Heterodimers as the Structural Unit of the T=1 Capsid of the Fungal Double-Stranded RNA Rosellinia necatrix Quadrivirus 1

Abstract

Most double-stranded RNA (dsRNA) viruses are transcribed and replicated in a specialized icosahedral capsid with a T‫1؍‬ lattice consisting of 60 asymmetric capsid protein (CP) dimers. These capsids help to organize the viral genome and replicative complex(es). They also act as molecular sieves that isolate the virus genome from host defense mechanisms and allow the passage of nucleotides and viral transcripts. Rosellinia necatrix quadrivirus 1 (RnQV1), the type species of the family Quadriviridae, is a dsRNA fungal virus with a multipartite genome consisting of four monocistronic segments (segments 1 to 4). dsRNA-2 and dsRNA-4 encode two CPs (P2 and P4, respectively), which coassemble into ϳ450-Å-diameter capsids. We used three-dimensional cryo-electron microscopy combined with complementary biophysical techniques to determine the structures of RnQV1 virion strains W1075 and W1118. RnQV1 has a quadripartite genome, and the capsid is based on a single-shelled T‫1؍‬ lattice built of P2-P4 dimers. Whereas the RnQV1-W1118 capsid is built of full-length CP, P2 and P4 of RnQV1-W1075 are cleaved into several polypeptides, maintaining the capsid structural organization. RnQV1 heterodimers have a quaternary organization similar to that of homodimers of reoviruses and other dsRNA mycoviruses. The RnQV1 capsid is the first T‫1؍‬ capsid with a heterodimer as an asymmetric unit reported to date and follows the architectural principle for dsRNA viruses that a 120-subunit capsid is a conserved assembly that supports dsRNA replication and organization. IMPORTANCEGiven their importance to health, members of the family Reoviridae are the basis of most structural and functional studies and provide much of our knowledge of dsRNA viruses. Analysis of bacterial, protozoal, and fungal dsRNA viruses has improved our understanding of their structure, function, and evolution, as well. Here, we studied a dsRNA virus that infects the fungus Rosellinia necatrix, an ascomycete that is pathogenic to a wide range of plants. Using three-dimensional cryo-electron microscopy and analytical ultracentrifugation analysis, we determined the structure and stoichiometry of Rosellinia necatrix quadrivirus 1 (RnQV1). The RnQV1 capsid is a T‫1؍‬ capsid with 60 heterodimers as the asymmetric units. The large amount of genetic information used by RnQV1 to construct a simple T‫1؍‬ capsid is probably related to the numerous virus-host and virus-virus interactions that it must face in its life cycle, which lacks an extracellular phase.

Acquisition of functions on the outer capsid surface during evolution of double-stranded RNA fungal viruses

“…The ~470 Å-diameter T = 1 capsid shows 12 outwardly protruding decamers at the pentameric positions ( Fig 1B , orange), each bearing five copies of P2 and P4 [ 25 ]. Each pentameric capsomer is formed by an inner ring of five P2 monomers ( Fig 1D , blue; inset) surrounded by an outer ring of five partially intercalated P4 monomers ( Fig 1D , yellow; inset), in which the asymmetric unit is a P2-P4 heterodimer ( Fig 1B and 1D ).…”

“…Whereas most dsRNA virus capsids are based on dimers of a single protein, the type species of the family Quadriviridae , Rosellinia necatrix Quadrivirus 1 (RnQV1), has a single-shelled T = 1 capsid formed by 60 heterodimers of two proteins of 1356 (P2) and 1059 residues (P4) [ 25 ]. RnQV1 is a mycovirus with a multipartite genome consisting of four monocistronic dsRNA segments (genome sizes range from 3.7 to 4.9 kbp), in which one or two dsRNA segments are encapsidated in a similar particle [ 26 ].…”

Capsid Structure of dsRNA Fungal Viruses

“…RnQV1 virus strains W1075 and W1118, isolated from different locations in Japan, have been analyzed by 3D cryo-EM and analytical ultracentrifugation [ 36 ]. Their P2 and P4 proteins co-assemble into isometric virus particles ~45 nm in diameter, which each package either one or two of the four genome segments.…”

“…Top row: T = 1 capsids of bluetongue virus (BTV), orthoreovirus, rice dwarf virus (RDV), cytoplasmic polyhedrosis virus (CPV), rotavirus, grass carp reovirus (GCRV), picobirnavirus (PBV), ϕ6 phage and ϕ8 phage, L-A virus of Saccharomyces cerevisiae (ScV-L-A), Penicillium chrysogenum virus (PcV), Penicillium stoloniferum virus F (PsV-F), and Rosellinia necatrix quadrivirus 1 (RnQV1), viewed along a two-fold axis of icosahedral symmetry (center row). BTV VP3 [ 2 ] (PDB accession number 2btv; 901 residues), λ1 [ 41 ] (1ej6; 1275 residues), P3 [ 42 ] (1uf2; 1019 residues), VP1 [ 19 ] (3cnf; 1333 residues), VP2 [ 43 ] (3kz4; 880 residues), GCRV VP3 [ 22 ] (3k1q; 1027 residues), PBV CP [ 23 ] (2vf1; 590 residues), ϕ6 P1 [ 44 ] (4btq; 769 residues) and ϕ8 P1 [ 45 ] (4btp; 792 residues), Gag [ 26 ] (1m1c; 680 residues), PcV CP domain A [ 46 ] (3j3i; 498 residues; complete CP 982 residues), PsV-F CP [ 29 ] (3es5; 420 residues) and RnQV1-W1118 P2 and P4 [ 36 ] (5nd1; 972 and 1005 residues, respectively), shown from top view. Bottom row: side views of the same structures (T = 1 shell exterior at right).…”

“…They have also been described for members of the family Cystoviridae —bacteriophages that infect the prokaryote Pseudomonas syringae [ 25 , 26 ]. Members of the Toti- [ 27 , 28 , 29 ], Partiti- [ 30 , 31 ], Megabirna- [ 32 ], Chryso- [ 33 , 34 , 35 ], and Quadriviridae [ 36 , 37 ] families, which infect unicellular and simple eukaryotes, such as fungi, protozoa, but also some plants, also have these capsids. Members of the family Birnaviridae, which infect vertebrates, mollusks, insects, and rotifers, are exceptions, since they lack the T = 1 core of 60 CP dimers [ 38 , 39 ].…”