Deletion of the C-terminal 33 amino acids of cucumber mosaic virus movement protein enables a chimeric brome mosaic virus to move from cell to cell

Abstract

The movement protein (MP) gene of brome mosaic virus (BMV) was precisely replaced with that of cucumber mosaic virus (CMV). Infectivity tests of the chimeric BMV on Chenopodium quinoa, a permissive host for cell-to-cell movement of both BMV and CMV, showed that the chimeric BMV failed to move from cell to cell even though it replicated in protoplasts. A spontaneous mutant of the chimeric BMV that displayed cell-to-cell movement was subsequently obtained from a local lesion during one of the experiments. A cloned cDNA representing the genomic RNA encoding the MP of the chimeric BMV mutant was analyzed and found to contain a mutation in the CMV MP gene resulting in deletion of the C-terminal 33 amino acids of the MP. Directed mutagenesis of the CMV MP gene showed that the C-terminal deletion was responsible for the movement capability of the mutant. When the mutation was introduced into CMV, the CMV mutant moved from cell to cell in C. quinoa, though the movement was less efficient than that of the wild-type CMV. These results indicate that the CMV MP, except the C-terminal 33 amino acids, potentiates cell-to-cell movement of both BMV and CMV in C. quinoa. In addition, since C. quinoa is a common host for both BMV and CMV, these results suggest that the CMV MP has specificity for the viral genomes during cell-to-cell movement of the virus and that the C-terminal 33 amino acids of the CMV MP are involved in that specificity. MATERIALS AND METHODS Plants. C. quinoa and tobacco (Nicotiana tabacum L. cv. Xanthi nc) plants were grown in commercially available soil mixtures in a plant growth room at 24 Ϯ 1ЊC with illumination for 16 h per day. cDNA clones. Plasmids pBTF1, pBTF2, and pBTF3W contain the full-length cDNAs of RNAs 1, 2, and 3 of the wild-type BMV, respectively, from which infectious transcripts can be produced by using T7 RNA polymerase (32-34). In vitro transcripts from these plasmids and their progenies will be referred to as the BMV KU2 strain. Plasmids pCY1-T7 and pCY2-T7 (generous gifts from S. Kuwata and M. Suzuki, Japan Tobacco Incorporated) contain the full-length cDNAs of CMV strain Y RNAs 1 and 2, respectively, and infectious viral RNA transcripts can be transcribed in vitro by T7 RNA polymerase from these plasmids (44). A full-length RNA 3 cDNA clone of the wild-type CMV (an isolate of the Y strain) was constructed by joining partial cDNA fragments from pCY32C1 and pCY78 (generous gifts from M. Nakayama, Takeda Chemical Industries, Ltd.). The full-length RNA 3 cDNA fragment was inserted into pUCT7 (34) to

Compatibility of the movement protein and the coat protein of cucumoviruses is required for cell-to-cell movement

“…Construction of RNA 3 chimeras and their infectivity in N. clevelandii protoplasts Prior to the generation of chimeric RNA 3 clones, restriction endonuclease recognition sites were introduced into the same position in pT3/NdeI and pR3/NdeI by silent mutagenesis. For the MP, the locations of the exchange points were defined after considering the results of Nagano et al (1997). The CMV MP can assist cell-to-cell movement in the presence of Brome mosaic virus (BMV) CP if the C-terminal 33 aa of the MP are absent.…”

“…No specific domains necessary for movement functions (RNA binding, incorporation into the plasmodesmata, promoting cell-to-cell movement) have been identified in the exchanged 29 aa (Li et al, 2001). According to Nagano et al (1997), the 33 aa C-terminal part of the CMV MP determined whether the CP was required for movement. If this region was missing, movement occurred in a CPindependent manner.…”

“…The 3a protein was shown to function as the MP of cucumoviruses; it has sequence and functional similarity to the MP of TMV (Melcher, 1990;Kaplan et al, 1995), traffics itself and viral RNA from cell to cell (Ding et al, 1995), binds singlestranded RNA (Li & Palukaitis, 1996) and associates with the cell wall, both in infected plants and in transgenic plants expressing the CMV 3a gene (Vaquero et al, 1994(Vaquero et al, , 1996Cooper & Dodds, 1995). The RNA-binding domain of the CMV MP has been localized between amino acids (aa) 174 and 233 (Vaquero et al, 1997), and the C-terminal 33 aa of the MP were shown to be responsible for specificity to the viral genome during cell-to-cell movement (Nagano et al, 1997). The functional domains of the MP have been analysed in detail (Li et al, 2001), while the precise role of the CP in cell-to-cell movement is still not known.…”

Viral cell-to-cell movement requires formation of cortical punctate structures containing Red clover necrotic mosaic virus movement protein

“…Deletion, or alanine-scanning mutation, in the C-terminal region of several MPs showed that this region is not essential for cell-to-cell movement. For example, more than 33 amino acids in the C-terminus of Alfalfa mosaic virus, Brome mosaic virus and CMV MPs are nonessential for virus cell-to-cell movement (Nagano et al, 1997(Nagano et al, , 2001Sánchez-Navarro and Bol, 2001;Takeda et al, 2004). These viruses belong to the family Bromoviridae and they require both MP and cognate CP for efficient cell-to-cell movement.…”

Host-Specific Cell-to-Cell and Long-Distance Movements of Cucumber Mosaic Virus Are Facilitated by the Movement Protein of Groundnut Rosette Virus

“…Again, the microinjected MP, which has been subjected to denaturation and renaturation, may contain a subpopulation with the appropriate conformation needed to facilitate movement in the absence of the function expressed by the CP. This model would also explain why the CMV MP did not function in place of the BMV MP in a BMV-CMV MP hybrid, whereas a variant of this hybrid, in which a deletion and frameshift in the CMV 3a gene resulted in a MP with a deletion of the C-terminal 33 amino acids, did function for movement (Nagano et al, 1997). That is, the BMV CP may not be able to facilitate the effects required for the CMV MP to function, but a deletion variant of the MP may already contain a subpopulation of MP with the conformation needed for trafficking.…”