Structure of filamentous alternanthera mosaic virus and mechanism of its activation

Abstract

Alternanthera mosaic virus (AltMV) is a filamentous plant virus belonging to the genus Potexvirus. The virus contains a single‐strand RNA and coat protein (CP) that self‐assembles around RNA into a flexible helical sheath. In contrast to rich information on the rigid rod plant viruses, structural information for flexible plant viruses has been lacking. High‐resolution structures are available only for papaya mosaic virus (Yang et al., 2012) and bamboo mosaic virus (DiMaio et al., 2015), and for some other Potexviruses low‐resolution structures exist. The aim of this work is to determine the structure of AltMV using cryo‐EM and image processing and to propose a mechanism of uncoating of viral RNA during infection. The virus particles were purified as described by Mukhamedzhanova et al. (2009) and quickly frozen in liquid ethane. The high resolution data were collected on Titan Krios electron microscope (FEI) at 300 kV acceleration voltage, using low dose condition. Images were captured using Falcon direct detector and image processing has been accomplished using software packages IMAGIC and Spider. All images (2000 single particles) were corrected for the CTF. The helical parameters were estimated and the obtained 3D map of viral particle had a resolution of 7Å which allowed to reveal the orientation of alpha helixes. The high radius region of AltMV was quite similar to that of PapMV and this could be explained by high (79.8%) homology of CPs of these viruses (Geering, 1999). The electron density revealed a central channel with ~18Å in diameter. The viral RNA may be positioned at 36Å radius of the particle. As crystal structure of AltMV is not known the homology modelling of AltMV coat protein was carried out. In order to interpret the obtained structure the homology model was fitted into 3D electron density map (Fig. 1). Like in other Potexviruses with known structure (Yang et al., 2012; DiMaio et al., 2015)N terminus of AltMV CP contacts with adjacent protein subunit. This contact likely plays a key role in maintaining of virion stability. Previously it was shown that encapsidated AltMV RNA is nontranslatable in vitro , but can be converted into a translatable form after phosphorylation of coat protein (Mukhamedzhanova et al., 2011). In order to understand the mechanism of uncoating of AltMV RNA after such phosphorylation a series of umbrella sampling simulations was carried out. We measured binding free energy of two AltMV CP subunits with all serine and threonine residues exposed on outer surface of a virion phosphorylated and compared it with binding free energy of AltMV CP subunits without phosphorylation. Our results indicate that phosphorylation considerably reduces binding energy of the complex and promotes its disassembly. The minimum on the energy profile of the non‐phosphorylated complex corresponded to a conformation where N terminus of one subunit was in a close contact with the adjacent protein subunit. For the phosphorylated complex the minimum was reached when N terminus moved 1 nm away from the neighboring protein subunit (Fig. 2). Thus the new evidence that the mechanism of AltMV activation during infection may include phosphorylation of CPs has been obtained. The phosphorylation causes destruction of the contact between N terminus of a CP and adjacent protein subunit. As a result, AltMV virion becomes unstable without the contact and uncoating of RNA starts.