Complete nucleotide sequence of Radish mosaic virus RNA polymerase gene and phylogenetic relationships in the genus Comovirus

The 3'-terminal part of RNA1 genome segment of Radish mosaic virus (RaMV) including complete RNA polymerase gene was sequenced. The 207 amino acids long polymerase is matured from a polyprotein precursor by cleavage at putative Q/H site by viral protease. The alignment of available amino acid sequences of RNA polymerase genes of comoviruses revealed a closest (55%) identity of RaMV to Red clover mottle virus (RCMV).     

Development of a full-length infectious clone of sunflower chlorotic mottle virus (SuCMoV)

A full-length cDNA clone (p35SuCMoV) of the sunflower chlorotic mottle virus common strain (SuCMoV-C) genomic RNA was constructed. Three cDNA fragments covering the whole genome of SuCMoV-C were cloned between a cauliflower mosaic virus 35S promoter and a nopaline synthase terminator. Mechanical inoculation of sunflower and Nicotiana occidentalis seedlings with p35SuCMoV DNA led to systemic infection. Symptoms induced by p35SuCMoV were similar to those caused by the wild-type SuCMoV-C but appeared four days later. Infection was confirmed by a western blot test, electron microscopy, RT-PCR and inoculation of progeny virions to sunflower seedlings. This is the first report about the construction of a biologically active, full-length cDNA copy of the SuCMoV-C RNA genome.     

Complete nucleotide sequence and construction of an infectious clone of Chinese yam necrotic mosaic virus suggest that macluraviruses have the smallest genome among members of the family Potyviridae

The complete nucleotide sequence of Chinese yam necrotic mosaic virus (CYNMV) was determined from cloned virus cDNA. The CYNMV genomic RNA is 8224 nucleotides in length, excluding the poly(A) tail, and contains one long open reading frame encoding a large polyprotein of 2620 amino acids. CYNMV has no counterpart to the P1 cistron and a short HC-Pro cistron located at the 5?? side of the potyvirus genome. A full-length cDNA clone, pCYNMV, was assembled under the control of the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator. Biolistic inoculation of Nagaimo plants with cDNA resulted in systemic necrotic mosaic symptoms typical of CYNMV infection. To our knowledge, this is the first report of the complete nucleotide sequence and construction of an infectious cDNA clone of a member of the genus Macluravirus.     

Construction of an infectious full-length cDNA clone of potato virus M

An infectious full-length cDNA clone of potato virus M (PVM) was produced. Total RNA was extracted from PVM-infected Nicotiana hesperis plants and used for cDNA synthesis. Subsequent RT-PCR produced two DNA fragments of about 5.5 and 3.2 kbp, which were ligated downstream of an enhanced 35S cauliflower mosaic virus promoter. After cloning of the enhanced 35S promoter with the PVM sequence into a modified pBIN19 plasmid and electroporation of Agrobacterium tumefaciens, the agroinoculated PVM full-length clone (pPVM-flc) led to systemic PVM infections in different host plants, causing symptoms indistinguishable from those caused by wild-type PVM.     

Genome sequence of a Japanese isolate of Radish mosaic virus: the first complete nucleotide sequence of a crucifer-infecting comovirus

Summary The complete nucleotide sequences of RNA1 and RNA2 of a Japanese isolate of Radish mosaic virus (RaMV-J), a crucifer-infecting comovirus, were determined. RNA1 is 6064 nucleotides long and encodes a 210-kDa polyprotein containing conserved motifs that are required for replication. RNA2 is 4020 nucleotides long and encodes a 123-kDa polyprotein containing the putative movement protein and two coat proteins. Comparisons of the encoded proteins confirmed that RaMV-J and a Czech RaMV isolate are isolates of the same species in the genus Comovirus. A phylogenetic analysis of RaMV-J and other comoviruses revealed that legume-infecting comoviruses constitute a single branch to which RaMV is distantly related.     

Mutational analysis of the putative catalytic triad of the cowpea mosaic virus 24K protease

To investigate the mechanism of action of the cowpea mosaic virus (CPMV) 24K protease, a full-length cDNA clone of bottom component (B) RNA has been constructed from which RNA can be transcribed in vitro using T7 RNA polymerase. Translation of the resulting RNA in rabbit reticulocyte lysate leads to the synthesis of a 200 kDa product (the 200K protein) which cleaves itself in a manner identical to that of the product translated from B RNA isolated from virions. Site-directed mutagenesis of the full-length clone was used to examine the effects of altering individual amino acids in the 24K protease on its activity. The results obtained are consistent with the prediction that the 24K protease is structurally similar to the trypsin-like family of serine proteases and suggest that His40, Glu76, and Cys166 comprise the active site. Substitution of Cys166 by a serine residue results in an enzyme with reduced catalytic activity.     

Biological and molecular characterization of Soybean yellow common mosaic virus, a new species in the genus Sobemovirus

A novel soybean-infecting sobemovirus termed Soybean yellow common mosaic virus (SYCMV) was characterized. The virus has a single, positive-strand RNA genome of 4152 nucleotides. The virus contains four putative open reading frames encoding P1 (78-566 nt), polyprotein ORF2a (524-2248 nt), polymerase domain ORF2b (1852-3417 nt), and CP (3227-4030 nt). The entire nucleotide sequence of SYCMV showed 31.2-71.3% nucleotide identity with the previously known eleven species of sobemovirus. In host range analysis of SYCMV, in which twenty one species and three different Nicotiana tabacum cultivars belonging to seven families were inoculated with the virus, SYCMV had a narrow host range, infecting only Glycine max and G. soja. Based on the obtained sequence, full-length clones of SYCMV were constructed. Symptoms produced by inoculation with clones were indistinguishable from those produced by inoculation with sap from symptomatic plants. Viral RNA accumulation of SYCMV was detected in the upper leaves by Northern blotting. This indicated that full-length clones of SYCMV were sufficient to produce disease symptoms. Genomic organization, the predicted amino acid sequence, and phylogenetic analyses with known sobemoviruses confirmed the assignment of SYCMV as a new member of the genus Sobemovirus.