Single-Molecule Force Spectroscopy Study on the Mechanism of RNA Disassembly in Tobacco Mosaic Virus

Abstract

To explore the disassembly mechanism of tobacco mosaic virus (TMV), a model system for virus study, during infection, we have used single-molecule force spectroscopy to mimic and follow the process of RNA disassembly from the protein coat of TMV by the replisome (molecular motor) in vivo, under different pH and Ca(2+) concentrations. Dynamic force spectroscopy revealed the unbinding free-energy landscapes as that at pH 4.7 the disassembly process is dominated by one free-energy barrier, whereas at pH 7.0 the process is dominated by one barrier and that there exists a second barrier. The additional free-energy barrier at longer distance has been attributed to the hindrance of disordered loops within the inner channel of TMV, and the biological function of those protein loops was discussed. The combination of pH increase and Ca(2+) concentration drop could weaken RNA-protein interactions so much that the molecular motor replisome would be able to pull and disassemble the rest of the genetic RNA from the protein coat in vivo. All these facts provide supporting evidence at the single-molecule level, to our knowledge for the first time, for the cotranslational disassembly mechanism during TMV infection under physiological conditions.

Elucidation of the viral disassembly switch of tobacco mosaic virus

“…In order to confirm that our batch of TMV presents a biologically active virus, we also showed experimentally that our sample is capable of infecting tobacco plants (Fig EV4). This shared Ca 2+ site provides a direct explanation of preferred capsid opening at the virion ends and cooperative weakening of RNA-coat protein interactions thus facilitating cotranslational virion disassembly by the replisome [7]. In the case of the 5 0 and 3 0 ends of the virion, the end subunits can only weakly interact at the shared Ca 2+ coordination site in the Ca 2+ /acidic pH condition as they lack the stabilizing neighboring subunit residues.…”

“…The proposed structural destabilization mechanism offers the possibility of a cooperative disassembly switch between subunits: the removal of a Ca 2+ from its coordination site at lower radius has an immediate effect on the neighboring Ca 2+ sites, which are located in close proximity of 10 Å . This is an important aspect of the CP plasticity, which only requires a subtle destabilization of the metastable switch to trigger cotranslational disassembly [26] and, at the same time, to be sufficiently stable to allow for reassembly of the virion after viral replication [7]. Upon drop of Ca 2+ concentration, the ends are even further destabilized and more easily accessible to the pulling replisome machinery.…”

“…The TMV capsid is primarily composed of a 17 kDa coat protein (CP) that is organized in a helical assembly thereby tightly enclosing the viral RNA of the genome [2][3][4][5]. The propagation of the virus is then triggered by a controlled opening of the viral capsid inside the host cell in order to facilitate cotranslational virion disassembly by the replisome [7]. The propagation of the virus is then triggered by a controlled opening of the viral capsid inside the host cell in order to facilitate cotranslational virion disassembly by the replisome [7].…”

“…The CP folds into a four-helix bundle that is commonly divided into three regions referring to the radial distance from the helical axis [13]: lower radius 20-40 Å , middle radius 40-60 Å , and higher radius 60-90 Å . Moreover, it has been shown by single molecule force spectroscopy, that upon Ca 2+ decrease the 5 0 end of the capsid becomes exposed and RNA-coat protein interactions are weakened at the rest of the virion [7], presumably to advance cotranslational disassembly by the replisome machinery. Moreover, it has been shown by single molecule force spectroscopy, that upon Ca 2+ decrease the 5 0 end of the capsid becomes exposed and RNA-coat protein interactions are weakened at the rest of the virion [7], presumably to advance cotranslational disassembly by the replisome machinery.…”

Single-Molecule Force Spectroscopy of an Artificial DNA Duplex Comprising a Silver(I)-Mediated Base Pair

“…Single-molecule force spectroscopy is a powerful technique for the investigation of intermolecular forces. [32][33][34] It has found wide application in the determination of the interaction of biomolecules, [35][36][37][38][39][40][41][42] including DNA base pairing. 43,44 Numerous DNA conformations have been investigated, including duplexes, [45][46][47][48] quadruplexes, [49][50][51] and even DNA-based nanostructures.…”