Functional analysis of the N-terminal basic motif of a eukaryotic satellite RNA virus capsid protein in replication and packaging

Abstract

Efficient replication and assembly of virus particles are integral to the establishment of infection. In addition to the primary role of the capsid protein (CP) in encapsidating the RNA progeny, experimental evidence on positive sense single-stranded RNA viruses suggests that the CP also regulates RNA synthesis. Here, we demonstrate that replication of Satellite tobacco mosaic virus (STMV) is controlled by the cooperative interaction between STMV CP and the helper virus (HV) Tobacco mosaic virus (TMV) replicase. We identified that the STMV CP-HV replicase interaction requires a positively charged residue at the third position (3R) in the N-terminal 13 amino acid (aa) motif. Far-Northwestern blotting showed that STMV CP promotes binding between HV-replicase and STMV RNA. An STMV CP variant having an arginine to alanine substitution at position 3 in the N-terminal 13aa motif abolished replicase-CP binding. The N-terminal 13aa motif of the CP bearing alanine substitutions for positively charged residues located at positions 5, 7, 10 and 11 are defective in packaging full-length STMV, but can package a truncated STMV RNA lacking the 3′ terminal 150 nt region. These findings provide insights into the mechanism underlying the regulation of STMV replication and packaging.

Effects of RNA branching on the electrostatic stabilization of viruses

“…While theoretical arguments suggest that the details of the RNA structure are important for its efficient pack-aging in the small volume of the virus capsid [13,[21][22][23][24][25], it remains overall poorly understood how the RNA sequence chemical composition together with its length affect the compactification and the packaging efficiency. Based on simple scaling arguments, it has been shown that genome secondary structures, or more specifically branching, lower the free energy of RNA encapsidation [21,22].…”

“…Quite interestingly, in a recent experiment on Satellite Tobacco Mosaic Virus (STMV), Sivanandam et al find that reducing the number of charges on the N-terminal section of capsid proteins through mutations results into the encapsidation of shorter RNAs than the wild type ones. However, unexpectedly a single mutation in one specific location along the N-terminal completely stops the self-assembly [13]. Investigating the nature of how and which structural details of RNA could be important for virus assembly is thus urgently required to ascertain on which point along the axis of "charge-matching" to "packaging signals" hypotheses the viruses actually drive and regulate their assembly.…”

“…However, besides the importance of electrostatics, packaging experiments suggest that there must exist a correlation between the specific details of the nucleic acid structure and the efficient virus assembly [10][11][12][13]. In a beautifully designed experiment Comas-Garcia et al [10] have set the viral RNA1 of Brome Mosaic Virus (BMV) and the RNA of Cowpea Chlorotic Mottle Virus (CCMV) to compete against each other for capsid proteins belonging to CCMV exclusively.…”

The different faces of mass action in virus assembly

“…Another issue that has not yet received a lot of attention in the theoretical virus physics literature is how the interaction between the polyanions and the RNA binding domains or ARMs is affected by the molecular weight of the former. Indeed, density functional theoretic calculations are usually done at the level of the ground-state approximation, which presumes all segments to be statistically equivalent [ 23 , 48 – 52 ]. This implies that end- or finite-size effects are ignored.…”

Amino acid changes in the capsid protein of a reassortant betanodavirus strain: Effect on viral replication in vitro and in vivo

“…However, another change was observed in the N‐terminal one‐third of the capsid protein (position 20). The N‐terminal region of capsid proteins can play important roles in different parts of the viral cycle, that is, replication and packaging as described in the Satellite tobacco mosaic virus (STMV) (Sivanandam, Mathews, Garmann, & Erdemci‐tandogan, ) or binding and internalization as reported in the parvovirus B19 (Leisi, Di Tommaso, Kempf, & Ros, ). Combining the three substitutions could be a promising option to generate attenuated virus for the future development of a vaccine against nodavirus.…”