Capsid protein structure, self-assembly, and processing reveal morphogenesis of the marine virophage mavirus Diana Borna, Lukas Reutera, Ulrike Mersdorfa, Melanie Muellera, Matthias G. Fischera, Anton Meinharta, and Jochen Reinsteina,1 aDepartment of Biomolecular Mechanisms, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany Edited by Michael G. Rossmann, Purdue University, West Lafayette, IN, and approved May 30, 2018 (received for review March 28, 2018) Virophages have the unique property of parasitizing giant viruses single jelly-roll (SJR) minor capsid protein called penton protein, within unicellular hosts. Little is understood about how they form a clan CE cysteine protease, and an FtsK/HerA-type ATPase (12, infectious virions in this tripartite interplay. We provide mechanistic 15). Cryo-electron microscopy (EM) uncovered the structure of insights into assembly and maturation of mavirus, a marine viro- the mature Sputnik particle with triangulation number T = 27, 260 phage, by combining structural and stability studies on capsomers, trimeric MCP capsomers, and 12 pentameric penton proteins “ ” virus-like particles (VLPs), and native virions. We found that the mavi- ( pentons ) (16, 17). MCP capsomers with quasi-sixfold symmetry ’ rus protease processes the double jelly-roll (DJR) major capsid protein form the capsid s facets, and pentons show pentagonal symmetry (MCP) at multiple C-terminal sites and that these sites are conserved to build the vertices as observed in other DJR viruses of the among virophages. Mavirus MCP assembled in Escherichia coli in the PRD1-adenovirus lineage (16, 18). The JR fold consists of two sandwiched β-sheets, each composed of four antiparallel strands absence and presence of penton protein, forming VLPs with defined E. coli labeled B to I, generating sheets BIDG and CHEF (19). Notably, size and shape. While quantifying VLPs in lysates, we found the Sputnik MCP was found to be 87 residues shorter at the C that full-length rather than processed MCP is the competent state for terminus than predicted from the gene sequence (16). It was capsid assembly. Full-length MCP was thermally more labile than proposed that MCP processing occurs during virophage assembly, truncated MCP, and crystal structures of both states indicate that but experimental evidence is lacking. full-length MCP has an expanded DJR core. Thus, we propose that Although the structure of the Sputnik capsid revealed the ar- the MCP C-terminal domain serves as a scaffolding domain by adding rangement of capsid proteins in the mature virion, little is known strain on MCP to confer assembly competence. Mavirus protease about how they assembled, whether assembly and processing in- processed MCP more efficiently after capsid assembly, which pro- volved giant virus or host factors, and how the conserved protease vides a regulation mechanism for timing capsid maturation. By anal- and ATPase are involved in morphogenesis. Here, we explored ogy to Sputnik and adenovirus, we propose that MCP processing virophage capsid assembly and maturation processing, including renders mavirus particles infection competent by loosening interac- effects on capsid stability and implications for host infection. In- tions between genome and capsid shell and destabilizing pentons sights into these potentially conserved processes are crucial to for genome release into host cells. The high structural similarity of understand the remarkable virophage life cycle and how it inter- mavirus and Sputnik capsid proteins together with conservation of acts with giant virus and host cell. We studied the marine viro- protease and MCP processing suggest that assembly and maturation phage mavirus that infects the widespread nanoflagellate Cafeteria mechanisms described here are universal for virophages. roenbergensis and exploits the Cafeteria roenbergensis virus (CroV) for replication (9). Its ability to integrate into host cell genomes virus assembly | capsid maturation | viral protease | crystallography | electron microscopy Significance irion morphogenesis is crucial for the viral life cycle. Dynamic Virophages are parasites of giant viruses within protists. They Vstages of the virus, including host infection, replication, and reduce giant virus production and increase host cell survival. They virion formation, were emphasized after the discovery of giant provide a defense system for protists against giant viruses in viruses, which spurred discussions to define the intracellular virion diverse environments, likely with ecological relevance for protist factory as actual “viral self” (1, 2). The interplay between giant virus populations. To understand the remarkable virophage life cycle, and host cell became even more complex when virophages were it is crucial to investigate how they assemble into infectious discovered as parasites of giant viruses (3). Giant viruses of the particles and which processes require interactions with giant vi- family Mimiviridae reproduce within cytoplasmic virion factories rus and host. We examined the marine virophage mavirus to (reviewed in ref. 4) that are exploited by virophages of the family show that its major and minor capsid proteins assemble into Lavidaviridae, likely for transcription (5). Research on virophages so virus-like particles in the absence of specific host or viral factors. far mainly focused on their isolation, cultivation, and genetic char- Subsequently, the virophage-encoded protease processes the acterization, which led to the description of Sputnik (3, 6, 7), major capsid protein to prepare virions for infection. Zamilon (8), and mavirus (9). They infect freshwater and marine Author contributions: D.B. and J.R. designed research; D.B., L.R., U.M., M.M., M.G.F., and protozoa, but metagenomic data suggest that virophages also infect A.M. performed research; D.B., U.M., M.M., M.G.F., A.M., and J.R. analyzed data; and D.B. algae (10–12). Their ∼70-nm icosahedral capsids enclose a linear or and J.R. wrote the paper. circular dsDNA genome of 17–19 kbp, carrying 20–21 ORFs. It was The authors declare no conflict of interest. further revealed that virophages can integrate into genomes of both This article is a PNAS Direct Submission. giant viruses (6) and host cells (13, 14); the latter is proposed to be a This open access article is distributed under Creative Commons Attribution-NonCommercial- defense strategy against giant viruses, potentially with ecological NoDerivatives License 4.0 (CC BY-NC-ND). relevance for protist populations (3, 9, 13, 14). Data deposition: The atomic coordinates and structure factors have been deposited in the Although dozens of virophage genomes are described, little is Protein Data Bank, www.wwpdb.org (PDB ID codes 6G41–6G45). understood about how they form infectious particles and which 1To whom correspondence should be addressed. Email: jochen.reinstein@mpimf- processes rely on interactions with the giant virus and the host cell. heidelberg.mpg.de. Interestingly, the four genes conserved among virophages are all This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. proposed to be involved in virion morphogenesis (5, 12). They 1073/pnas.1805376115/-/DCSupplemental. code for a double jelly-roll (DJR) major capsid protein (MCP), a Published online June 25, 2018. 7332–7337 | PNAS | July 10, 2018 | vol. 115 | no. 28 www.pnas.org/cgi/doi/10.1073/pnas.1805376115 and to act as a defense system against CroV has been proposed to concentration of available native mavirus virions, but we can also be of ecological importance for zooplankton populations (14). not exclude that MCP C-terminal fragments are absent in these virions. In addition, five further native mavirus gene products Results were identified: MV02 (integrase), two cleavage fragments of Mavirus Protease Processes MCP at Multiple Sites in Vitro. The MV13 (predicted α/β hydrolase), MV14 (unknown function), virophage protease is suggested to be involved in capsid matu- MV15 (FtsK/HerA-type ATPase), and MV17 (penton protein) ration, as observed for other eukaryotic viruses of the PRD1- (9). The SDS/PAGE fragments at ∼20 kDa and ∼60 kDa corre- adenovirus lineage (15, 20). To investigate its function in the spond to the N- and C-terminal part, respectively, of the 80.6-kDa virophage mavirus, we recombinantly expressed the mavirus MV13. Interestingly, the MV13 sequence between MV13N and cysteine protease (MVP), MCP, and penton protein. Purified MV13C that was not covered by tryptic peptide masses (res. MVP was present as a dimer, MCP as a trimeric capsomer, and 476–499) contains a potential MVP cleavage site, GK j G. We penton protein as a pentamer (SI Appendix, Fig. S1 A–C). MVP also identified MVP in native mavirus virions by immunoblotting cleaved recombinant MCP at multiple sites but not recombinant (Fig. 1C, Right), supporting the hypothesis that it is the rele- penton protein (Fig. 1A and SI Appendix, Fig. S1E). We used vant protease for processing not only MCP but also MV13 in electrospray-ionization quadrupole time-of-flight mass spectrome- mavirus virions. try (MS) to identify MCP cleavage fragments from this in vitro turnover (SI Appendix,Fig.S1F and Table S1). The largest MCP MCP Processing Results in Jelly-Roll Core Compaction and Tower fragment comprised residues (res.) 1–516 of full-length MCP Expansion. To elucidate the effect of processing on mavirus (flMCP), which we termed MCPΔC (Fig. 1B). The C-terminal MCP regarding structure and stability, we recombinantly gen- domain (CTD) of flMCP (res. 517–606) seems to be cleaved from erated MCPΔC and compared its properties with flMCP. Both flMCP first in this in vitro experiment and was further processed at proteins form trimers in solution (SI Appendix, Fig. S1 A and D), – three additional sites, generating MCPC1 (res. 517 567), MCPC2 but MCPΔC showed higher melting temperatures (T ) than – – – m (res. 568 580), MCPC3 (res. 581 592) and MCPC4 (res. 593 606). flMCP (SI Appendix, Fig. S3A), indicating higher thermal sta- Three of four cleavage sites in mavirus MCP are preceded by a bility of MCPΔC. To answer whether processing alters the MCP diglycine motif (GG j X, Fig.