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Molecular Biology and Structure of a Novel Penaeid Shrimp Densovirus Elucidate Convergent Parvoviral Host Capsid Evolution

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Molecular Biology and Structure of a Novel Penaeid Shrimp Densovirus Elucidate Convergent Parvoviral Host Capsid Evolution Molecular biology and structure of a novel penaeid shrimp densovirus elucidate convergent parvoviral host capsid evolution Judit J. Pénzesa,b, Hanh T. Phama, Paul Chipmanb, Nilakshee Bhattacharyac, Robert McKennab, Mavis Agbandje-McKennab,1,2, and Peter Tijssena,1,2 aInstitut Armand-Frappier, Institut national de la recherche scientifique-Institut Armand-Frappier, Laval, QC H7V 1B7, Canada; bThe McKnight Brain Institute, University of Florida, Gainesville, FL 32610; and cInstitute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306 Edited by Kenneth I. Berns, University of Florida College of Medicine, Gainesville, FL, and approved July 8, 2020 (received for review April 27, 2020) The giant tiger prawn (Penaeus monodon) is a decapod crustacean isolated from both proto- and deuterostome invertebrates, mostly widely reared for human consumption. Currently, viruses of two insects and other arthropods (6–18). distinct lineages of parvoviruses (PVs, family Parvoviridae; subfamily To date, parvovirus crustacean pathogens comprise three Hamaparvovirinae) infect penaeid shrimp. Here, a PV was isolated distinct lineages with divergent genome organizations and tran- and cloned from Vietnamese P. monodon specimens, designated scription patterns. Cherax quadricarinatus DV, isolated from the Penaeus monodon metallodensovirus (PmMDV). This is the first freshwater red-clawed crayfish (Cherax quadricarinatus), has an member of a third divergent lineage shown to infect penaeid ambisense genome with a PLA2 domain and is now an assigned decapods. PmMDV has a transcription strategy unique among in- member of genus Aquambidensovirus of the Densovirinae (19). vertebrate PVs, using extensive alternative splicing and incorpo- Genera Hepanhamaparvovirus and Penstylhamaparvovirus are rating transcription elements characteristic of vertebrate-infecting members of subfamily Hamaparvovirinae, each with one species PVs. The PmMDV proteins have no significant sequence similarity that lacks a PLA2 domain. They both infect penaeid shrimps, with other PVs, except for an SF3 helicase domain in its nonstruc- including Penaeus monodon and Litopenaeus stylirostris (20–26). tural protein. Its capsid structure, determined by cryoelectron mi- Hepanhamaparvoviruses possess a larger genome ∼6.3 kb with croscopy to 3-Å resolution, has a similar surface morphology to 220-nt-long ITRs that form hairpins. The Penstylhamaparvovirus MICROBIOLOGY Penaeus stylirostris densovirus, despite the lack of significant cap- genome is only 3.9-kb long and instead of hairpins harbors direct sid viral protein (VP) sequence similarity. Unlike other PVs, PmMDV terminal repeats (27), an exception among PVs. folds its VP without incorporating a βA strand and displayed unique In contrast to the ∼100 capsid structures determined for multimer interactions, including the incorporation of a Ca2+ cation, members of the Parvovirinae, there are only four high-resolution attaching the N termini under the icosahedral fivefold symmetry structures for invertebrate-infecting PVs (28). These include three axis, and forming a basket-like pentamer helix bundle. While the members of the Densovirinae,GalleriamellonellaDV(GmDV)of PmMDV VP sequence lacks a canonical phospholipase A2 domain, genus Protoambidensovirus at a resolution of 3.7 Å (29), Acheta the structure of an EDTA-treated capsid, determined to 2.8-Å res- domestica DV (AdDV) of genus Scindoambidensovirus at 3.5-Å olution, suggests an alternative membrane-penetrating cation- resolution (30), and Bombyx mori DV 1 (BmDV1) of Iteradensovirus dependent mechanism in its N-terminal region. PmMDV is an ob- served example of convergent evolution among invertebrate PVs Significance with respect to host-driven capsid structure and unique as a PV show- ing a cation-sensitive/dependent basket structure for an alternative Parvoviruses (PVs) are ssDNA viruses, with T = 1 icosahedral endosomal egress. symmetry, infecting deuterostome and protostome animals. Most PVs have a highly conserved phospholipase A2 domain Crustacea | capsid structure | Parvoviridae | densovirus | (PLA2) in the N-terminal region of their minor capsid protein. convergent evolution Under acidic pH, during endosomal/lysosomal egress, the PLA2 domain is activated to disrupt vesicle membranes. However, ensoviruses (DVs) are autonomous parvoviruses (PVs) of certain PVs lack the PLA2 and thus must use a different escape Dthe family Parvoviridae infecting invertebrates. Until re- mechanism. Our study offers insight into this enigma, showing cently, all known PVs infecting invertebrate hosts were members how a recently discovered PV of marine crustacean has evolved of Densovirinae; however, they have recently been divided into a cation-dependent mechanism to accomplish this task. We two separate subfamilies: Densovirinae, composed of exclusively also show how host-driven convergent evolution pushed two invertebrate-infecting PVs, and Hamaparvovirinae, which infect PVs, infecting the same host species, to adopt strikingly similar both invertebrates and vertebrates (1). The third Parvoviridae surface morphologies, despite distinct multimer interactions subfamily, Parvovirinae, contains exclusively vertebrate-infecting and lack of sequence similarity. PVs. All PVs are nonenveloped, single-stranded DNA (ssDNA) viruses, with an approximate capsid diameter of 21.5 to 25 nm Author contributions: J.J.P., M.A.-M., and P.T. designed research; J.J.P. and H.T.P. per- (2). They package relatively small genomes 3.9 to 6.3 kb, flanked formed research; H.T.P., P.C., and N.B. contributed new reagents/analytic tools; J.J.P., by two inverted terminal repeat (ITR)-containing palindromic R.M., M.A.-M., and P.T. analyzed data; and J.J.P., M.A.-M., and P.T. wrote the paper. sequences forming various hairpin-shaped secondary structures. The authors declare no competing interest. The genome organization is conserved and includes two major This article is a PNAS Direct Submission. ORF expression cassettes. Conventionally these are referred to Published under the PNAS license. as rep, which encodes the nonstructural (NS) proteins, and cap, 1M.A.-M. and P.T. contributed equally to this work. which encodes the capsid viral proteins (VPs), which may have 2To whom correspondence may be addressed. Email: [email protected] or peter.tijssen@ different N-terminal extensions (2). Most Parvoviridae contain a iaf.inrs.ca. phospholipase A2 (PLA2) domain in the N-terminal region of This article contains supporting information online at https://www.pnas.org/lookup/suppl/ their VP1, which breach the endosomal membrane during cellular doi:10.1073/pnas.2008191117/-/DCSupplemental. trafficking (3, 4). DVs are pathogenic for their hosts (5) and were www.pnas.org/cgi/doi/10.1073/pnas.2008191117 PNAS Latest Articles | 1of12 Downloaded by guest on September 24, 2021 at 3.1-Å resolution (31), and one member of the Hamaparvovirinae, Results Penaeus stylirostris DV (PstDV) of genus Penstylhamaparvovirus,ata Virus Detection and Cloning. Deceased P. monodon specimens resolution of 2.5 Å (32). All PVs structures are T = 1icosahedral showing clinical signs of a red telson, uropodia, and pleopods, capsids (point group operator 5.3.2), consisting of 60 VP subunits. were acquired from a farm in the South Vietnam. Discoloration The VP core is structurally conserved with a jellyroll fold (33) of the cephalothorax was observed, suggesting an underlying flanked by loops inserted between the β-strands of the jellyroll, viral infection. Negative-staining electron microscopy (EM) from and strands and helices, forming the surface morphology. In the homogenized tissue revealed uniform ∼21-nm icosahedral particles case of the Parvoviridae, the BIDG sheet of the jellyroll is com- (Fig. 1A). Consequently, extracted DNA was blunt-ended, cloned, plemented with an additional β-strand, strand A (28). The PV and sequenced; it contained a previously unknown crustacean DV, fivefold axis assembly forms a channel-like opening reported to designated PmMDV. aid genome packaging and uncoating, and PLA2 domain exter- Complete Genome Characterization of the Crustacean DV. The com- nalization when required (34, 35). plete genome sequence of PmMDV was deposited into GenBank This study reports the complete genome sequence, expression under the accession number of MK028683 (Fig. 1B). Its length strategy, and near-atomic 3D structure of a DV, designated was 4,374 nt, flanked by ITRs of 416 nt, of which 161 nt fold into a P. monodon metallodensovirus (PmMDV), isolated from P. monodon regular, T-shaped hairpin (Fig. 1B) (36). There was a single nu- shrimp. Its relationship to other PVs by phylogenetic inference, cleotide insertion in the stem of the right ITR, which was present transcription mapping, and expression analysis were also charac- in all three clones sequenced. The overall GC content of the genome terized. The PmMDV and PstDV capsids have convergently evolved was 45.6%, with 76.4% at the termini, presumably stabilizing the similar morphologies. However, PmMDV incorporates unique secondary structure. In silico analysis revealed three ORFs, with a strategies to stabilize its capsid and it has possibly evolved an length greater than 100 nt, as well as a fourth without a canonical alternative membrane-penetrating mechanism in the absence of start codon (summarized in SI Appendix, Table S1). The leftmost the PLA2 that is dependent on divalent cations. Furthermore, ORF, ORF1 (516 aa), displayed sequence identity with the major PmMDV, as the third distinct lineage
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