Structure of saguaro cactus virus 3' translational enhancer mimics 5' cap for eIF4E binding.

The genomes of several plant viruses contain RNA structures at their 3' ends called cap-independent translation enhancers (CITEs) that bind the host protein factors such as mRNA 5' cap-binding protein eIF4E for promoting cap-independent genome translation. However, the structural basis of such 5' cap-binding protein recognition by the uncapped RNA remains largely unknown. Here, we have determined the crystal structure of a 3' CITE, panicum mosaic virus-like translation enhancer (PTE) from the saguaro cactus virus (SCV), using a Fab crystallization chaperone. The PTE RNA folds into a three-way junction architecture with a pseudoknot between the purine-rich R domain and pyrimidine-rich Y domain, which organizes the overall structure to protrude out a specific guanine nucleotide, G18, from the R domain that comprises a major interaction site for the eIF4E binding. The superimposable crystal structures of the wild-type, G18A, G18C, and G18U mutants suggest that the PTE scaffold is preorganized with the flipped-out G18 ready to dock into the eIF4E 5' cap-binding pocket. The binding studies with wheat and human eIF4Es using gel electrophoresis and isothermal titration calorimetry, and molecular docking computation for the PTE-eIF4E complex demonstrated that the PTE structure essentially mimics the mRNA 5' cap for eIF4E binding. Such 5' cap mimicry by the uncapped and structured viral RNA highlights how viruses can exploit RNA structures to mimic the host protein-binding partners and bypass the canonical mechanisms for their genome translation, providing opportunities for a better understanding of virus-host interactions and non-canonical translation mechanisms found in many pathogenic RNA viruses.

New World Cactaceae Plants Harbor Diverse Geminiviruses.

The family Cactaceae comprises a diverse group of typically succulent plants that are native to the American continent but have been introduced to nearly all other continents, predominantly for ornamental purposes. Despite their economic, cultural, and ecological importance, very little research has been conducted on the viral community that infects them. We previously identified a highly divergent geminivirus that is the first known to infect cacti. Recent research efforts in non-cultivated and asymptomatic plants have shown that the diversity of this viral family has been under-sampled. As a consequence, little is known about the effects and interactions of geminiviruses in many plants, such as cacti. With the objective to expand knowledge on the diversity of geminiviruses infecting cacti, we used previously acquired high-throughput sequencing results to search for viral sequences using BLASTx against a viral RefSeq protein database. We identified two additional sequences with similarity to geminiviruses, for which we designed abutting primers and recovered full-length genomes. From 42 cacti and five scale insects, we derived 42 complete genome sequences of a novel geminivirus species that we have tentatively named Opuntia virus 2 (OpV2) and 32 genomes of an Opuntia-infecting becurtovirus (which is a new strain of the spinach curly top Arizona virus species). Interspecies recombination analysis of the OpV2 group revealed several recombinant regions, in some cases spanning half of the genome. Phylogenetic analysis demonstrated that OpV2 is a novel geminivirus more closely related to viruses of the genus Curtovirus, which was further supported by the detection of three recombination events between curtoviruses and OpV2. Both OpV2 and Opuntia becurtoviruses were identified in mixed infections, which also included the previously characterized Opuntia virus 1. Viral quantification of the co-infected cactus plants compared with single infections did not show any clear trend in viral dynamics that might be associated with the mixed infections. Using experimental Rhizobium-mediated inoculations, we found that the initial accumulation of OpV2 is facilitated by co-infection with OpV1. This study shows that the diversity of geminiviruses that infect cacti is under-sampled and that cacti harbor diverse geminiviruses. The detection of the Opuntia becurtoviruses suggests spill-over events between viruses of cultivated species and native vegetation. The threat this poses to cacti needs to be further investigated.

First identification and molecular characterization of a novel cavemovirus infecting Epiphyllum spp.

A new virus with sequence similarities to members of the genus Cavemovirus in the family Caulimoviridae was identified in an Epiphyllum hybrid. The complete genome of the virus, tentatively named "epiphyllum virus 4" (EpV-4), was determined to be 7,296 nucleotides long. Its circular genome organization is typical of cavemoviruses, containing four open reading frames. This virus and the two known cavemoviruses share 67-69% and 72-75% overall nucleotide sequence identity in the replicase gene. Phylogenetic analysis placed EpV-4 in a same cluster with the two recognized cavemoviruses. Thus, EpV-4 should be considered a representative of a third species of the genus Cavemovirus. The virus was transmitted by grafting.

A Novel Divergent Geminivirus Identified in Asymptomatic New World Cactaceae Plants.

Cactaceae comprise a diverse and iconic group of flowering plants which are almost exclusively indigenous to the New World. The wide variety of growth forms found amongst the cacti have led to the trafficking of many species throughout the world as ornamentals. Despite the evolution and physiological properties of these plants having been extensively studied, little research has focused on cactus-associated viral communities. While only single-stranded RNA viruses had ever been reported in cacti, here we report the discovery of cactus-infecting single-stranded DNA viruses. These viruses all apparently belong to a single divergent species of the family Geminiviridae and have been tentatively named Opuntia virus 1 (OpV1). A total of 79 apparently complete OpV1 genomes were recovered from 31 different cactus plants (belonging to 20 different cactus species from both the Cactoideae and Opuntioideae clades) and from nine cactus-feeding cochineal insects (Dactylopius sp.) sampled in the USA and Mexico. These 79 OpV1 genomes all share > 78.4% nucleotide identity with one another and < 64.9% identity with previously characterized geminiviruses. Collectively, the OpV1 genomes display evidence of frequent recombination, with some genomes displaying up to five recombinant regions. In one case, recombinant regions span ~40% of the genome. We demonstrate that an infectious clone of an OpV1 genome can replicate in Nicotiana benthamiana and Opuntia microdasys. In addition to expanding the inventory of viruses that are known to infect cacti, the OpV1 group is so distantly related to other known geminiviruses that it likely represents a new geminivirus genus. It remains to be determined whether, like its cactus hosts, its geographical distribution spans the globe.

Complete genome sequence of a new member of the genus Badnavirus from red pitaya (Hylocereus polyrhizus).

Here, we report a circular double-stranded DNA virus from red pitaya (Hylocereus polyrhizus). The complete genome sequence is 7,837 nt in length and shares 98.7% nucleotide sequence identity with epiphyllum mottle-associated virus (EpMoaV) and 40.4-54.6% with other members of the genus Badnavirus. It has four open reading frames (ORFs), encoding putative proteins of 19.9, 14.8, 225.7 and 14.2 kDa, respectively. The reverse transcriptase (RT)-ribonuclease H (RNase H) region exhibits less than 70.5% nucleotide sequence identity to RT-RNase H of other badnaviruses, and 99.7% to that of EpMoaV. Phylogenetic analysis revealed that the virus from this study and EpMoaV form a single group. Consequently, we propose this virus as a new member of the genus Badnavirus in the family Caulimoviridae and have named it "pitaya badnavirus 1" (PiBV1). PiBV1 and EpMoaV should be considered two isolates of a badnavirus that infects members of the family Cactaceae.

Molecular characterization and detection of two carlaviruses infecting cactus.

Two large contigs with sequence similarities to different carlaviruses were identified by high-throughput sequencing in samples from a cactus plant. The complete genomes of the two viruses, tentatively named "cactus carlavirus 1" (CCV-1) and "cactus carlavirus 2" (CCV-2), were determined to be 8,441 and 8,396 nucleotides long, respectively, excluding the poly(A) tail. These viruses have the typical genomic organization of members of the genus Carlavirus. CCV-1 appears to be a cactus isolate of the carlavirus HSO-2016a, with 90.1% nucleotide sequence identity between the two virus genomes, whereas CCV-2 may be classified as a member of a new species. The sequences of CCV-2 and other carlaviruses are 48.9-60.0% identical at the whole-genome level.

The complete genome sequence of a member of a new species of tobamovirus (rattail cactus necrosis-associated virus) isolated from Aporcactus flagelliformis.

In this study, we identified a new tobamovirus from diseased Aporcactus flagelliformis cactus plants, named it rattail cactus necrosis-associated virus (RCNaV), and determined its complete genome sequence. The full RCNaV genome consisted of 6,506 nucleotides and contained four open reading frames coding for proteins of M(r) 128 kDa (3,441 nt), 185 kDa (4,929 nt), 55 kDa (1452 nt), 36 kDa (1,005 nt) and 19 kDa (513 nt) from the 5' to 3' end, respectively. The overall similarities for the four ORFs of RCNaV were from 32.5% to 64.1% and from 17.0% to 67.3% to those of the other tobamoviruses, at the nucleotide and amino acid level, respectively. Comparison of the coding and non-coding regions of the virus with those of other tobamoviruses showed that RCNaV is the most closely related to cactus mild mottle virus.

Long-distance kissing loop interactions between a 3' proximal Y-shaped structure and apical loops of 5' hairpins enhance translation of Saguaro cactus virus.

Circularization of cellular mRNAs is a key event prior to translation initiation. We report that efficient translation of Saguaro cactus virus (SCV) requires a 3' translational enhancer (PTE) located partially in coding sequences. Unlike a similar PTE reported in the 3' UTR of Pea enation mosaic virus that does not engage in an RNA:RNA interaction (Wang Z. et al., J. Biol. Chem. 284, 14189-14202, 2009), the SCV PTE participates in long distance RNA:RNA interactions with hairpins located in the p26 ORF and in the 5' UTR of one subgenomic RNA. At least two additional RNA:RNA interactions are also present, one of which involves the p26 initiation codon. Similar PTE can be found in six additional carmoviruses that can putatively form long-distance interactions with 5' hairpins located in comparable positions.

Complete sequence and genome structure of cactus mild mottle virus.

We have completed the genomic sequence of a tobamovirus, cactus mild mottle virus (CMMoV), and compared it to those of other known tobamoviruses. The complete genome sequence of CMMoV consists of 6,449 nucleotides. The genome RNA of the virus contains four open reading frames, encoding, from the 5' to the 3' end, the 120-kDa viral replicase, the 186-kDa viral polymerase, the 33-kDa movement protein and the 18-kDa coat protein. Overall amino acid similarities for the four viral proteins of CMMoV ranged from 16.3 to 44.4% compared to those of 20 other tobamoviruses. Phylogenetic analysis of the viral replicases and MP revealed that CMMoV is closely related to cucurbit-infecting tobamoviruses, while the CMMoV CP is more closely related to brassica- and solanaceous-infecting tobamoviruses.

Cactus mild mottle virus is a new cactus-infecting tobamovirus.

A new cactus-infecting tobamovirus, Cactus mild mottle virus (CMMoV), was isolated from diseased grafted cactus, Gymnocalycium mihanovichii and its molecular properties were characterized. CMMoV is distantly related to known species of the genus Tobamovirus on the basis of serological and sequence analyses. Western blot analysis showed that CMMoV is serologically unrelated to Sammon's Opuntia virus, which is the only known species of the genus Tobamovirus found in cactus plants. The 3'-terminal 2,910 nucleotides of CMMoV have been sequenced. The coat protein (CP) and movement protein (MP) genes encode 161 and 306 amino acids residues, respectively, and the 3' untranslated region (UTR) consists of 229 nucleotides long. The nucleotide and amino acid sequences of the CP of CMMoV were 39.6% to 49.2% and 25.8% to 40.3% identical to other seventeen tobamoviruses, respectively. The MP shared 34.9% to 40.6% and 16.3% to 27.0% and 44.6% to 63.4% identities, respectively, at the amino acid and nucleotide levels with other members of the genus. Percentage identities of nucleotides of the 3' UTR ranged from 42.5% to 63.4%. Phylogenetic tree analyses of the CP and MP suggest the existence of the fifth cactus-infecting subgroup in the genus Tobamovirus. Sequence analyses of these two viral proteins revealed that the highest amino acid sequence identity between the virus and seventeen other tobamoviruses was 40.6%, supporting the view that CMMoV is a new definite species of the genus Tobamovirus.

Molecular characterisation of potexviruses isolated from three different genera in the family Cactaceae.

The genome properties of three potexviruses which previously had been isolated from different genera in the family Cactaceae and had been found to be only distantly related serologically have been studied. The sequence of the 3040 3' terminal nucleotides of the genomic RNA of isolate K11 from Schlumbergera bridgesii and the complete RNA sequences of isolates B1 and CC10 from Zygocactus sp. and Opuntia sp., respectively, were determined. Starting sequences were obtained by means of immunocapture reverse transcription PCR using primers derived from highly conserved sequences in other potexviral RNAs. The known parts of the sequences were extended by means of random-primed cDNAs and specific primers derived from the known parts of the sequences. The genome structure of the three viruses resembles that of other potexviruses. The conserved motifs typical for replication-associated proteins, triple gene block (TGB) proteins and coat proteins of potexviruses were readily identified in the translation products of the five open reading frames. The 3' untranslated regions of the three RNAs are folded into secondary structures containing three characteristic hairpins. Rather low percentages of amino acid sequence identities ranging from 62% to 76% for the coat proteins and 41% to 49% for TGB proteins 3 suggest that these viruses should be regarded as distinct virus species for which the names Zygocactus virus X, Schlumbergera virus X and Opuntia virus X are proposed. It is also suggested that the name Cactus virus X which originally was coined for all three virus isolates should no longer be used.

Complete nucleotide sequence and genome organization of a Cactus virus X strain from Hylocereus undatus (Cactaceae).

The complete nucleotide sequence of a strain of Cactus virus X (CVX-Hu) isolated from Hylocereus undatus (Cactaceae) has been determined. Excluding the poly(A) tail, the sequence is 6614 nucleotides in length and contains seven open reading frames (ORFs). The genome organization of CVX is similar to that of other potexviruses. ORF1 encodes the putative viral replicase with conserved methyltransferase, helicase, and polymerase motifs. Within ORF1, two other ORFs were located separately in the +2 reading frame, we call these ORF6 and ORF7. ORF2, 3, and 4, which form the "triple gene block" characteristic of the potexviruses, encode proteins with molecular mass of 25, 12, and 7 KDa, respectively. ORF5 encodes the coat protein with an estimated molecular mass of 24 KDa. Sequence analysis indicated that proteins encoded by ORF1-5 display certain degree of homology to the corresponding proteins of other potexviruses. Putative product of ORF6, however, shows no significant similarity to those of other potexviruses. Phylogenetic analyses based on the replicase (the methyltransferase, helicase, and polymerase domains) and coat protein demonstrated a closer relationship of CVX with Bamboo mosaic virus, Cassava common mosaic virus, Foxtail mosaic virus, Papaya mosaic virus, and Plantago asiatica mosaic virus.