Structures of L-BC virus and its open particle provide insight into Totivirus capsid assembly.

L-BC virus persists in the budding yeast Saccharomyces cerevisiae, whereas other viruses from the family Totiviridae infect a diverse group of organisms including protists, fungi, arthropods, and vertebrates. The presence of totiviruses alters the fitness of the host organisms, for example, by maintaining the killer system in yeast or increasing the virulence of Leishmania guyanensis. Despite the importance of totiviruses for their host survival, there is limited information about Totivirus structure and assembly. Here we used cryo-electron microscopy to determine the structure of L-BC virus to a resolution of 2.9 Å. The L-BC capsid is organized with icosahedral symmetry, with each asymmetric unit composed of two copies of the capsid protein. Decamers of capsid proteins are stabilized by domain swapping of the C-termini of subunits located around icosahedral fivefold axes. We show that capsids of 9% of particles in a purified L-BC sample were open and lacked one decamer of capsid proteins. The existence of the open particles together with domain swapping within a decamer provides evidence that Totiviridae capsids assemble from the decamers of capsid proteins. Furthermore, the open particles may be assembly intermediates that are prepared for the incorporation of the virus (+) strand RNA.

Acquired Functional Capsid Structures in Metazoan Totivirus-like dsRNA Virus.

Non-enveloped icosahedral double-stranded RNA (dsRNA) viruses possess multifunctional capsids required for their proliferation. Whereas protozoan/fungal dsRNA viruses have a relatively simple capsid structure, which suffices for the intracellular phase in their life cycle, metazoan dsRNA viruses have acquired additional structural features as an adaptation for extracellular cell-to-cell transmission in multicellular hosts. Here, we present the first atomic model of a metazoan dsRNA totivirus-like virus and the structure reveals three unique structural traits: a C-terminal interlocking arm, surface projecting loops, and an obstruction at the pore on the 5-fold symmetry axis. These traits are keys to understanding the capsid functions of metazoan dsRNA viruses, such as particle stability and formation, cell entry, and endogenous intraparticle transcription of mRNA. On the basis of molecular dynamics simulations of the obstructed pore, we propose a possible mechanism of intraparticle transcription in totivirus-like viruses, which dynamically switches between open and closed states of the pore(s).

Complete genome sequence of a variant of maize-associated totivirus from Ecuador.

The complete genomic sequence of a variant of the recently reported maize-associated totivirus (MATV) from China was obtained from commercial maize in Ecuador. The genome of MATV-Ec (Ecuador) (4,998 bp) is considerably longer than that of MATV-Ch (China) (3,956 bp), the main difference due to a ≈ 1-kb-long capsid-protein-encoding fragment that is completely absent from the Chinese genome. Sequence alignments between MATV-Ec and MATV-Ch showed an overall identity of 82% at the nucleotide level, whereas at the amino acid level, the viruses exhibited 95% and 94% identity for the putative capsid protein and the RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis of the viral RdRp domain indicated that MATV-Ec and MATV-Ch share a common ancestor with other plant-associated totiviruses, with Panax notoginseng virus A as the closest relative. MATV-Ec was detected in 46% (n = 80) of maize plants tested in this study, but not in endophytic fungi isolated from plants positive for the virus.

Three-dimensional structure of victorivirus HvV190S suggests coat proteins in most totiviruses share a conserved core.

Double-stranded (ds)RNA fungal viruses are currently assigned to six different families. Those from the family Totiviridae are characterized by nonsegmented genomes and single-layer capsids, 300-450 Å in diameter. Helminthosporium victoriae virus 190S (HvV190S), prototype of recently recognized genus Victorivirus, infects the filamentous fungus Helminthosporium victoriae (telomorph: Cochliobolus victoriae), which is the causal agent of Victoria blight of oats. The HvV190S genome is 5179 bp long and encompasses two large, slightly overlapping open reading frames that encode the coat protein (CP, 772 aa) and the RNA-dependent RNA polymerase (RdRp, 835 aa). To our present knowledge, victoriviruses uniquely express their RdRps via a coupled termination-reinitiation mechanism that differs from the well-characterized Saccharomyces cerevisiae virus L-A (ScV-L-A, prototype of genus Totivirus), in which the RdRp is expressed as a CP/RdRp fusion protein due to ribosomal frameshifting. Here, we used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structures of HvV190S virions and two types of virus-like particles (capsids lacking dsRNA and capsids lacking both dsRNA and RdRp) at estimated resolutions of 7.1, 7.5, and 7.6 Å, respectively. The HvV190S capsid is thin and smooth, and contains 120 copies of CP arranged in a "T = 2" icosahedral lattice characteristic of ScV-L-A and other dsRNA viruses. For aid in our interpretations, we developed and used an iterative segmentation procedure to define the boundaries of the two, chemically identical CP subunits in each asymmetric unit. Both subunits have a similar fold, but one that differs from ScV-L-A in many details except for a core α-helical region that is further predicted to be conserved among many other totiviruses. In particular, we predict the structures of other victoriviruses to be highly similar to HvV190S and the structures of most if not all totiviruses including, Leishmania RNA virus 1, to be similar as well.

Three-dimensional structure and stoichiometry of Helmintosporium victoriae190S totivirus.

Most double-stranded RNA viruses have a characteristic capsid consisting of 60 asymmetric coat protein dimers in a so-called T = 2 organization, a feature probably related to their unique life cycle. These capsids organize the replicative complex(es) that is actively involved in genome transcription and replication. Available structural data indicate that their RNA-dependent RNA polymerase (RDRP) is packaged as an integral capsid component, either as a replicative complex at the pentameric vertex (as in reovirus capsids) or as a fusion protein with the coat protein (as in some totivirus). In contrast with members of the family Reoviridae, there are two well-established capsid arrangements for dsRNA fungal viruses, exemplified by the totiviruses L-A and UmV and the chrysovirus PcV. Whereas L-A and UmV have a canonical T = 2 capsid, the PcV capsid is based on a T = 1 lattice composed of 60 capsid proteins. We used cryo-electron microscopy combined with three-dimensional reconstruction techniques and hydrodynamic analysis to determine the structure at 13.8 A resolution of Helminthosporium victoriae 190S virus (Hv190SV), a totivirus isolated from a filamentous fungus. The Hv190SV capsid has a smooth surface and is based on a T = 2 lattice with 60 equivalent dimers. Unlike the RDRP of some other totiviruses, which are expressed as a capsid protein-RDRP fusion protein, the Hv190SV RDRP is incorporated into the capsid as a separate, nonfused protein, free or non-covalently associated to the capsid interior.

Purification, crystallization, and preliminary X-ray analysis of L-A: a dsRNA yeast virus.

TheL-A virus (LAV) particle is a specialized compartment for the transcription and replication of double-stranded RNA. It is 390 A in diameter and infects yeast. The particle is formed by a capsid containing 120 copies of a 680-residue gene product arranged with T = 1 icosahedral symmetry, approximately two copies of an RNA-directed RNA polymerase, and a 4.6-kb linear, duplex RNA. LAV crystals diffracting to at least 4.5-A resolution were grown in a combination of polyethylene glycol 8000, ethylene glycol, and lithium chloride. Following crystallization the reservoir solution was replaced by a 2x concentrated reservoir solution in order for ethylene glycol to function as a cryoprotectant even though initial crystals would not grow at sufficiently high concentrations of ethylene glycol for cryoprotection. A complete data set was collected to 6-A resolution from a frozen crystal obtained with this procedure. The crystals belong to space group P2(1). The unit cell dimensions are a = 406.7 A, b = 403.3 A, c = 572.5 A, beta = 90.3 degrees with two virus particles in the unit cell. The particle orientation was determined with the rotation function and the particle center was estimated on the basis of packing considerations.