A conserved region in the Closterovirus 1a polyprotein drives extensive remodeling of endoplasmic reticulum membranes and induces motile globules in Nicotiana benthamiana cells.

In infected plant cells, closterovirus replicative polyproteins 1a and 1ab drive membrane remodeling and formation of multivesicular replication platforms. Polyprotein 1a contains a variable Central Region (CR) between the methyltransferase and helicase domains. In a previous study, we have found that transient expression of the Beet yellows virus CR-2 segment (aa 1305-1494) in Nicotiana benthamiana induces the formation of ~1µm mobile globules originating from the ER membranes. In the present study, sequence analysis has shown that a part of the CR named the "Zemlya region" (overlapping the CR-2), is conserved in all members of the Closterovirus genus and contains a predicted amphipathic helix (aa 1368-1385). By deletion analysis, the CR-2 region responsible for the induction of 1-µm globules has been mapped to aa 1368-1432. We suggest that the conserved membrane-modifying region of the BYV 1a may be involved in the biogenesis of closterovirus replication platforms.

Critical regions and residues for self-interaction of grapevine leafroll-associated virus 2 protein p24.

The 24-kDa protein (p24) encoded by grapevine leafroll-associated virus 2 (GLRaV-2) is an RNA-silencing suppressor. In this work, a yeast two-hybrid system (YTHS) and bimolecular fluorescence complementation analyses showed that GLRaV-2 p24 can interact with itself, and that this interaction occurs in the cytoplasm of Nicotiana benthamiana cells. To identify the functional region(s) and crucial amino acid residues required for p24 self-interaction, various truncated and substitution mutants were generated. YTHS assay showed that in both homologous pairing and pairing with the wild-type p24, the functional regions mapped to aa 10-180 or 1-170 which contain, respectively, all seven α-helices or the first six α-helices and the N-terminal end (aa 1-9) of the protein. When only the full-length p24 was an interaction partner, the functional region of aa 1-170 could be further mapped to aa 1-140 which contains four α-helices plus most of the fifth α-helix. Further analysis with substitution mutants demonstrated that hydrophobic residues I35/F38/V85/V89/W149 and V162/L169/L170, which may, respectively, mediate the inter-domain interaction of the same p24 monomer and the tail-to-tail association between two p24 counterparts, are crucial for homotypic p24-p24 interaction. In addition, substitution of two basic residues-R2 or R86-of p24, which may play important functional roles in RNA binding, did not seem to affect self-interaction of the mutants in yeast but had obvious effects in plant cells. Taken together, our results demonstrate the functional regions and crucial amino acids for p24 self-interaction.

Citrus tristeza virus transmission by the Toxoptera citricida vector: in vitro acquisition and transmission and infectivity immunoneutralization experiments.

Citrus tristeza virus (CTV) is transmitted by several aphid species in a semi-persistent manner with Toxoptera citricida, the brown citrus aphid (BrCA), being the most efficient. As yet, the molecular interactions between the virus and its aphid vectors have not been determined. This is the first report of aphids acquiring CTV from preparations through an artificial membrane and then transmitting it to receptor plants. The BrCA fed across artificial membranes on crude tissue preparations made from CTV-infected bark tissue were able to transmit CTV to virus-free receptor plants at low rates. CTV p20, p27 and p25 proteins, detected by Western blots, were present in all crude tissue preparations from CTV-infected plants. Partially purified CTV preparations were not transmitted by the BrCA in this manner. Infectivity immunoneutralization experiments were conducted where aphids were forced to feed in vitro on three CTV-specific antibodies (p25, p27 and p20) before being placed on receptor plants following a 48h acquisition feed on CTV-infected source plants. There were no differences in transmission rates among the majority of treatments and the control treatments. However, in one infectivity immunoneutralization experiment, the CTV p20 antibodies significantly enhanced CTV transmission compared to buffer only, pre-immune antiserum or no antibody control treatments. This suggests the inactivity of CTV p20 aids BrCA transmission of virions.

A novel class of large and infectious defective RNAs of Citrus tristeza virus.

Citrus tristeza virus (CTV)-infected plants contain one or more populations of defective RNAs (dRNAs), mostly with a size range of ca. 2.0 to 5.0 kb. Several CTV dRNAs have been characterized and found to consist mainly of the two termini of the genomic RNA, with extensive internal deletions. The present paper describes a new class of large ( approximately 12.0 kb) dRNAs from three different CTV isolates with two unusual features. First is their composition with intact replicase genes. These dRNAs contained a large 5' portion of the genomic RNA terminus, which apparently corresponded to the recently described 5' large single-stranded subgenomic RNA (sgRNA) of ORF1a+1b (Che et al., 2001). The 3' portion of the large dRNAs varied among the 10 different cDNA clones examined in this work. In 2 dRNAs this portion consisted of truncated ORF10 (p20), and in 5 dRNAs it contained truncated ORF11 (p23). Two dRNA molecules were found with a 3' portion that started in the exact 5' position of the intergenic region between the p20 and p23 ORFs. In one dRNA, this portion coincided with the full-length sgRNA corresponding to ORF10. The second unusual feature was their ability to be readily transmitted mechanically to citrus plants by stem slashing and also to Nicotiana benthamiana protoplasts. The possibility that these dRNAs may be encapsidated and be capable of self-replication is discussed.

Cell-to-cell movement of potato virus X involves distinct functions of the coat protein.

Complementation of movement-deficient potato virus X (PVX) coat protein (CP) mutants, namely PVX.CP-Xho lacking the 18 C-terminal amino acid residues and PVX.DeltaCP lacking the entire CP gene, was studied by transient co-expression with heterologous proteins. These data demonstrated that the potyvirus CPs and both the major and minor CPs of beet yellows closterovirus could complement cell-to-cell movement of PVX.CP-Xho but not PVX.DeltaCP. These data also indicated that the C-terminally truncated PVX CP lacked a movement function which could be provided in trans by the CPs of other filamentous viruses, whereas another movement determinant specified by some region outside the most C-terminal part of the PVX CP could not be complemented either by potyvirus or closterovirus CPs. Surprisingly, the CP of spherical cocksfoot mottle sobemovirus rescued all of the PVX CP movement functions, complementing the spread of PVX.CP-Xho and, to a lesser extent, PVX.DeltaCP. Both these mutants were also rescued by the tobacco mosaic virus (TMV) movement protein (MP). To shed light on the movement function of PVX CP, attempts were made to complement PVX.CP-Xho by a series of TMV MP mutants. An internal deletion abolished complementation, suggesting that the internal region of TMV MP, which includes a number of overlapping functional domains important for cell-to-cell transport, provides an activity complementing movement determinant(s) specified by the C-terminal region of PVX CP.

Closterovirus encoded HSP70 homolog and p61 in addition to both coat proteins function in efficient virion assembly.

Assembly of the viral genome into virions is a critical process of the virus life cycle often defining the ability of the virus to move within the plant and to be transmitted horizontally to other plants. Closteroviridae virions are polar helical rods assembled primarily by a major coat protein, but with a related minor coat protein at one end. The Closteroviridae is the only virus family that encodes a protein with similarity to cellular chaperones, a 70-kDa heat-shock protein homolog (HSP70h). We examined the involvement of gene products of Citrus tristeza virus (CTV) in virion formation and found that the chaperone-like protein plus the p61 and both coat proteins were required for efficient virion assembly. Competency of virion assembly of different CTV mutants was assayed by their ability to be serially passaged in Nicotiana benthamiana protoplasts using crude sap as inoculum, and complete and partial virus particles were analyzed by serologically specific electron microscopy. Deletion mutagenesis revealed that p33, p6, p18, p13, p20, and p23 genes were not needed for virion formation. However, deletion of either minor- or major-coat protein resulted in formation of short particles which failed to be serially transferred in protoplasts, suggesting that both coat proteins are required for efficient virion assembly. Deletion or mutation of HSP70h and/or p61 dramatically reduced passage and formation of full-length virions. Frameshift mutations suggested that the HSP70h and p61 proteins, not the RNA sequences, were needed for virion assembly. Substitution of the key amino acid residues in the ATPase domain of HSP70h, Asp(7) to Lys or Glu(180) to Arg, reduced assembly, suggesting that the chaperone-like ATPase activity is involved in assembly. Both HSP70h and p61 proteins appeared to contribute equally to assembly, consistent with coordinate functions of these proteins in closterovirus virion formation. The requirement of two accessory proteins in addition to both coat proteins for efficient assembly is uniquely complex for helical virions.

[Grapevine viruses in Tunisia]. / Les viroses de la vigne en Tunisie.

Tunisian grapevine culture is affected by many viruses caused by some phytovirus belonging to nepovirus, closterovirus and trichovirus groups. The present work deal with the economically important viroses identified in tunisian grapevines. We present here the development methods to detect these viruses in propagating material. The important viruses biologically, biochemically, serologically and using molecular techniques, characterised are: GFLV, GLRaV3 and GVB. The genetic polymorphism analysis was also carried and tunisian isolates were compared to previously described ones in literature.