The fd phage and a peptide derived from its p8 coat protein interact with the HIV-1 Tat-NLS and inhibit its biological functions.

Filamentous fd bacteriophages are used to construct phage-display peptide libraries, which have been instrumental in selecting peptides that interact with specific domains within target molecules. Here we demonstrate that the fd bacteriophage itself, as well as NTP8 - a synthetic peptide derived from it and bearing amino acids 1-20 of the phage p8 protein - interact with the nuclear localization signal (NLS) of the HIV-1 Tat protein. Accordingly, fd bacteriophage and the NTP8 peptide inhibit binding mediated by the Tat-NLS to the nuclear-import receptor importin beta and Tat-NLS-mediated translocation into cell nuclei. The NTP8 peptide, at 100 microM concentration, also caused about 50% inhibition of HIV-1 propagation in cultured cells. The fd bacteriophage prevents heparan sulfate proteoglycans-mediated uptake of extracellular Tat by target cells and consequently transactivation of a chloramphenicol acetyltransferase (CAT) reporter gene. A BSA-NTP8 conjugate inhibits Tat-NLS-mediated binding to heparin immobilized on a BIAcore surface. BLAST analysis of the NTP8 amino-acid sequence revealed similarity to sequences in several human proteins, including ADA2 and CD53.

Regulation of plasmodesmal transport by phosphorylation of tobacco mosaic virus cell-to-cell movement protein.

Cell-to-cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata, is mediated by a specialized viral movement protein (MP). In vivo studies using transgenic tobacco plants showed that MP is phosphorylated at its C-terminus at amino acid residues Ser258, Thr261 and Ser265. When MP phosphorylation was mimicked by negatively charged amino acid substitutions, MP lost its ability to gate plasmodesmata. This effect on MP-plasmodesmata interactions was specific because other activities of MP, such as RNA binding and interaction with pectin methylesterases, were not affected. Furthermore, TMV encoding the MP mutant mimicking phosphorylation was unable to spread from cell to cell in inoculated tobacco plants. The regulatory effect of MP phosphorylation on plasmodesmal permeability was host dependent, occurring in tobacco but not in a more promiscuous Nicotiana benthamiana host. Thus, phosphorylation may represent a regulatory mechanism for controlling the TMV MP-plasmodesmata interactions in a host-dependent fashion.

Cell-to-cell movement of tobacco mosaic virus: enigmas and explanations.

Abstract Tobacco mosaic virus (TMV) spreads between cells through plant intercellular connections, the plasmodesmata. This transport process is mediated by a specialized virus-encoded movement protein, TMV MP. Recent advances in two major aspects of TMV MP function highlight the limits of our current knowledge and promise exciting future developments. First, findings that TMV MP interacts with cytoskeletal elements and cell wall proteins suggest potential mechanisms for TMV MP targeting from the cell cytoplasm to plasmodesmal channels. Second, indications that TMV MP phosphorylation plays a regulatory role in several activities of TMV MP begin to unravel molecular pathways that control TMV cell-to-cell transport. TMV systemic movement that follows its initial cell-to-cell spread, on the other hand, may be controlled through two different pathways used for viral entry into and exit from the host plant vascular tissue.

Plasmodesmata: gateways for information transfer.

Intercellular communication in plants has evolved to occur via elongated cytoplasmic bridges, called plasmodesmata, that traverse the thick cell walls that surround plant cells. Historically, plasmodesmata have been assigned the mostly passive role of creating cytoplasmic continuity between plant cells enabling free transport of the wealth of small plant metabolites and growth hormones under 1 kDa. When it was discovered that plant viruses pirate plasmodesmata for movement of viral genomes during infection, it was proposed that viruses modified plasmodesmata for transport of very large molecules. Now, there is compelling evidence that plasmodesmata are inherently dynamic, rapidly altering their dimensions to increase their transport capabilities, upon contact with viral as well as developmentally important plant proteins. Further, the study of intercellular transport has prompted analyses of intracellular transport pathways, implicating the cytoskeleton as a major tracking system to plasmodesmata. Thus, plasmodesmata form a three-dimensional network of transportation channels and major checkpoints for information transfer. In the following, current knowledge about structure and function of these connective organelles, and about routing of molecules towards plasmodesmata, will be summarized.

Ultrastructural analysis of leaf trichome plasmodesmata reveals major differences from mesophyll plasmodesmata.

Functional studies on molecular transport through plasmodesmata in leaf mesophyll and trichome cells revealed significant differences in their basal size-exclusion limits and their response to microinjected tobacco mosaic virus movement protein (E. Waigmann et al., 1994, Proc. Natl. Acad. Sci. USA 91: 1433-1437; E. Waigmann and P. Zambryski, 1995, Plant Cell 7; 2069-2079). To address the basis for these functional differences, Nicotiana clevelandii trichome and mesophyll plasmodesmata were compared ultrastructurally. Trichome plasmodesmata increase in ultrastructural complexity from the tip to the base cell. Their neck regions, thought to control molecular traffic through plasmodesmata, are clearly distinct from necks of mesophyll plasmodesmata. In contrast to the electrondense desmotubular area in mesophyll plasmodesmata, trichome plasmodesmata contain an electron-translucent circle in their center, surrounded by an electrondense ring. This latter ring is connected to the inner leaflet of the plasma membrane by multiple spokes or filaments. Two monoclonal antibodies raised against a maize plasmodesmal protein preparation (A. Turner et al., 1994, J Cell Sci. 107: 3351-3361) interact with both trichome and mesophyll N. clevelandii plasmodesmata. Based on the localization pattern and the high degree of cross-reactivity, both antibodies likely recognize a conserved structural component of plasmodesmata, and may be useful to mark plasmodesma in a variety of plants and tissues.

Characterization of a novel arginine/serine-rich splicing factor in Arabidopsis.

Many splicing factors in vertebrate nuclei belong to a class of evolutionarily conserved proteins containing arginine/serine (RS) or serine/arginine (SR) domains. Previously, we demonstrated the existence of SR splicing factors in plants. In this article, we report on a novel member of this splicing factor family from Arabidopsis designated atRSp31. It has one N-terminal RNA recognition motif and a C-terminal RS domain highly enriched in arginines. The RNA recognition motif shows significant homology to all animal SR proteins identified to date, but the intermediate region does not show any homology to any other known protein. Subsequently, we characterized two cDNAs from Arabidopsis that are highly homologous to atRSp31 (designated atRSp35 and atRSp41). Their deduced amino acid sequences indicate that these proteins constitute a new family of RS domain splicing factors. Purified recombinant atRSp31 is able to restore splicing in SR protein-deficient human S100 extracts. This indicates that atRSp31 is a true plant splicing factor and plays a crucial role in splicing, similar to that of other RS splicing factors. All of the three genes are differentially expressed in a tissue-specific manner. The isolation of this new plant splicing factor family enlarges the essential group of RS domain splicing factors. Furthermore, because no animal equivalent to this protein family has been identified to date, our results suggest that these proteins play key roles in constitutive and alternative splicing in plants.

Tobacco mosaic virus movement protein-mediated protein transport between trichome cells.

Tobacco mosaic virus movement protein (TMV MP) is required to mediate viral spread between plant cells via plasmodesmata. Plasmodesmata are cytoplasmic bridges that connect individual plant cells and ordinarily limit molecular diffusion to small molecules and metabolites with a molecular mass up to 1 kD. Here, we characterize functional properties of Nicotiana clevelandii trichome plasmodesmata and analyze their interaction with TMV MP. Trichomes constitute a linear cellular system and provide a predictable pathway of movement. Their plasmodesmata are functionally distinct from plasmodesmata in other plant cel types; they allow cell-to-cell diffusion of dextrans with a molecular mass up to 7 kD, and TMV MP does not increase this size exclusion limit for dextrans. In contrast, the 30-kD TMV MP itself moves between trichome cells and specifically mediates the translocation of a 90-kD beta-glucuronidase (GUS) reporter protein as a GUS::TMV MP fusion. Neither GUS by itself nor GUS in the presence of TMV MP moves between cells. These data imply that a plasmodesmal transport signal resides within TMV MP and is essential for movement. This signal confers selectivity to the translocated protein and cannot function in trans to support movement of other molecules.

Plasmodesmata. Gateways for rapid information transfer.

Viruses spread their genomes throughout infected plants by exploiting plasmodesmata, the cytoplasmic bridges that wire plant cells into a three-dimensional network and rapidly transport a variety of molecules.

Direct functional assay for tobacco mosaic virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmal permeability.

Plasmodesmata are cytoplasmic bridges between plant cells thought to generally allow only the passage of small molecules and metabolites. However, large structures such as plant viruses also move from cell to cell via plasmodesmata. In tobacco mosaic virus (TMV) infection a viral movement protein (TMV-MP) mediates viral spread. Here, a microinjection assay is used to monitor the dynamics of TMV-MP function directly in wild-type plants. The results indicate that TMV-MP interacts with an endogenous plant pathway increasing plasmodesmal size exclusion limit to permit passage of 20-kDa dextrans. Furthermore, TMV-MP influences plasmodesmal size exclusion limit several cells distant from the injection site, indicating either that TMV-MP itself crosses plasmodesmata or that TMV-MP induces a diffusable signal capable of dilating microchannels of plasmodesmata. The region of TMV-MP responsible for increasing plasmodesmal size exclusion limit was mapped to the carboxyl-terminal part of the 268-amino acid residue protein between amino acid residues 126 and 224.

Cell-to-cell movement of plant viruses.

To establish an infection, most plant viruses move from cell to cell in the plant. Virus-encoded movement proteins mediate this process and appear to use two mechanisms for transport. Both mechanisms involve interaction with and potential modification of plant intercellular connections, the plasmodesmata. Thus, although viral movement proteins are a diverse group, they share an ability to interact with specific plant components.

Processing of chimeric introns in dicot plants: evidence for a close cooperation between 5' and 3' splice sites.

Splice sites of vertebrate introns are generally not recognized in plant cells. Several lines of evidences have led to the proposal that the mechanism of 3' splice site selection differs in plants and animals (K. Wiebauer, J.J. Herrero, and W. Filipowicz, Mol. Cell. Biol. 8:2042-2051, 1988). To gain a better insight into the mechanistic differences between plant and animal splicing, we constructed chimeric introns consisting partly of dicotyledonous plant and partly of animal intron sequences. Splicing of these chimeric introns was analyzed in transiently transfected tobacco protoplasts. The results show that there are no principal sequence or structural differences between the 3' splice regions of plants and animals. Furthermore, evidence is provided that cooperation between 5' and 3' splice sites takes place and influences their mutual selection.