Phase separation of a plant virus movement protein and cellular factors support virus-host interactions

Abstract

Both cellular and viral proteins can undergo phase separation and form membraneless compartments that concentrate biomolecules. The p26 movement protein from single-stranded, positive-sense Pea enation mosaic virus 2 (PEMV2) separates into a dense phase in nucleoli where p26 and related orthologues must interact with fibrillarin (Fib2) as a pre-requisite for systemic virus movement. Using in vitro assays, viral ribonucleoprotein complexes containing p26, Fib2, and PEMV2 genomic RNAs formed droplets that may provide the basis for self-assembly in planta. Mutating basic p26 residues (R/K-G) blocked droplet formation and partitioning into Fib2 droplets or the nucleolus and prevented systemic movement of a Tobacco mosaic virus (TMV) vector in Nicotiana benthamiana. Mutating acidic residues (D/E-G) reduced droplet formation in vitro, increased nucleolar retention 6.5-fold, and prevented systemic movement of TMV, thus demonstrating that p26 requires electrostatic interactions for droplet formation and charged residues are critical for nucleolar trafficking and virus movement. p26 readily partitioned into stress granules (SGs), which are membraneless compartments that assemble by clustering of the RNA binding protein G3BP following stress. G3BP is upregulated during PEMV2 infection and over-expression of G3BP restricted PEMV2 RNA accumulation >20-fold. Deletion of the NTF2 domain that is required for G3BP condensation restored PEMV2 RNA accumulation >4-fold, demonstrating that phase separation enhances G3BP antiviral activity. These results indicate that p26 partitions into membraneless compartments with either proviral (Fib2) or antiviral (G3BP) factors.

Liaisons dangereuses: Intrinsic Disorder in Cellular Proteins Recruited to Viral Infection-Related Biocondensates

“…2.5.6. Fibrillarin-2 and G3BP-like SG Nucleator from N. benthamiana An interesting mechanism of virus-induced LLPS was described in N. benthamiana cells infected with single-stranded, positive-sense RNA Pea enation mosaic virus 2 (PEMV2) [72].…”

“…Here, dense and poorly dynamic condensates containing PEMV2 p26, a protein required for the trafficking of viral RNA through the vascular system of infected plants [184]), were observed in the nucleolus of infected cells. These condensates, in addition to viral p26, contain nucleolar protein fibrillarin (Fib2) and PEMV2 genomic RNAs [72]. The recruitment of Fib2 into droplets and the ability to systemically traffic a virus vector requires p26 s ability to phase separate, as both of these activities are suppressed in phase separationdeficient p26 mutants [72].…”

“…These condensates, in addition to viral p26, contain nucleolar protein fibrillarin (Fib2) and PEMV2 genomic RNAs [72]. The recruitment of Fib2 into droplets and the ability to systemically traffic a virus vector requires p26 s ability to phase separate, as both of these activities are suppressed in phase separationdeficient p26 mutants [72]. Fib2, which is involved in the systemic trafficking of viral ribonucleoprotein complexes [185,186], itself forms the dense fibrillar component of the nucleolus [187], indicating that it acts as a scaffold responsible for recruiting client proteins into the nucleolus [72].…”

“…The recruitment of Fib2 into droplets and the ability to systemically traffic a virus vector requires p26 s ability to phase separate, as both of these activities are suppressed in phase separationdeficient p26 mutants [72]. Fib2, which is involved in the systemic trafficking of viral ribonucleoprotein complexes [185,186], itself forms the dense fibrillar component of the nucleolus [187], indicating that it acts as a scaffold responsible for recruiting client proteins into the nucleolus [72]. Although there is no structural information on N. benthamiana Fib2, its counterpart from Arabidopsis thaliana was shown to contain an N-terminal intrinsically disordered glycine-and arginine-rich (GAR) domain (Fib2 GAR ) that is sufficient, per se, to drive Fib2 phase separation [72].…”

“…Fib2, which is involved in the systemic trafficking of viral ribonucleoprotein complexes [185,186], itself forms the dense fibrillar component of the nucleolus [187], indicating that it acts as a scaffold responsible for recruiting client proteins into the nucleolus [72]. Although there is no structural information on N. benthamiana Fib2, its counterpart from Arabidopsis thaliana was shown to contain an N-terminal intrinsically disordered glycine-and arginine-rich (GAR) domain (Fib2 GAR ) that is sufficient, per se, to drive Fib2 phase separation [72]. In line with these data, we predicted three short IDRs and a GAR domain (residues 1-83) in N. benthamiana Fib2 (see Table 1).…”