Involvement of the nucleolus in plant virus systemic infection.

The nucleolus is a prominent subnuclear domain and is classically regarded as the site of transcription of rRNA, processing of the precursor rRNAs and biogenesis of pre-ribosomal particles. In addition to these traditionally recognized activities, the nucleolus also participates in many other aspects of cell function. The umbravirus-encoded ORF3 protein is a multifunctional RNA-binding protein involved in long-distance RNA movement, and protection of viral RNA from RNase attack, including possibly small interfering RNA-guided RNA silencing. In addition to its presence in cytoplasmic ribonucleoprotein particles containing viral RNA, the umbraviral ORF3 protein accumulates in nuclei, preferentially targeting nucleoli. The ORF3 protein domains involved in the localization of the protein to the nucleolus were identified. Functional analysis of the mutants revealed the correlation between the ORF3 protein nucleolar localization and its ability to form the cytoplasmic ribonucleoprotein particles and transport viral RNA long distances via the phloem. Possible mechanisms of the nucleolar involvement in systemic virus infection are discussed.

Umbravirus-encoded proteins both stabilize heterologous viral RNA and mediate its systemic movement in some plant species.

The proteins encoded by open reading frame 3 (ORF3) of the umbraviruses pea enation mosaic virus-2 and tobacco mottle virus, like that of groundnut rosette virus, mediated the movement of viral RNA through the phloem of infected Nicotiana benthamiana or N. clevelandii plants when they were expressed from chimeric tobacco mosaic virus in place of the coat protein. However, these chimeras did not move systemically in N. tabacum. In lysates of N. benthamiana or N. tabacum protoplasts, the chimeric RNAs were more stable than was RNA of tobacco mosaic virus lacking the coat protein gene. The chimeric viruses also protected the latter in trans, suggesting that the ORF3 proteins can increase the stability of heterologous viral RNA. Umbraviral ORF3 proteins contain a conserved arginine-rich domain, and the possible roles of this motif in the functions of the proteins are discussed.

Umbravirus gene expression helps potato leafroll virus to invade mesophyll tissues and to be transmitted mechanically between plants.

Potato leafroll virus (PLRV) was mechanically transmissible when inocula also contained the umbravirus Pea enation mosaic virus-2 (PEMV-2). In plants infected with PLRV and PEMV-2, PLRV accumulated in clusters of mesophyll cells in both inoculated and systemically infected leaves. No transmissions were obtained by coinoculation with Potato virus Y, Potato virus X (PVX), Tobacco mosaic virus, or Cucumber mosaic virus (CMV), although PLRV was transmissible from mixtures with CMV(ORF4) (a recombinant that contained the movement protein (MP) gene of the umbravirus Groundnut rosette virus (GRV) in place of the CMV MP gene). In contrast, neither a recombinant PVX that expressed GRV MP nor a mutant of CMV(ORF4), in which the CMV 2b gene was untranslatable, was able to help PLRV transmission. Possibly both a cell-to-cell movement function and counterdefense mechanisms such as those that block posttranscriptional gene silencing are involved in movement of PLRV within plants and its mechanical transmission between plants.

Tagging potato leafroll virus with the jellyfish green fluorescent protein gene.

A full-length cDNA corresponding to the RNA genome of Potato leafroll virus (PLRV) was modified by inserting cDNA that encoded the jellyfish green fluorescent protein (GFP) into the P5 gene near its 3' end. Nicotiana benthamiana protoplasts electroporated with plasmid DNA containing this cDNA behind the 35S RNA promoter of Cauliflower mosaic virus became infected with the recombinant virus (PLRV-GFP). Up to 5% of transfected protoplasts showed GFP-specific fluorescence. Progeny virus particles were morphologically indistinguishable from those of wild-type PLRV but, unlike PLRV particles, they bound to grids coated with antibodies to GFP. Aphids fed on extracts of these protoplasts transmitted PLRV-GFP to test plants, as shown by specific fluorescence in some vascular tissue and epidermal cells and subsequent systemic infection. In plants agroinfected with PLRV-GFP cDNA in pBIN19, some cells became fluorescent and systemic infections developed. However, after either type of inoculation, fluorescence was mostly restricted to single cells and the only PLRV genome detected in systemically infected tissues lacked some or all of the inserted GFP cDNA, apparently because of naturally occurring deletions. Thus, intact PLRV-GFP was unable to move from cell to cell. Nevertheless, PLRV-GFP has novel potential for exploring the initial stages of PLRV infection.

Host-specific cell-to-cell and long-distance movements of cucumber mosaic virus are facilitated by the movement protein of groundnut rosette virus.

The cucumovirus, cucumber mosaic virus (CMV), requires both the 3a movement protein (MP) and the capsid protein (CP) for cell-to-cell movement. Replacement of the MP of CMV with the MP of the umbravirus, groundnut rosette virus (GRV), which does not encode a CP, resulted in a hybrid virus, CMV(ORF4), which could move cell to cell in Nicotiana tabacum and long distance in N. benthamiana. After replacement of the CMV CP in CMV(ORF4) with the gene encoding the green fluorescent protein (GFP), the hybrid virus, CMV(ORF4.GFP), expressing both the GRV MP and the GFP, could move cell to cell but not systemically in either Nicotiana species. Immunoelectron microscopic analysis of cells infected by the hybrid viruses showed different cellular barriers in the vasculature preventing long-distance movement of CMV(ORF4) in N. tabacum and CMV(ORF4.GFP) in N. benthamiana. Thus the GRV MP, which shows limited sequence similarity to the CMV MP, was able to support CP-independent cell-to-cell movement of the hybrid virus, but CP was still required for long-distance movement and entry of particular vascular cells required functions encoded by different proteins.

A plant virus-encoded protein facilitates long-distance movement of heterologous viral RNA.

Transport of plant viruses from cell to cell typically involves one or more viral proteins that supply specific cell-to-cell movement functions. Long-distance transport of viruses through the vascular system is a less well understood process with requirements different from those of cell-to-cell movement. Usually viral coat protein (CP) is required for long-distance movement, but groundnut rosette umbravirus (GRV) does not code for a CP. However, this virus moves efficiently from cell to cell and long distance. We demonstrate here that the protein encoded by ORF3 of GRV can functionally replace the CP of tobacco mosaic virus (TMV) for long-distance movement. In spite of low levels of virus RNA accumulation in infected cells, chimeric TMV with a replacement of the CP gene by GRV ORF3 was able to move rapidly through the phloem. Moreover, this chimeric virus complemented long-distance movement of another CP-deficient TMV derivative expressing the gene encoding the green fluorescent protein. Thus, the GRV ORF3-encoded protein represents a class of trans-acting long-distance movement factors that can facilitate trafficking of an unrelated viral RNA.

Satellite RNA is essential for encapsidation of groundnut rosette umbravirus RNA by groundnut rosette assistor luteovirus coat protein.

Groundnut rosette disease is caused by a complex of agents comprising groundnut rosette umbravirus (GRV), GRV satellite RNA (sat-RNA)groundnut rosette assistor luteovirus (GRAV). Both GRAV and GRV sat-RNA are needed for GRV to be aphid transmissible. To understand the role of GRAVGRV sat-RNA in the aphid transmission of GRV, encapsidation of GRV genomicsatellite RNAs has been studied using transgenic Nicotiana benthamiana plants expressing GRAV coat protein (CP). GRAV CP expressed from a transgene was shown to package GRV genomicsatellite RNAs efficiently, giving a high yield of transcapsidated virus particles. GRV sat-RNA was absolutely essential for this process. GRV genomic RNA was not encapsidated by GRAV CP in the absence of the sat-RNA. Using different mutants of GRV sat-RNA, it was found that some property of full-length satellite RNA molecules, such as size or specific conformation rather than potential open reading frames, was required for the production of virus particles. A correlation between the ability of sat-RNA to stimulate encapsidation of GRV RNA by GRAV CPits capacity to promote aphid transmission of GRV was observed.

Intracellular location of two groundnut rosette umbravirus proteins delivered by PVX and TMV vectors.

The proteins encoded by open reading frames (ORF) 3 and 4 of groundnut rosette umbravirus (GRV) were expressed in Nicotiana benthamiana as fusions with green fluorescent protein (GFP) from modified potato virus X (PVX) and tobacco mosaic virus (TMV) vectors. Regardless of which plant virus vector was used, GFP fused to the ORF3 protein accumulated in large cytoplasmic inclusion bodies and in nucleoli, whereas GFP fused to the ORF4 protein was found in cell walls close to plasmodesmata. Cell-to-cell movement of PVX requires three proteins encoded by the triple gene block (TGB) and also the coat protein (CP). However, when GRV ORF4 was substituted for the PVX CP gene, the hybrid virus was able to move normally in inoculated leaves but not into noninoculated leaves. In contrast, when GRV ORF4 was substituted for the TGB, or for both the TGB and the CP gene, movement of the hybrid viruses was limited to a few epidermal cells neighboring the infection site. Thus, the GRV ORF4 protein can replace the movement proteins of PVX for some of their functions.

Polyclonal antibodies against human gamma-tubulin stain centrioles in mammalian cells from different tissues.

Rabbit polyclonal antibodies were raised against the C-terminal fragment (amino acid residues 318-451) of human gamma-tubulin. These antibodies were used to stain cultured cells of various tissues (epithelium, nervous tissue, fibroblasts) from different animals (human, monkey, pig, rat, kangaroo rat, mouse, hamster, chicken, triton). The antibodies specifically stained centrioles in the interphase and mitotic cells of mammals, but not birds (chicken) or amphibians (newt). In the interphase cells, centrioles were stained as a pair of dots (or as a double dot) in 96-97% of the cells. The distances between the maternal and filial centrioles varied in different cultures. Procentrioles were stained in certain cells, but with less intensity than mature centrioles. In mitotic cells, the antibodies revealed two spots corresponding to two mitotic poles. The spots in mitosis were significantly larger than the interphase dots, but the staining was more faint. In spontaneous tripolar mitoses, only two poles were stained. Thus, it was shown that, on the one hand, gamma-tubulin is associated with centrioles irrespective of whether or not they serve as the microtubule organizing centres and, on the other hand, gamma-tubulin might not be an essential component of the microtubule organizing centres.

Use of highly conserved motifs in plant virus RNA polymerases as the tags for specific detection of carmovirus-related RNA-dependent RNA polymerase genes.

Two highly degenerate primers for sequence-specific amplification and cloning of a 510-nucleotide-long segment of RNA-dependent RNA-polymerase (RdRp) genes were selected and synthesized on the basis of available plant carmovirus-like viral RdRp sequences. These primers were shown to be efficient in PCR screening of different RdRp genes including those of carmoviruses, dianthoviruses, and tombusviruses. In particular, they were used for amplification, cloning, and sequencing of an RdRp gene fragment of an isometric plant virus with unknown evolutionary relationships, pelargonium flower break virus (PFBV). Alignment of the respective nucleotide and amino acid sequences indicates a very close similarity between PFBV and carnation mottle virus, the type member of carmoviruses.

Nucleotide sequence of carnation ringspot dianthovirus RNA-1.

The nucleotide sequence of carnation ringspot virus (CRSV) RNA-1, the type member of the dianthovirus genus, has been determined. The 3756 nucleotide genomic RNA-1 contains three large open reading frames (ORFs), capable of encoding 27K, 54K and 38K polypeptides. In addition, a small ORF encoding a 10K polypeptide at the 3' terminus of the RNA has been identified. The gene organization of CRSV RNA-1 is similar to those of red clover necrotic mosaic (RCNMV) and sweet clover necrotic mosaic (SCNMV) dianthoviruses with the exception that CRSV RNA-1 contains the additional 3'-terminal ORF. The 27K and 54K proteins possess significant sequence similarity to corresponding polypeptides of the other dianthoviruses. The 54K protein also contains the conserved RNA-dependent RNA polymerase motif. The identification of a shifty heptanucleotide preceding the p27 ORF termination codon and a predicted secondary structure following the terminator suggest that a translational frameshifting event allows translation to continue past the p27 ORF into the p54 ORF, which is in the -1 frame, generating an 88K fusion protein. Amino acid sequence alignment of the 38K protein with the corresponding RCNMV and SCNMV polypeptides indicate that this is the viral capsid protein.

Nucleotide sequence of shallot virus X RNA reveals a 5'-proximal cistron closely related to those of potexviruses and a unique arrangement of the 3'-proximal cistrons.

The 8890 nucleotide RNA sequence of shallot virus X (ShVX), a new virus isolated from shallot, has been determined. The sequence contains six open reading frames (ORFs) which encode putative proteins (in the 5' to 3' direction) of M(r) 194528 (ORF1), 26333 (ORF2), 11245 (ORF3), 42209 (ORF4), 28486 (ORF5) and 14741 (ORF6). The ORF1 protein was found to be highly homologous to the putative potexvirus RNA replicases; ORF2, -3, -5 and -6 proteins also have analogues among the potex- and/or carlavirus-encoded proteins. ORF3 is followed by an AUG-lacking frame coding for an amino acid sequence homologous to that of the 7K to 8K proteins of the triple gene block of the above-mentioned viruses. The putative ORF4 protein has no reliable homology with proteins in the database. The results obtained testify that, except for the unique 42K protein gene, the ShVX genome combines a number of elements typical of both carla- and potexviruses.

Diverse groups of plant RNA and DNA viruses share related movement proteins that may possess chaperone-like activity.

Amino acid sequences of plant virus proteins mediating cell-to-cell movement were compared to each other and to protein sequences in databases. Two families of movement proteins have been identified, the members of which show statistically significant sequence similarity. The first, larger family (I) encompasses the movement proteins of tobamo-, tobra-, caulimo- and comoviruses, apple chlorotic leaf spot virus (ACLSV) and geminiviruses with bipartite genomes. Thus this family includes viruses which move by two methods, those requiring the coat protein for the cell-to-cell spread (comoviruses) and those not having this requirement (tobamoviruses). The previously unsuspected relationship between the movement proteins of RNA and DNA viruses having no RNA stage in their life cycle (geminiviruses) suggested that their movement mechanisms might be similar. The second, smaller family (II) consists of the movement proteins of tricornaviruses (bromoviruses, cucumoviruses, alfalfa mosaic virus and tobacco streak virus) and dianthoviruses. Alignment of the sequences of family I movement proteins highlighted two motifs, centred at conserved Gly and Asp residues, respectively, which are assumed to be crucial for the movement protein function(s). Screening the amino acid sequence database revealed another conserved motif that is shared by a large subset of family I movement proteins (those of caulimo- and comoviruses, and ACLSV) and the family of cellular 90K heat shock proteins (HSP90). Based on the analogy to HSP90, it is speculated that many plant virus movement proteins may mediate virus transport in a chaperone-like manner.