A novel procedure for the localization of viral RNAs in protoplasts and whole plants

Analysis of virus spread using co-expressed reporter proteins has provided important details on cell-to-cell and long-distance movement of viruses in plants. However, most viruses cannot tolerate insertion of large non-viral segments or loss of any open-reading frames, procedures required to detect viruses non-evasively. A technique used to localize mRNAs intracellularly in yeast has been modified for detection of viral RNAs in whole plants. The technique makes use of the binding of the coat protein of MS2 bacteriophage (CPMS2) to a 19 base hairpin (hp). A fusion protein, consisting of the CPMS2, green fluorescent protein (GFP), and a nuclear localization signal (NLS), was nuclear-localized upon transient expression in protoplasts. However, addition of the hp to the 3' untranslated region of Turnip crinkle virus (TCV-hp) and co-transfection of the virus and fusion protein construct into protoplasts resulted in the re-location of GFP to the cytoplasm. Neither the insertion of the hp nor the interaction with the fusion protein impaired any viral functions. Transgenic plants expressing the GFP-NLS-CPMS2 fusion protein were generated, and GFP was detected in nuclei of young plant cells. Foci of GFP cytoplasmic fluorescence were detected in TCV-hp-inoculated leaves at 2 days post-inoculation. Later, GFP was detected in young leaves near the midvein and in the base (support) cells of trichomes in the vicinity of secondary and tertiary veins. In older leaves, cytoplasmic GFP could be visualized throughout many of the leaves. This technique should be amenable for detection of any virus with a transformable plant (or animal) host and may also prove useful for localizing properly engineered host RNAs.     

A Begomovirus DNAbeta-encoded protein binds DNA, functions as a suppressor of RNA silencing, and targets the cell nucleus

Our previous results demonstrated that the DNAbeta satellite (Y10beta) associated with Tomato yellow leaf curl China virus Y10 isolate (TYLCCNV-Y10) is essential for induction of leaf curl symptoms in plants and that transgenic expression of its betaC1 gene in Nicotiana plants induces virus-like symptoms. In the present study, in vitro DNA binding activity of the betaC1 proteins of Y10beta and DNAbeta (Y35beta) found in the Tobacco curly shoot virus Y35 isolate (TbCSV-Y35) were studied following their expression as six-His fusion proteins in Escherichia coli. Electrophoretic mobility shift assays and UV cross-linking experiments revealed that betaC1 proteins could bind both single-stranded and double-stranded DNA without size or sequence specificity. Suppression of green fluorescent protein (GFP) transgene silencing was observed with the new leaves of GFP-expressing Nicotiana benthamiana plants coinoculated by TYLCCNV-Y10 plus Y10beta or by TbCSV-Y35 plus Y35beta. In a patch agroinfiltration assay, the transiently expressed betaC1 gene of Y10beta or Y35beta was able to suppress host RNA silencing activities and permitted the accumulation of high levels of GFP mRNA in the infiltrated leaf patches of GFP transgenic N. benthamiana plants. The betaC1 protein of Y10beta accumulated primarily in the nuclei of plant and insect cells when fused with beta-glucuronidase or GFP and immunogold labeling showed that the betaC1 protein is present in the nuclei of infected N. benthamiana plants. A mutant version of Y10beta carrying the mutations within the putative nuclear localization sequence of the Y10 betaC1 protein failed to induce disease symptoms, suppress RNA silencing, or accumulate in the nucleus, suggesting that nuclear localization of the betaC1 protein is a key requirement for symptom induction and silencing suppression.     

Matrix‐glycoprotein interactions required for budding of a plant nucleorhabdovirus and induction of inner nuclear membrane invagination

Nucleorhabdoviruses such as Sonchus yellow net virus (SYNV) replicate in the nuclei and undergo morphogenesis at the inner nuclear membrane (IM) in plant cells. Mature particles are presumed to form by budding of the Matrix (M) protein‐nucleocapsid complexes through host IMs to acquire host phospholipids and the surface glycoproteins (G). To address mechanisms underlying nucleorhabdovirus budding, we generated recombinant SYNV G mutants containing a truncated amino‐terminal (NT) or carboxyl‐terminal (CT) domain. Electron microscopy and sucrose gradient centrifugation analyses showed that the CT domain is essential for virion morphogenesis whereas the NT domain is also required for efficient budding. SYNV infection induces IM invaginations that are thought to provide membrane sites for virus budding. We found that in the context of viral infections, interactions of the M protein with the CT domain of the membrane‐anchored G protein mediate M protein translocation and IM invagination. Interestingly, tethering the M protein to endomembranes, either by co‐expression with a transmembrane G protein CT domain or by artificial fusion with the G protein membrane targeting sequence, induces IM invagination in uninfected cells. Further evidence to support functions of G‐M interactions in virus budding came from dominant negative effects on SYNV‐induced IM invagination and viral infections that were elicited by expression of a soluble version of the G protein CT domain. Based on these data, we propose that cooperative G‐M interactions promote efficient SYNV budding.     

Subcellular localization and targeting of glucocorticoid receptor protein fusions expressed in transgenic Arabidopsis thaliana

An animal system of inducible activation of protein fusions with the binding domain of glucocorticoid receptor (BDGR) was tested in Arabidopsis thaliana by monitoring dexamethasone (DEX)-induced nuclear targeting of reporter constructs. Two constructs containing green fluorescent protein (GFP), human homeobox protein Hanf-1 and Xenopus laevis BDGR were used, GFP/Hanf-1/BDGR and GFP/BDGR. The control construct contained GFP alone. In the absence of DEX both fusion proteins were uniformly distributed in the cytoplasm of root cells, but showed strong association with plastids in plant aerial parts. DEX treatment of roots prompted a strong and reversible nuclear accumulation of GFP/Hanf-1/BDGR, but not GFP/BDGR. Thus, in roots, the specific nuclear translocation of GFP/Hanf-1/BDGR was driven by Hanf-1 and tightly regulated by BDGR. However, in plant aerial parts treated with DEX, nuclear translocation of GFP/Hanf-1/BDGR was observed only in a few cases, and most part of the fusion protein was incorrectly and irreversibly targeted to plastids. Protease X digestion of isolated chloroplasts showed that BDGR fusion proteins were translocated into the chloroplast envelope and bound to envelope membranes, probably due to association with the chloroplast import apparatus. Thus, for efficient use of the glucocorticoid-inducible system in plants, it will be necessary to modify BDGR structure to prevent incorrect targeting of fusion proteins.     

Nucleocytoplasmic partitioning of the plant photoreceptors phytochrome A,B, C,D, and E is regulated differentially by light and exhibits a diurnal rhythm

The phytochrome family of plant photoreceptors has a central role in the adaptation of plant development to changes in ambient light conditions. The individual phytochrome species regulate different or partly overlapping physiological responses. We generated transgenic Arabidopsis plants expressing phytochrome A to E:green fluorescent protein (GFP) fusion proteins to assess the biological role of intracellular compartmentation of these photoreceptors in light-regulated signaling. We show that all phytochrome:GFP fusion proteins were imported into the nuclei. Translocation of these photoreceptors into the nuclei was regulated differentially by light. Light-induced accumulation of phytochrome species in the nuclei resulted in the formation of speckles. The appearance of these nuclear structures exhibited distinctly different kinetics, wavelengths, and fluence dependence and was regulated by a diurnal rhythm. Furthermore, we demonstrate that the import of mutant phytochrome B:GFP and phytochrome A:GFP fusion proteins, shown to be defective in signaling in vivo, is regulated by light but is not accompanied by the formation of speckles. These results suggest that (1) the differential regulation of the translocation of phytochrome A to E into nuclei plays a role in the specification of functions, and (2) the appearance of speckles is a functional feature of phytochrome-regulated signaling.     

大麦黄矮病毒运动蛋白是RNA沉默抑制因子

大麦黄矮病毒(barley yellow dwarf virus,BYDV)属黄症病毒科家族,其基因组包含6个开放阅读框(open reading frames,ORFs).将BYDV的6个基因分别克隆到pWEIMING101载体上,得到重组基因.电击转化农杆菌后,利用农杆菌瞬时表达方法渗透注射转GFP基因的本氏烟草16c植株的叶片,在长波长紫外灯下观察GFP的表达,并通过Northern blot证明所得现象.研究结果表明,BYDV的PAV株系ORF4编码的运动蛋白(movement protein,MP)是RNA沉默抑制因子,其表达可以抑制局部和系统RNA沉默.BYDV-MP与GFP的双链RNA(dsGFP)共表达后仍能抑制RNA沉默,荧光强度与叶片中GFP的mRNA和其沉默降解形成的siRNA的量有对应关系,其N端核定位序列对抑制局部基因沉默起主要作用,第5、6位氨基酸是抑制基因沉默的关键氨基酸.BYDV-MP单独渗透注射的部位均产生细胞死亡.     

Developing reverse genetics systems of northern cereal mosaic virus to reveal superinfection exclusion of two cytorhabdoviruses in barley plants

Recently, reverse genetics systems of plant negative‐stranded RNA (NSR) viruses have been developed to study virus–host interactions. Nonetheless, genetic rescue of plant NSR viruses in both insect vectors and monocot plants is very limited. Northern cereal mosaic virus (NCMV), a plant cytorhabdovirus, causes severe diseases in cereal plants through transmission by the small brown planthopper (SBPH, Laodelphax striatellus) in a propagative manner. In this study, we first developed a minireplicon system of NCMV in Nicotiana benthamiana plants, and then recovered a recombinant NCMV virus (rNCMV‐RFP), with a red fluorescent protein (RFP) insertion, in SBPHs and barley plants. We further used rNCMV‐RFP and green fluorescent protein (GFP)‐tagged barley yellow striate mosaic virus (rBYSMV‐GFP), a closely related cytorhabdovirus, to study superinfection exclusion, a widely observed phenomenon in dicot plants rarely studied in monocot plants. Interestingly, cellular superinfection exclusion of rBYSMV‐GFP and rNCMV‐RFP was observed in barley leaves. Our results demonstrate that two insect‐transmitted cytorhabdoviruses are enemies rather than friends at the cellular level during coinfections in plants.     

Sonchus Yellow Net Rhabdovirus Nuclear Viroplasms Contain Polymerase-Associated Proteins

We have initiated a study of the cytopathology of nucleorhabdoviruses by analyzing the subcellular localization of sonchus yellow net virus (SYNV) genomic and antigenomic RNAs and the encoded polymerase proteins. In situ hybridizations demonstrated that the minus-strand genomic RNA sequences are restricted to the nuclei of infected cells, while the complementary plus-strand antigenomic RNA sequences are present in both the nuclei and the cytoplasm. Immunofluorescence and immunogold labeling experiments also revealed that the nucleocapsid (N) protein and phosphoprotein (M2) are primarily localized to discrete regions within the nuclei and in virus particles that accumulate in perinuclear spaces. The N protein antiserum specifically labeled the nuclear viroplasms, whereas the M2 antiserum was more generally distributed throughout the nuclei. Antibody detection also indicated that the polymerase (L) protein is present in small amounts in the viroplasm. When the N and M2 proteins were expressed individually from the heterologous potato virus X (PVX) vector, both proteins preferentially accumulated in the nuclei. In addition, viroplasm-like inclusions formed in the nuclei of cells infected with the PVX vector containing the N gene. Fusions of the carboxy terminus of β-glucuronidase to N and M2 resulted in staining of the nuclei of infected cells following expression from the PVX vector. Deletion analyses suggested that multiple regions of the N protein contain signals that are important for nuclear localization.     

Escape of a plant virus from amplicon-mediated RNA silencing is associated with biotic or abiotic stress

Strong RNA silencing was induced in plants transformed with an amplicon consisting of full-length cDNA of potato leafroll virus (PLRV) expressing green fluorescent protein (GFP), as shown by low levels of PLRV-GFP accumulation, lack of symptoms and accumulation of amplicon-specific short interfering RNAs (siRNAs). Inoculation of these plants with various viruses known to encode silencing suppressor proteins induced a striking synergistic effect leading to the enhanced accumulation of PLRV-GFP, suggesting that it had escaped from silencing. However, PLRV-GFP escape also occurred following inoculation with viruses that do not encode known silencing suppressors and treatment of silenced plants with biotic or abiotic stress agents. We propose that viruses can evade host RNA-silencing defences by a previously unrecognized mechanism that may be associated with a host response to some types of abiotic stress such as heat shock.     

HCF-dependent nuclear import of VP16.

Transactivation by VP16 requires the formation of a multicomponent complex, the TAATGAAAT recognition factor complex (TRF.C), that contains in addition to VP16, two cellular proteins, Oct-1 and HCF. HCF binds directly to VP16 and this promotes subsequent interaction of the VP16-HCF complex with the POU DNA-binding domain of Oct-1 and selective assembly onto target sites. Here we demonstrate a novel role of HCF in the intracellular compartmentalization of VP16. We show that while VP16 does not contain a consensus nuclear localization signal (NLS) and is largely cytoplasmic, co-expression with HCF resulted in VP16 nuclear accumulation. A candidate NLS within the C-terminus of HCF was identified and insertion of this motif into green fluorescent protein (GFP) promoted nuclear accumulation. Conversely, removal of this signal from HCF (HCFDeltaNLS) resulted in its cytoplasmic accumulation. Co-expression of HCFDeltaNLS with wild-type (wt) VP16, or of wt HCF with VP16 mutants lacking HCF-binding activity failed to promote the nuclear enrichment of VP16. These results indicate that in addition to its role in stabilizing TRF.C, HCF acts as a nuclear import factor for VP16.     

Gene C2 of the monopartite geminivirus tomato yellow leaf curl virus-China encodes a pathogenicity determinant that is localized in the nucleus

Expression of the Tomato yellow leaf curl virus-China (TYLCV-C) C2 protein and green fluorescent protein (GFP) fused to the C2 protein (C2-GFP) in Nicotiana benthamiana from a Potato virus X (PVX) vector induced necrotic ringspots on inoculated leaves as well as necrotic vein banding and severe necrosis on systemically infected leaves. The localization of GFP fluorescence in plant cells infected with PVX/C2-GFP and in insect cells transfected with Baculovirus expressing C2-GFP indicates that the TYLCV-C C2 protein is capable of shuttling GFP into plant and insect cell nuclei. Our data demonstrate that the TYLCV-C C2 protein may contribute to viral pathogenicity in planta and is nuclear localized.     

A critical domain of Sweet potato chlorotic fleck virus nucleotide‐binding protein (NaBp) for RNA silencing suppression,nuclear localization and viral pathogenesis

RNA silencing is an important mechanism of antiviral defence in plants. To counteract this resistance mechanism, many viruses have evolved RNA silencing suppressors. In this study, we analysed five proteins encoded by Sweet potato chlorotic fleck virus (SPCFV) for their abilities to suppress RNA silencing using a green fluorescent protein (GFP)‐based transient expression assay in Nicotiana benthamiana line 16c plants. Our results showed that a putative nucleotide‐binding protein (NaBp), but not other proteins encoded by the virus, could efficiently suppress local and systemic RNA silencing induced by either sense or double‐stranded RNA (dsRNA) molecules. Deletion mutation analysis of NaBp demonstrated that the basic motif (an arginine‐rich region) was critical for its RNA silencing suppression activity. Using confocal laser scanning microscopy imaging of transfected protoplasts expressing NaBp fused to GFP, we showed that NaBp accumulated predominantly in the nucleus. Mutational analysis of NaBp demonstrated that the basic motif represented part of the nuclear localization signal. In addition, we demonstrated that the basic motif in NaBp was a pathogenicity determinant in the Potato virus X (PVX) heterogeneous system. Overall, our results demonstrate that the basic motif of SPCFV NaBp plays a critical role in RNA silencing suppression, nuclear localization and viral pathogenesis.     

The UL14 tegument protein of herpes simplex virus type 1 is required for efficient nuclear transport of the alpha transinducing factor VP16 and viral capsids

The protein encoded by the UL14 gene of herpes simplex virus type 1 (HSV-1) and HSV-2 is expressed late in infection and is a minor component of the virion tegument. An UL14-deficient HSV-1 mutant (UL14D) forms small plaques and exhibits an extended growth cycle at low multiplicities of infection (MOI) compared to wild-type virus. Although UL14 is likely to be involved in the process of viral maturation and egress, its precise role in viral replication is still enigmatic. In this study, we found that immediate-early viral mRNA expression was decreased in UL14D-infected cells. Transient coexpression of UL14 and VP16 in the absence of infection stimulated the nuclear accumulation of both proteins. We intended to visualize the fate of VP16 released from the infected virion and constructed UL14-null (14D-VP16G) and rescued (14R-VP16G) viruses that expressed a VP16-green fluorescent protein (GFP) fusion protein. Synchronous high-multiplicity infection of the viruses was performed at 4°C in the absence of de novo protein synthesis. We found that the presence of UL14 in the virion had an enhancing effect on the nuclear accumulation of VP16-GFP. The lack of UL14 did not significantly alter virus internalization but affected incoming capsid transport to the nuclear pore. These observations suggested that UL14 (i) enhanced VP16 nuclear localization at the immediately early phase, thus indirectly regulating the expression of immediate-early genes, and (ii) was associated with efficient nuclear targeting of capsids. The tegument protein UL14 could be part of the machinery that regulates HSV-1 replication.