Pest Management in Horticultural Ecosystems, Vol. 21, No. 2 pp 233-235 (2015)
SCIENTIFIC CORRESPONDENCE Cytosolic vibrations initiate the assembly of capsid and genetic material of tobacco mosaic virus – A theoretical perspective TEJAS THONDEHAALMUTT1 and VIVEK KEMPRAJ2* 1
Department of Biological Sciences, Jnanabharathi Campus, Bengaluru University, Bengaluru, Karnataka– 560 056, India. Division of Entomology and Nematology, ICAR-Indian Institute of Horticultural Research, Hesserghatta, Bengaluru, Karnataka – 560 089, India. *Email:
[email protected] 2
ABSTRACT: The tobacco mosaic virus (TMV) is a positive-sense single stranded RNA virus that infects a wide range of horticultural crops.TMV is extensively studied and most of its molecular working has been deduced. However, not much is known about how the capsids and the genetic material self-assemble to form the virus. In this study, we give a theoretical perspective and proofs that cytosolic vibrations are involved in the assembly of the TMV. During the replication of TMV, a –RNA (negative-sense RNA) is synthesized using the genomic +RNA (positive-sense RNA) as a template, followed by the synthesis of +RNA using –RNA as a template. This replication process gives raise to electrostatic repulsion between the RNA’s. The negative charge of a RNA molecule works against its folding, whereas, positive ions promote folding by reducing the repulsion between the RNA’s phosphates thus stabilizing the viral RNAs. Stabilization of the viral RNAs by ionic interactions releases energy that causescytosolic vibrations, thus initiating the assembly of the virus. A detailed theoretical perspective is elaborated. Keywords: cytosolic vibration, tobacco mosaic virus
INTRODUCTION
VIRUS ENTRY AND REPLICATION
Tobacco mosaic virus (TMV) (Fig 1) is one of the simplest plant viruses and is a classical example of a rod-shaped virus. Its rod shape results from the arrangement of helical arrays of identical protein subunits, embedded with a single molecule of a RNA helix (Klug, 1999). However, knowledge on the arrangement of the viral particle is inadequate and here we provide a theoretical perspective on the same. For an immaculate understanding, we explain the complete process from entry to assembly.
TMV enters a plant cell through mechanical wounds that either transiently open the plasma membrane or allow pinocytosis (Liu and Nelson, 2013; Cheng et al., 2000). Soon after entering, the virus begins to disassemble within 3 min of entry followed by the translation of viral RNA (Liu & Nelson, 2013; Cheng et al., 2000). The replication of TMV RNA involves synthesis of a –RNA using the genomic +RNA as a template, followed by the synthesis of +RNA on the –RNA templates. The negative strand aids in the synthesis of sub-genomic mRNAs that
Figure 1. Illustration of the tobacco mosaic virus. 1. The genome of TMV is a positive-sense single strand RNA. 2. The capsomer proteins that cover the genetic material. 3. The Capsid.
Figure 2. Molecular structure of the monomeric unit of the tobacco mosaic virus coat protein. 233
Tejas Thondehaalmutt and Vivek Kempraj
hairpin structure of the viral RNA before encapsidation (Butcher and Pyle 2011; Grilley et al., 2006).
code for motor proteins and coat proteins. The motorproteins influence the intracellular movement of otherproteins that aids in the formation of the virus particle (Buck, 1999; Cheng et al., 2000). Whereas, the +RNA synthesize mRNAs for the production of a 126-kD protein with its N-terminal region possessing methyl transferase like activity and its C-terminal possesses helicase like activity. The 183-kD protein consists of a polymerase domain (RNA dependent RNA polymerase) (Hagiwara et al., 2003; Heinlein et al., 1998; Más and Beachy, 1999). Both these proteins are involved in the replication of viral RNA (Cheng et al., 2000;Osman & Buck, 1996). All these processes take place in the endoplasmic reticulum (ER), suggesting that the replication of the genome of the TMV takes place in proximal association with the ER (Cheng et al., 2000; Draper, 2004).
CYTOPLASMIC VIBRATION The microtubules are a key components of the cytoskeleton and are essential in performing a variety of functions including chromosome movement during cell division, intracellular transport of materials, movement of organelles and intracellular tracking (Salitz and Schmitz, 1989). When microtubule inhibitors like cytochalasin D and oryzalin were injected, efficient inhibition took place and thus the cytoplasmic streaming rapidly stopped and when they reduced the inhibitors concentration the streaming enhanced to 100%, thus we can say that microtubules are very essential for the generation of the force for particle movement (Turner et al., 1998). There is a stress field generated in the cytosol due to the vibrating microtubules that help in particle movement (Salitz and Schmitz, 1989). For the vibration of the microtubules to take place, anenergy source is a prerequisite. There are three possible sources for the excitations of the microtubules (Daneshmand, 2012) viz., Hydrolysis of GTP, motor proteins interaction with the microtubules and Energy from the mitochondria.
RNA STABILITY After the replication of many TMV genomes, there occurs an electrostatic repulsion between the genomes (RNAs) owing to the negative charge of the RNA phosphate. However, positive ions in plant cells reduce the repulsion between the RNA phosphate groups (Draper, 2004 & 2008). The positive ions are mostly monovalent cation or divalent cation (K+ or Mg++). Mg++ is considered more efficient in stabilizing RNA for simple entropic reasons first explained by Manning (1978). Mg++ stabilizes the electrostatic stress generated by the repulsions between the RNA phosphate as same as 2 K+ ions at a lower entropic cost as there are fewer cations confined near the RNA. Thus Mg++ is more efficient in stabilizing the negatively charged RNA (Draper, 2004 and 2008; Grilley et al., 2006; Chu et al., 2008). Before encapsidation of the vRNA, the RNA forms a secondary structure called the ‘stem loop hairpin’. This secondary structure of the vRNA gets into the central hole of the protein disk between 2 layers of the capsids. Then the protein subunits are added to elongate the 2 layered proteins to form a complete capsid for the new viral RNA (Turner et al., 1998). Thus the RNA has to attain the secondary structure (stem loop hairpin structure) for encapsidation. The Mg++ ions interact with the vRNA on the basis of specific energetics namely diffusely bound or site bound (Misra and Draper, 2001). In diffusely bound type of interaction, the ion binds to the negative-charged pockets (due to the improper arrangement of the molecule) and in site bound interaction, the ion interacts at a distinct location on the vRNA containing electronegative ligands. However, the stem loop hairpin structure is stabilized by the diffusely bound Mg++ ion (Butcher and Pyle, 2011). Due to the stabilization of the RNA and decrease in the Gibbs free energy (negative value of the ÄG), there is a release in energy during the stabilization process of the stem loop Pest Management in Horticultural Ecosystems, Vol. 21, No. 2 pp 233-235 (2015)
These are the only possible ways by which the microtubules may be excited and the particle movement in the cytosol may take place. At later stages of infection, the association of vRNA with motor proteins was observed (Mása and Beachya, 1999). The motor proteins coded by the virus, associate with the microtubules and help the intracellular transport of various necessary proteins and also aid in the transportation of TMV to adjacent cells (Ashby et al., 2006; Seemanpillai et al., 2006; Niehl et al., 2013). Thus, the energy released during the stabilization of secondary structure of the vRNA is transferred to the microtubule via the motor proteins causing the excitation of microtubules in ways similar to the excitation caused by the energy liberated by hydrolysis of GTP (Mása and Beachy, 2000; Pokorný, 2004). This excitation of microtubule instigates cytosolic vibrations resulting in the movement of particles in the cytoplasm. Owing to motion of particles there occurs a probability where the stable secondary structure of vRNA comes in contact with the two layered disk of protein subunit which when entraps the hairpin loop structure of vRNA, starts the process of capsidation forming the virions. The virions are then spread from one cell to other via the plasmodesmata with the help of the virus encoded motor proteins (Chen et al., 2000). We conclude that this theoretical perspective may provide insights on future research into controlling TMV at a molecular level. Controlling this devastating virus or similar plant viruses may help the world save millions of dollars. Apart, our study may also be an impetus for development of new futuristic methods for controlling this devastating virus. 234
Assembly of capsid a theoretical perspective
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MS Received : 11 Novermber 2015 MS Accepted : 24 December 2015
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