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The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Both cell-to-cell and long-distance movement of White clover mosaic virus in which individual, combinations, or all movement functions were mutated could be rescued by transgenic Nicotiana benthamiana expressing complementary viral products. To address the importance of TGB functions in vascular transport, we used an experimental system based on grafted plants and trans-complementation, to define co-translocated viral products and the minimal requirements for viral exit from the plant vasculature. Evidence is presented that TGBp1 is co-translocated with viral RNA and CP and that, once viral RNA is loaded into the phloem translocation stream, it can exit in sink tissues and replicate in the absence of TGBp2-3. These results are discussed in the context of the recent finding that TGBp1 can mediate the suppression of signaling involved in systemic gene silencing., (Copyright 2001 Academic Press.)

The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Separate models have been proposed for intercellular movement of two of these viruses, transport of intact virions, or a ribonucleoprotein complex (RNP) comprising genomic RNA, TGBp1, and the CP. At issue therefore, is the form(s) in which RNA transport occurs and the roles of TGBp1-3 and the CP in movement. Evidence is presented that, based on microprojectile bombardment studies, TGBp1 and the CP, but not TGBp2 or TGBp3, are co-translocated between cells with viral RNA. In addition, cell-to-cell movement and encapsidation functions of the CP were shown to be separable, and the rate-limiting factor of potexvirus movement was shown not to be virion accumulation, but rather, the presence of TGBp1-3 and the CP in the infected cell. These findings are consistent with a common mode of transport for potexviruses, involving a non-virion RNP, and show that TGBp1 is the movement protein, whereas TGBp2 and TGBp3 are either involved in intracellular transport or interact with the cellular machinery/docking sites at the plasmodesmata.

The sequence of the 3'-terminal 2424 nucleotides of RNA-2 of the flowering cherry strain of strawberry latent ringspot virus (SLRV) was determined from cDNA clones. The sequence contains a reading frame in the virus-sense strand of 2070 nucleotides, a 3' untranslated region of 552 nucleotides and a 3'-terminal poly(A) tract. The positions of the two coat proteins of SLRV within the reading frame were determined from sequence data obtained by N-terminal sequencing using Edman degradation. The larger coat protein with an M(r) of 43K is located 5' of the smaller coat protein of 27K, and the two proteins are apparently cleaved at a Ser-Gly bond. Although there are numerous similarities between SLRV and the nepoviruses and comoviruses, there is no significant homology between the SLRV coat proteins and the coat proteins of either group. Furthermore, the hydropathy profiles of the SLRV coat proteins are unlike those of either group. No comparisons could be made with the fabaviruses owing to lack of sequencing information. This lack of homology suggests that SLRV is more distantly related to the nepoviruses and comoviruses than has been considered previously.

Functions of the coat protein of white clover mosaic potexvirus (WCIMV) were investigated using C-terminal deletion mutants. Whereas plants inoculated with RNA transcripts of a full-length wild-type clone of WCIMV produced typical infections, plants inoculated with transcripts of each mutant did not produce symptoms, and viral RNA species were not detected by Northern analysis. The mutants were able to replicate in protoplasts, although, relative to the wild-type RNA profile, the level of genomic RNA, but not subgenomic RNA, was reduced. These results indicate a role for the coat protein in efficient cell-to-cell transport in plants. Virus-like particles were detected in protoplast extracts inoculated with transcripts of a mutant in which the coat protein was truncated by 31 amino acids. This result suggests that the lack of detectable transport in plants was not due solely to a failure of the mutants to form virus particles. Possible roles for the coat protein in transport and replication are discussed. A 6-kDa open reading frame, internal to the coat protein gene, was shown by mutational analysis not to be essential for replication or transport.

The sequence of the 3'-terminal 1492 nucleotides of the genome of a New Zealand isolate of clover yellow vein potyvirus (CYVV) has been determined. This sequence encodes a large open reading frame of 1314 nucleotides, the start of which was not identified, but which encodes a putative 272 amino acid coat protein. Downstream of the coat protein coding region is a 177 nucleotide untranslated sequence terminated by a polyadenylate tract. Comparison of the deduced CYVV-NZ coat protein amino acid sequence with two other strains of CYVV showed 86-93% similarity, suggesting CYVV-NZ should be regarded as a separate CYVV strain. CYVV-NZ shares with other CYVV strains a direct repeat of 14-16 nucleotides that is capable of forming a stem-loop structure. Examination of 35 strains of 15 other potyviruses showed a similar stem-loop structure conserved in all cases. A possible role in replication is hypothesized for the structure.

The sequence of 1612 nucleotides of the 3'-terminal region of white clover mosaic virus (WCIMV) has been determined from cDNA clones. The viral sense RNA contains four open reading frames of Mr 20,684, Mr 7219, Mr 12,989, and at least Mr 17,000. The latter begins 5' to the sequence determined. The amino acid sequence of the open reading frame encoding the 20,684 polypeptide shows marked homology to the coat proteins of three other potexviruses. The putative coat protein gene was subcloned in a T7 transcription plasmid and RNAs produced by in vitro transcription were translated in the rabbit reticulocyte lysate system. The polypeptide products comigrated on SDS-polyacrylamide gels with one of those synthesized by the in vitro translation of viral RNA, and were immunoprecipitable with antiserum raised against WCIMV, confirming the location of the coat protein gene.