yellow vein virus (BNYVV) particles localize transiently to the cytosolic surfaces of mitochondria. To understand the molecular basis and significance of this localization, we analyzed the targeting and membrane insertion properties of the viral proteins. ORF1 of BNYVV RNA-2 encodes the 21-kDa major coat protein, while ORF2 codes for a 75-kDa minor coat protein (P75) by readthrough of the ORF1 stop codon. Bioinformatic analysis highlighted a putative mitochondrial targeting sequence (MTS) as well as a major (TM1) and two minor (TM3 and TM4) transmembrane regions in the N-terminal part of the P75 readthrough domain. Deletion and gain-of-function analyses based on the localization of green fluorescent protein (GFP) fusions showed that the MTS was able to direct a reporter protein to mitochondria but that the protein was not persistently anchored to the organelles. GFP fused either to MTS and TM1 or to MTS and TM3-TM4 efficiently and specifically associated with mitochondria in vivo. The actual role of the individual domains in the interaction with the mitochondria seemed to be determined by the folding of P75. Anchoring assays to the outer membranes of isolated mitochondria, together with in vivo data, suggest that the TM3-TM4 domain is the membrane anchor in the context of full-length P75. All of the domains involved in mitochondrial targeting and anchoring were also indispensable for encapsidation, suggesting that the assembly of BNYVV particles occurs on mitochondria. Further data show that virions are subsequently released from mitochondria and accumulate in the cytosol.Viruses recruit a variety of structures and pathways in infected host cells to develop their life cycle. These processes are critical for the development of infection and are mediated by viral-encoded polypeptides. Characterizing the subcellular localization of viral proteins and establishing a relationship between localization and activity are therefore of fundamental relevance. Many viruses, especially eukaryotic positive-sense single-stranded RNA viruses, transiently interact with cellular membranes. Depending on the virus, a variety of membrane systems can be targeted (7). Among these, mitochondria can play an important role and have received increasing interest during the last few years in view of their role in apoptosis. The previously described interactions between viruses and mitochondria involve anchoring of the replication complex to the organelle outer membrane (20, 34, 55) or molecular processes which might be related to host cell death (3, 18, 51). Like regular organelle proteins, viral proteins that associate with mitochondria typically carry transmembrane domains and mitochondrial targeting signals. This is the case for protein A of betanodaviruses (20), for the RNA polymerase of Flock house virus (34), and for the core protein of Hepatitis C virus (51).Concerning plant viruses, it has recently been reported that Carnation Italian ringspot virus, a Tombusvirus, encodes a transmembrane domain-contai...

Beet necrotic yellow vein virus accumulates inside resting spores and zoosporangia of its vector Polymyxa betae BNYVV infects P. betae

“…P15 was mainly in the storage body and central body of the spore. RTD, P13, and P15 are transmembrane proteins with different properties [19-21]. Perhaps both RTD and P15 associated with membranes of the storage body.…”

Three-Dimensional Analysis of the Association of Viral Particles with Mitochondria during the Replication of Rice Gall Dwarf Virus

“…Plant viruses, such as Tobacco rattle virus and BNYVV, have been observed at the cytosolic surfaces of mitochondria in infected plant cells. [3][4][5] In cells infected with African swine fever virus, virions do not adhere to organelles, but mitochondria are recruited to the margins of viral assembly sites to supply the energy required for viral morphogenetic processes. 29 During the assembly of animal reoviruses, such as Bluetongue virus, a viral protein (VP6 in the case of Bluetongue virus) binds ATP.…”

“…1,2 Localization of some plant viruses, such as Tobacco rattle virus and Beet necrotic yellow vein virus (BNYVV), has been observed on the cytosolic side of mitochondrial outer membranes in plant cells during viral infection. [3][4][5] Furthermore, infection by some viruses, such as African swine fever virus, results in the concentration of mitochondria near so-called viral factories to meet the energy requirements of viral replication. 6,7 Rice gall dwarf virus (RGDV), Rice dwarf virus (RDV), and Wound tumor virus, which are members of the genus Phytoreovirus in the family Reoviridae, multiply both in plants and in invertebrate insect vectors.…”

Gene Therapy of the Other Genome: The Challenges of Treating Mitochondrial DNA Defects

“…There does not however seem to be much interest in the application of viral systems to mitochondrial gene therapy. The viral life cycle is well known to be closely associated with mitochondrial function (81) and it is also known that certain viral particles display a marked mitochondrial localization (82,83). We believe that it is not unreasonable to speculate on the eventual availability of a viral gene delivery vector for transfection of mammalian mitochondria.…”