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zmfzj至尊木虫 (著名写手)
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[交流]
续“Maize chlorotic mottle virus”
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Replication The replication strategy of MCMV has not been completely determined, but inoculation of maize protoplasts with transcripts from wild type and mutant versions of an infectious cDNA has provided some information. Transcripts with mutations in the 3′ third of the genome that stop expression of one or more of the proteins encoded on sgRNA1 are capable of replication. Additionally, mutations just upstream of the sgRNA1 transcription start site that stop expression of sgRNA1 but do not alter the sequence of p111 are capable of replication, indicating that none of the proteins encoded on sgRNA1 are necessary for replication. Based on the replication mechanisms of other tombusvirus family members it is likely that after virion disassembly, MCMV viral RNA is translated to produce the viral replicase which then synthesizes the negative strand of genomic RNA after recognizing sequences and structures located at the viral 3′terminus that have sequence and structural similarities to the promoters of carmoviruses. The complementary strand is then used as template for synthesis of progeny viral RNA strands. sgRNA synthesis mechanisms differ between genera in the family Tombusviridae, and it is not known which mechanism is used by MCMV. sgRNA1 synthesis may initiate by replicase binding internally to the sgRNA promoter on the genomic complementary strand. Alternatively, occasional premature termination of viral complementary strand synthesis at a specific location may produce separate complementary strand copies of sgRNA1 that are used as templates to synthesize many copies of sgRNA1. Although sgRNA2 accumulates in infected maize plants and inoculated protoplasts, its function and method of transcription are not known. Serologic Relationships and Variability The complete relationship of MCMV isolates from countries where MCMV has been found on a noticeable scale has not been determined. Two serotypes from Kansas (K1 and K2) and one from Peru have been compared, and they can be differentiated by agar double-diffusion assays. Most isolates from Nebraska and Kansas are similar to MCMV-K1, which is the source used for the MCMV sequence in GenBank (X14736) and the infectious transcript cDNA. Sequence comparisons have been done using reverse transcription-polymerase chain reaction (RT-PCR) amplification of the CP shell domain ORF from 47 isolates collected from Nebraska/Kansas, Hawaii, and Peru. Alignment of the 200 bp fragments indicates that each geographic region can be identified by a predominant unique genotype. The Hawaiian isolates show the least intrapopulational divergence, consistent with the recent appearance of the virus in Hawaii before the sample collection period. The Hawaiian isolates are more closely related to Peru isolates than to those from Nebraska/Kansas. The complete sequence of the CP gene of MCMV isolated in Thailand (AY587605) shows 96% identity to MCMV-K1 at the nucleotide level and 97% identity at the protein level. When the corresponding 200 nt region is compared to the data in the larger study, the Thai sequence groupswithNebraska/Kansas sequences. These data suggest that Peru was the source for MCMV in Hawaii while the source of virus in Thailand was Nebraska/Kansas. Genetics Viral RNA or uncapped transcripts of a full-length cDNA are sufficient for replication in protoplasts and infection of plants. The first ORF encodes a highly acidic 32 kDa protein with no similarity to any other protein in databases. The location of ORF1 suggests that it is a protein needed early in the viral life cycle, but its function is not known. ORF2 and ORF3 comprise a 50 kDa protein and a readthrough protein of 111 kDa containing the ‘GDD’ motif found in almost all RNA-dependent RNA polymerases (Figure 2). Sequence similarities of the carboxyl end of p50 and the p111 read through region to viral replicase proteins of other members of the family Tombusviridae suggestthatthesearesimilarly involved in replication of MCMV. p50 is much larger than the pre-readthrough proteins encoded by most of the monopartite viruses in the same family (27–33 kDa) and the sequence similarity only extends to the carboxyl third of p50. The similarity between p50 and the 48 kDa protein from PMV encompasses about 60% of the carboxyl end. Along with other members of the family, no regions identifiable as a helicase domain or a methyltransferase domain are foundinp111.LikePMVandoatchloroticstuntvirus(genus Avenavirus), MCMV produces a single sgRNA to express a cluster ofORFs in the 3′ third of the genome, and none of the proteins encoded in this region are required for replication. The genes for p7a and p7b are upstream of the CP ORF, similar to the location of small ORFs in PMV, necroviruses, and carmoviruses. p7a and p7b have similar hydrophobic/hydrophilic characteristics and some sequence similarity to the peptides encoded in the corresponding locations in PMV, necroviruses, and carmoviruses, with the greatest sequence similarity occurring in the C-terminal region of p7a. These small peptides are required for cell-to-cell movement for PMV, necroviruses, and carmoviruses, so it is likely that p7a and p7b have similar functions. Database searches with the protein encoded in ORF5 do not identify any proteins with related sequence. The most 3′ORF encodes the viral CP. |
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zmfzj
至尊木虫 (著名写手)
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