Structure of Cowpea mottle virus: a consensus in the genus Carmovirus.
(33/132)Cowpea mottle virus (CPMoV) is a T = 3 virus that belongs to Carmovirus genus of the Tombusviridae family. Here, we report the crystal structure of CPMoV determined to a resolution of 7.0 angstroms. The structures and sequences of three Carmoviruses, CPMoV, Turnip crinkle virus (TCV), and Carnation mottle virus (CarMV) have been compared to TBSV from the Tombusvirus genus. CPMoV, TCV, and CarMV all have a deletion in betaC strand in the S domain relative to TBSV that may be distinctive to the genus. Although CPMoV has an elongated C-terminus like TBSV, it does not interact with the icosahedrally related P domain as observed in TBSV. In CPMoV, the termini of A and B interact with the icosahedrally related shell domains of A and C, respectively, to form a chain of interactions around the 5-fold axes. The C subunit terminus does not, however, interact with the B subunit because of quasi-equivalent differences in the P domain orientations. (+info)
The Arabidopsis cucumovirus multiplication 1 and 2 loci encode translation initiation factors 4E and 4G.
(34/132)The cum1 and cum2 mutations of Arabidopsis thaliana inhibit cucumber mosaic virus (CMV) multiplication. In cum1 and cum2 protoplasts, CMV RNA and the coat protein accumulated to wild-type levels, but the accumulation of the 3a protein of CMV, which is necessary for cell-to-cell movement of the virus, was strongly reduced compared with that in wild-type protoplasts. In cum2 protoplasts, the accumulation of turnip crinkle virus (TCV)-related RNA and proteins was also reduced. Positional cloning demonstrated that CUM1 and CUM2 encode eukaryotic translation initiation factors 4E and 4G, respectively. Unlike most cellular mRNA, the CMV RNA lacks a poly(A) tail, whereas the TCV RNA lacks both a 5'-terminal cap and a poly(A) tail. In vivo translation analyses, using chimeric luciferase mRNA carrying the terminal structures and untranslated sequences of the CMV or TCV RNA, demonstrated that these viral untranslated sequences contain elements that regulate the expression of encoded proteins positively or negatively. The cum1 and cum2 mutations had different effects on the action of these elements, suggesting that the cum1 and cum2 mutations cause inefficient production of CMV 3a protein and that the cum2 mutation affects the production of TCV-encoded proteins. (+info)
Significance of the 3'-terminal region in minus-strand RNA synthesis of Hibiscus chlorotic ringspot virus.
(35/132)RNA-dependent RNA polymerase (RdRp) was solubilized from crude extracts of Hibiscus cannabinus infected by Hibiscus chlorotic ringspot virus (HCRSV), a member of the Carmoviridae. After treatment of the extracts with micrococcal nuclease to remove the endogenous templates, the full-length genomic RNA and the two subgenomic RNAs were efficiently synthesized by the partially purified RdRp complex in vitro. When the full-length RNAs of Potato virus X, Tobacco mosaic virus, Odontoglossum ringspot virus and Cucumber mosaic virus were used as templates, no detectable RNA was synthesized. Synthesis of HCRSV minus-strand RNA was shown to initiate opposite the 3'-terminal two C residues at the 3' end in vitro and in vivo. The CCC-3' terminal nucleotide sequence was optimal and nucleotide variations from CCC-3' diminished minus-strand synthesis. In addition, two putative stem-loops (SLs) located within the 3'-terminal 87 nt of HCRSV plus-strand RNA were also essential for minus-strand RNA synthesis. Deletion or disruption of the structure of these two SLs severely reduced or abolished RNA synthesis. HCRSV RNA in which the two SLs were replaced with the SLs of Turnip crinkle virus could replicate in kenaf protoplasts, indicating that functionally conserved structure, rather than nucleotide sequence, plays an important role in the minus-strand synthesis of HCRSV. Taken together, the specific sequence CCC at the 3' terminus and the two SLs structures located in the 3'UTR are essential for efficient minus-strand synthesis of HCRSV. (+info)
Molecular characterization of a Melon necrotic spot virus strain that overcomes the resistance in melon and nonhost plants.
(36/132)Resistance of melon (Cucumis melo L.) to Melon necrotic spot virus (MNSV) is inherited as a single recessive gene, denoted nsv. No MNSV isolates described to date (e.g., MNSV-Malpha5), except for the MNSV-264 strain described here, are able to overcome the resistance conferred by nsv. Analysis of protoplasts of susceptible (Nsv/-) and resistant (nsv/nsv) melon cultivars inoculated with MNSV-264 or MNSV-Malpha5 indicated that the resistance trait conferred by this gene is expressed at the single-cell level. The nucleotide sequence of the MNSV-264 genome has a high nucleotide identity with the sequences of other MNSV isolates, with the exception of its genomic 3'-untranslated region (3'-UTR), where less than 50% of the nucleotides are shared between MNSV-264 and the other two MNSV isolates completely sequenced to date. Uncapped RNAs transcribed from a full-length MNSV-264 cDNA clone were infectious and caused symptoms indistinguishable from those caused by the parental viral RNA. This cDNA clone allowed generation of chimeric mutants between MNSV-264 and MNSV-Malpha5 through the exchange of the last 74 nucleotides of their coat protein (CP) open reading frames and the complete 3'-UTRs. Analysis of protoplasts of susceptible and resistant melon cultivars inoculated with chimeric mutants clearly showed that the MNSV avirulence determinant resides in the exchanged region. The carboxy-termini of the CP of both isolates are identical; therefore, the avirulence determinant likely consists of the RNA sequence itself. We also demonstrated that this genomic region contains the determinant for the unique ability of the isolate MNSV-264 to infect noncucurbit hosts (Nicotiana benthamiana and Gomphrena globosa). (+info)
Repression and derepression of minus-strand synthesis in a plus-strand RNA virus replicon.
(37/132)Plus-strand viral RNAs contain sequences and structural elements that allow cognate RNA-dependent RNA polymerases (RdRp) to correctly initiate and transcribe asymmetric levels of plus and minus strands during RNA replication. cis-acting sequences involved in minus-strand synthesis, including promoters, enhancers, and, recently, transcriptional repressors (J. Pogany, M. R. Fabian, K. A. White, and P. D. Nagy, EMBO J. 22:5602-5611, 2003), have been identified for many viruses. A second example of a transcriptional repressor has been discovered in satC, a replicon associated with turnip crinkle virus. satC hairpin 5 (H5), located proximal to the core hairpin promoter, contains a large symmetrical internal loop (LSL) with sequence complementary to 3'-terminal bases. Deletion of satC 3'-terminal bases or alteration of the putative interacting bases enhanced transcription in vitro, while compensatory exchanges between the LSL and 3' end restored near-normal transcription. Solution structure analysis indicated that substantial alteration of the satC H5 region occurs when the three 3'-terminal cytidylates are deleted. These results indicate that H5 functions to suppress synthesis of minus strands by sequestering the 3' terminus from the RdRp. Alteration of a second sequence strongly repressed transcription in vitro and accumulation in vivo, suggesting that this sequence may function as a derepressor to free the 3' end from interaction with H5. Hairpins with similar sequence and/or structural features that contain sequence complementary to 3'-terminal bases, as well as sequences that could function as derepressors, are located in similar regions in other carmoviruses, suggesting a general mechanism for controlling minus-strand synthesis in the genus. (+info)
Biased hypermutagenesis associated with mutations in an untranslated hairpin of an RNA virus.
(38/132)The mutation frequency of Turnip crinkle virus can increase 12-fold without inducing error catastrophe. Lesions in a hairpin repressor frequently reverted and led to second-site alterations biased for specific mutations. These results suggest that the hairpin may also function as an RNA chaperone to properly fold the RNA-dependent RNA polymerase. (+info)
Analysis of a viral replication repressor: sequence requirements for a large symmetrical internal loop.
(39/132)Nearly all members of the Carmovirus genus contain a structurally conserved 3' proximal hairpin (H5) with a large internal symmetrical loop (LSL). H5 has been identified as a repressor of minus-strand synthesis in a satellite RNA (satC), which shares partial sequence similarity with its helper virus Turnip crinkle virus (TCV). Repression was due to sequestration of the 3' end mediated by base pairing between 3' end sequence and the 3' side of the LSL (G. Zhang, J. Zhang and A. E. Simon, J. Virol., in press). Single site mutational analysis and in vivo genetic selection (SELEX) of the 14 base satC H5 LSL indicated specific sequences in the middle and upper regions on both sides of the LSL are necessary for robust satC accumulation in plants and protoplasts. Fitness of wild-type satC and satC LSL mutants to accumulate in plants, however, did not necessarily correlate with the ability of these RNAs to replicate in protoplasts. This suggests that the LSL might be involved in processes in addition to repression of minus-strand synthesis. (+info)
RNA silencing-suppressor function of Turnip crinkle virus coat protein cannot be attributed to its interaction with the Arabidopsis protein TIP.
(40/132)The interaction of the coat protein (CP) of Turnip crinkle virus (TCV) with a host protein, TCV-interacting protein (TIP), from Arabidopsis thaliana has been reported previously. This interaction correlates with the ability of TCV CP to elicit the resistance response that is mediated by the resistance gene HRT in Arabidopsis ecotype Di-17. It has also been established that TCV CP is a suppressor of RNA silencing, a process by which the host plant targets viral RNA for degradation. These results have led to the speculation that TIP might be a component of the RNA-silencing pathway and that TCV CP suppresses RNA silencing through its interaction with TIP. In the current report, a number of TCV CP mutants have been investigated for their ability to suppress RNA silencing. These mutants include single amino acid substitution mutants that are known to have lost their ability to interact with TIP, as well as deletion mutants of TCV CP that are of different sizes and from different regions of the protein. Results showed that each of the single amino acid substitution mutants tested retained high levels of RNA silencing-suppressor activity. In addition, a mutant containing a 5 aa deletion in the region that is known to be critical for TIP interaction retained the ability to suppress RNA silencing significantly. Larger deletions in all regions of TCV CP abolished silencing-suppressor activity. It can be concluded from these results that the RNA silencing-suppressor activity of TCV CP cannot be attributed to its ability to interact directly with TIP. (+info)