INTRODUCTION
Birnaviridae viruses are disease-causative agents to vertebrates, encompassing a few species in the genus
Aquabirnavirus infecting fishes and genus
Avibirnavirus infecting birds. Recently, putative birnaviruses have also been identified in pigs (
1). The infectious pancreatic necrosis virus (IPNV), a member of the
Aquabirnavirus, can infect diverse salmonid species, leading to lethal outcomes characterized by pancreatic necrosis and catarrhal enteritis (
2,
3). Because of its negative impact on farmed fish, control measures, such as vaccination and genetic selection, have been implemented to combat this virus (
4,
5). Another important birnavirus pathogen, the infectious bursal disease virus (IBDV), infects chickens and turkeys, leading to severe immunosuppression (
5,
6).
Birnaviruses have a bi-segmented dsRNA genome (
7,
8). Segment A encodes a large polyprotein consisting of pVP2-VP3-VP4, and, in some species, a short VP5 protein (
9,
10). Segment B encodes the RNA-dependent RNA polymerase (RdRp), VP1 (
9). The preVP2–VP3–VP4 polyprotein undergoes self-cleavage by the VP4 protease, generating preVP2 and VP3 (
11). During particle assembly, preVP2 is further processed to mature VP2 and short peptides by cleaving its C-terminal region (
12). The disintegrated birnavirus particle releases bi-segmented dsRNA filaments, which provide the evidence of the formation of ribonucleoprotein complexes (RNPs) (
13,
14). In these RNPs, hundreds of VP3s are bound to the viral dsRNA filaments, likely contributing to the compaction of the viral genomes (
14). VP1 exists either in a free form or covalently linked to each segmented genome (
15,
16). VP3 interacts with VP1 to package the segmented genomes inside the particle (
17–19). On the contrary, VP2s assemble into a 60–70 nm T=13 icosahedral single-layer capsid to protect the RNPs (
20,
21). The precursor N- and C-terminal regions of VP2 and the C-terminal peptide of VP3 are critical for the formation of infectious large T=13 particles since expressing only the matured form of VP2 results in the formation of non-infectious small ~23 nm T=1 subviral particles (
21–26). Short peptide fragments from the cleaved C-terminal region of the preVP2 are present in IPNV particles and also play a critical role in assembling T=13 virus particles (
27,
28).
The crystal structures of T=1 subviral particles from both IBDV and IPNV have been determined, revealing three structural domains within VP2: the P, S, and B domains (
21,
23,
24,
29). The S domain employs a typical jelly-roll fold of icosahedral viruses, which implies an evolutionary link between the +ssRNA nodaviruses and tetraviruses and the dsRNA birnaviruses and reoviruses (
21,
30). The P domain unique to birnaviruses is considerably variable in amino acid sequence and forms surface protrusions (
21,
24). Structural variations in the surface loops of the P domain between the IBDV and IPNV subviral particles likely play a role in regulating virulence and tropism (
24). Certainly, mutations in the P domain often result in less virulent strains (
31–34). The B domain located inside the virus capsid is believed to play a role in genome encapsulation through its arrangement of α-helices, as described in other ssDNA and +ssRNA viruses that employ the jelly roll fold (
30,
35–37). The T=1 icosahedron contains only one VP2 subunit in the asymmetric unit, and the structure of this VP2 has been resolved in a single conformation (
24), limiting the understanding of the functional structures of the infectious T=13 capsid. This limitation also hinders the comprehension of possible mechanisms of the T=13 particle assembly.
Another uncertain aspect of the birnavirus capsid structure is the surface pore at each 5-fold axis. This pore possibly has a role in facilitating the synthesis of virus transcripts inside the capsid by incorporating nucleoside triphosphates (NTPs) and releasing +ssRNA virus transcripts. This process, known as intraparticle genome transcription, occurs in other icosahedral dsRNA viruses, such as reoviruses and totiviruses (
38–46). However, in birnaviruses, the surface pore might be obstructed by unresolved surface loops of VP2 at the 5-fold axis (
20,
21). Previous studies have described the capability of purified birnavirus particles to perform intraparticle genome synthesis (
47,
48). In contrast, it has also been suggested that the interior RNPs could be released from the capsid during the endocytosis and synthesize viral transcripts in cellular virus factories (
49–51). Whether the RNP functions as a capsid-independent transcription complex
in situ remains a pending question.
Considering the importance and unrevealed multifunctionality of the birnavirus capsid, it is critical to determine its infectious large T=13 particles using cryogenic electron microscopy (cryo-EM) single particle analysis. To date, only the cryo-EM structure of IBDV has been resolved (
20). In this study, we present the first cryo-EM structure of the salmonid IPNV, providing new insights into the structural mechanisms of VP2 that are essential for infection, particle formation, and intraparticle genome synthesis.
ACKNOWLEDGMENTS
The data were collected at the Cryo-EM Swedish National Facility funded by the Knut and Alice Wallenberg, Erling Persson Family, and Kempe Foundations, SciLifeLab, Stockholm University, and Umeå University. We thank Dustin Morado for help with data acquisition and Jakob Klefenberg for help with data analysis.
Funding was provided by the following agencies: Research Council of Norway (to Ø.E., grant numbers 324266 and 301083), the Swedish Research Council (to K.O., grant numbers 2018-03387 and 2023-01857; to A.M., grant number 2022-00236), the Swedish Foundation for International Cooperation in Research and Higher Education (STINT) (to K.O., grant number JA2014-5721), FORMAS research grant from the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (to K.O., grant numbers 2018-00421 and 2022-02347), Royal Swedish Academy of Sciences (to K.O., grant number BS2018-0053). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.