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Members of the Caulimoviridae family are important plant pathogens. These circular double-stranded DNA viruses may integrate into the host genome, although this integration is not required for the viral replication cycle. Here, we describe three complete genomes belonging to a new putative Caulimoviridae genus discovered through air metagenomics., Community of Madrid (Spain), under the AIRBIOTA-CM Program (S2013/MAE-2874).

Endogenous viral elements (paleoviruses) provide “molecular fossils” for studying the deep history and macroevolution of viruses. Endogenous plant pararetroviruses (EPRVs) are widespread in angiosperms, but little is known about EPRVs in earlier-branching plants. Here we use a large-scale phylogenomic approach to investigate the diversity and macroevolution of plant pararetroviruses (formally known as Caulimoviridae ). We uncover an unprecedented and unappreciated diversity of EPRVs within the genomes of gymnosperms and ferns. The known angiosperm viruses constitute only a minor part of the Caulimoviridae diversity. By characterizing the distribution of EPRVs, we show that no major euphyllophyte lineages escape the activity of Caulimoviridae , raising the possibility that many exogenous Caulimoviridae remain to be discovered in euphyllophytes. We find that the copy numbers of EPRVs are generally high, suggesting that EPRVs might define a unique group of repetitive elements and represent important components of euphyllophyte genomes. Evolutionary analyses suggest an ancient origin of Caulimoviridae and at least three independent origins of Caulimoviridae in angiosperms. Our findings reveal the remarkable diversity of Caulimoviridae and have important implications for understanding the origin and macroevolution of plant pararetroviruses. IMPORTANCE Few viruses have been documented in plants outside angiosperms. Viruses can occasionally integrate into host genomes, forming endogenous viral elements (EVEs). Endogenous plant pararetroviruses (EPRVs) are widespread in angiosperms. In this study, we performed comprehensive comparative and phylogenetic analyses of EPRVs and found that EPRVs are present in the genomes of gymnosperms and ferns. We identified numerous EPRVs in gymnosperm and fern genomes, revealing an unprecedented depth in the diversity of plant pararetroviruses. Plant pararetroviruses mainly underwent cross-species transmission, and angiosperm pararetroviruses arose at least three times. Our study provides novel insights into the diversity and macroevolution of plant pararetroviruses.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Viruses frequently use insect vectors to effect rapid spread through host populations. In plant viruses, vector transmission is the major mode of transmission, used by nearly 80% of species described to date. Despite the importance of this phenomenon in epidemiology, the specificity of the virus-vector relationship is poorly understood at both the molecular and the evolutionary level, and very limited data are available on the precise viral protein motifs that control specificity. Here, using the aphid-transmitted Cauliflower mosaic virus (CaMV) as a biological model, we confirm that the "noncirculative" mode of transmission dominant in plant viruses (designated "mechanical vector transmission" in animal viruses) involves extremely specific virus-vector recognition, and we identify an amino acid position in the "helper component" (HC) protein of CaMV involved in such recognition. Site-directed mutagenesis revealed that changing the residue at this position can differentially affect transmission rates obtained with various aphid species, thus modifying the spectrum of vector species for CaMV. Most interestingly, in a virus line transmitted by a single vector species, we observed the rapid appearance of a spontaneous mutant specifically losing its transmissibility by another aphid species. Hence, in addition to the first identification of an HC motif directly involved in specific vector recognition, we demonstrate that change of a virus to a different vector species requires only a single mutation and can occur rapidly and spontaneously.

Rice tungro bacilliform virus (RTBV) is a reverse-transcribing DNA virus which, in association with an RNA virus, Rice tungro spherical virus (RTSV), is responsible for rice tungro disease (22), the most important viral disease of rice in South and Southeast Asia. In rice tungro, RTBV induces most of the symptoms (yellowing and reddening of the leaves, stunting of rice plants) and RTSV is mainly involved in the transmission of both viruses via the green leafhopper Nephotettix virescens (5). RTBV is the type and only known member of the “RTBV-like viruses” genus, which has been classified in the Caulimoviridae family comprising caulimoviruses, badnaviruses, and two other genera (29, 31). The plant viruses which belong to this family have many features in common with retroviruses and are also often referred to, together with the human and animal hepadnaviruses, as pararetroviruses (23, 40, 42). The bacilliform RTBV particles are elongated icosahedrons with a diameter of 30 nm and a length of approximately 130 nm, which varies with the virus isolate (22). The RTBV genome is a circular double-stranded DNA molecule of about 8 kbp, containing two site-specific discontinuities resulting from the replication process by reverse transcription and four large open reading frames (ORFs) (Fig. ​(Fig.1A)1A) (1, 17, 39). The corresponding proteins, P1, P2, P3, and P4, are synthesized by specialized translation mechanisms (10–12) from a pregenomic RNA which is used as the template for viral replication and also serves as a polycistronic mRNA (22). FIG. 1 Schematic representations of the RTBV genome and P3 polyprotein. (A) Genome organization. Viral DNA is represented by a thin double line with the sites of the two discontinuities (Δ1 and Δ2) indicated. The thick arrows outside the DNA ... The roles of P1 (24 kDa) and P4 (46 kDa) are still unknown. P3 is a large polyprotein of 196 kDa (Fig. ​(Fig.1B).1B). Sequence comparisons with retroviral and other pararetroviral proteins suggest that P3 contains domains corresponding to the movement protein (MP), coat protein (CP), aspartic protease (PR), reverse transcriptase (RT), and RNase H (RH), ordered from the N terminus to the C terminus (17, 26, 39, 45). The viral protease is at least partly responsible for the processing of P3. The cleavage sites at the N- and C-terminal extremities of the RT-RH domain have been characterized. It has been demonstrated that the PR-RT-RH polyprotein can be processed to yield two proteins of 55 and 62 kDa (p55 and p62) when expressed in insect cells from the 3′ part of gene III (27). First reports indicated that RTBV particles contain two major CP species of 33 and 37 kDa (p33 and p37) (39). The N terminus of p33 was determined to be at amino acid 502. Considering its size and the position of its N-terminal residue within P3, p33 should contain, in its C-terminal region, the basic domain and the Cys-His motif which are conserved in plant pararetrovirus CPs. This motif is the equivalent of the zinc finger motif of retroviral Gag proteins and consequently is thought to be involved in specific RNA binding during packaging of the pregenomic RNA into virions (40). Recently, Marmey et al. (30) showed that RTBV virions contain only a single coat protein species of 37 kDa, with the second peptide (of 34 kDa) most probably being a degradation product of the 37-kDa protein generated during virus purification. Amino acids 477 and 791 of P3 were deduced, from mass spectral analysis, to correspond to the N- and C-terminal residues, respectively, of the 37-kDa coat protein (p37). ORF II encodes a 12-kDa protein (P2) for which no definite function has been assigned. P2 of RTBV and of the badnavirus commelina yellow mottle virus (CoYMV) were shown to be associated with purified virions (3, 22; A. Druka and R. Hull, personal communication). P2 of RTBV and of the badnavirus cacao swollen shoot virus (CSSV) were also described as sequence-nonspecific nucleic acid binding proteins (24, 25). The C termini of RTBV and CSSV P2, which possess basic, hydrophobic, and proline residues, support the nucleic acid binding activity. Such residues are also present at the C termini of caulimovirus gene III products and of bacterial histone-like proteins (34). Moreover, the C-terminal extremity of cauliflower mosaic virus (CaMV) P3 possesses a nonspecific nucleic acid binding activity (33, 34), suggesting a common role for this protein and the P2 of RTBV or badnaviruses in their respective life cycles. To investigate the role of RTBV P2, we searched for possible interactions between this protein and other RTBV proteins. P2 was shown to interact with the CP domain of P3 both in the yeast two-hybrid system and in vitro. We have characterized this interaction and identified peptide motifs involved in the binding on both proteins. To evaluate the importance of this interaction in the context of viral infection, we introduced point mutations within gene II of the RTBV genome and investigated the infectivity of these mutants by agroinoculation of rice plants. Our results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3.