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Results for “viral complexity”

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PubMed1985

In vitro phosphorylation of NS protein by the L protein of vesicular stomatitis virus.

The structural proteins L and NS of vesicular stomatitis virus were obtained from purified viral ribonucleoprotein complex followed by phosphocellulose column chromatography and assayed for protein kinase activity using [gamma-32P]ATP as the phosphate donor. The fractions containing purified L protein phosphorylated NS protein in vitro. 8-Azido-ATP, a photoreactive analogue of ATP, was also used as the phosphate donor for phosphorylation of NS protein by the L protein. In the presence of ultraviolet light, only L protein was specifically cross-linked with 8-azido-[gamma-32P]ATP. In the absence of u.v. light 8-azido ATP did no inhibit RNA transcription in a reconstituted reaction or substitute ATP for RNA synthesis in vitro. The above results, taken together, suggest that 8-azido-ATP was bound to the kinase site and phosphorylation of NS protein was mediated by the L protein. Exogenous phosphate acceptor proteins such as phosvitin and casein were also phosphorylated by the L protein fraction. However, addition of an excess of phosvitin failed to compete with the phosphorylation of NS by L, indicating that the protein kinase activity possessed higher affinity for NS. The phosphorylation of NS was strongly inhibited by photoreaction of L protein with 8-azido-ATP with concomitant inhibition of transcription in vitro. These results suggest that phosphorylation of NS protein by L may have a role in the regulation of the virus genome transcription in vitro.

Adenosine Triphosphate↗

PubMed1997

Inhibition of early and late events of the HIV-1 replication cycle by cytoplasmic Fab intrabodies against the matrix protein, p17.

BACKGROUND: The HIV-1 matrix (MA) protein, p17, contains two subcellular localization signals that facilitate both nuclear import of the viral preintegration complex early during infection and virus particle assembly late in infection. The dual role of MA in both the afferent and efferent arms of the HIV-1 life cycle makes it an important target for intracellular immunization-based gene therapy strategies. MATERIALS AND METHODS: Here we report, using a new bicistronic vector, that an intracellular Fab antibody, or Fab intrabody, directed against a carboxy-terminal epitope of MA from the Clade B HIV-1 genotype, can inhibit HIV-1 infection when expressed in the cytoplasm of actively dividing CD4+ T cells. RESULTS: Marked inhibition of proviral gene expression occurred when single-round HIV-1 CAT virus was used for infections. In challenge experiments using both laboratory strains and syncytium-inducing primary isolates of HIV-1, a substantial reduction in the infectivity of virions released from the cells was also observed. CONCLUSIONS: This novel strategy of simultaneously blocking early and late events of the HIV-1 life cycle may prove useful in clinical gene therapy approaches for the treatment of HIV-1 infection and AIDS, particularly when combined with genetic or pharmacologic-based strategies that inhibit other HIV-1 target molecules simultaneously.

Animals↗

PubMed2002

Phosphorylation of VP30 impairs ebola virus transcription.

Transcription of the highly pathogenic Ebola virus (EBOV) is dependent on VP30, a constituent of the viral nucleocapsid complex. Here we present evidence that phosphorylation of VP30, which takes place at six N-terminal serine residues and one threonine residue, is of functional significance. Replacement of the phosphoserines by alanines resulted in an only slightly phosphorylated VP30 (VP30(6A)) that is still able to activate EBOV-specific transcription in a plasmid-based minigenome system. VP30(6A), however, did not bind to inclusions that are induced by the major nucleocapsid protein NP. Three intracellular phosphatases (PP1, PP2A, and PP2C) have been determined to dephosphorylate VP30. The presence of okadaic acid (OA), an inhibitor of PP1 and PP2A, had the same negative effect on transcription activation by VP30 as the substitution of the six phosphoserines for aspartate residues. OA, however, did not impair transcription when VP30 was replaced by VP30(6A). In EBOV-infected cells, OA blocked virus growth dose-dependently. The block was mediated by the extensive phosphorylation of VP30, which is evidenced by the result that expression of VP30(6A), in trans, led to the progression of EBOV infection in the presence of OA. In conclusion, phosphorylation of VP30 was shown to regulate negatively transcription activation and positively binding to the NP inclusions.

Animals↗

PubMed2006

Characterization of membrane association domains within the Tomato ringspot nepovirus X2 protein, an endoplasmic reticulum-targeted polytopic membrane protein.

Replication of nepoviruses (family Comoviridae) occurs in association with endoplasmic reticulum (ER)-derived membranes. We have previously shown that the putative nucleoside triphosphate-binding protein (NTB) of Tomato ringspot nepovirus is an integral membrane protein with two ER-targeting sequences and have suggested that it anchors the viral replication complex (VRC) to the membranes. A second highly hydrophobic protein domain (X2) is located immediately upstream of the NTB domain in the RNA1-encoded polyprotein. X2 shares conserved sequence motifs with the comovirus 32-kDa protein, an ER-targeted protein implicated in VRC assembly. In this study, we examined the ability of X2 to associate with intracellular membranes. The X2 protein was fused to the green fluorescent protein and expressed in Nicotiana benthamiana by agroinfiltration. Confocal microscopy and membrane flotation experiments suggested that X2 is targeted to ER membranes. Mutagenesis studies revealed that X2 contains multiple ER-targeting domains, including two C-terminal transmembrane helices and a less-well-defined domain further upstream. To investigate the topology of the protein in the membrane, in vitro glycosylation assays were conducted using X2 derivatives that contained N-glycosylation sites introduced at the N or C termini of the protein. The results led us to propose a topological model for X2 in which the protein traverses the membrane three times, with the N terminus oriented in the lumen and the C terminus exposed to the cytoplasmic face. Taken together, our results indicate that X2 is an ER-targeted polytopic membrane protein and raises the possibility that it acts as a second membrane anchor for the VRC.

Amino Acid Sequence↗

PubMed2000

Identification of nucleocapsid binding sites within coronavirus-defective genomes.

The coronavirus nucleocapsid (N) protein is a major structural component of virions that associates with the genomic RNA to form a helical nucleocapsid. N appears to be a multifunctional protein since data also suggest that the protein may be involved in viral RNA replication and translation. All of these functions presumably involve interactions between N and viral RNAs. As a step toward understanding how N interacts with viral RNAs, we mapped high-efficiency N-binding sites within BCV- and MHV-defective genomes. Both in vivo and in vitro assays were used to study binding of BCV and MHV N proteins to viral and nonviral RNAs. N-viral RNA complexes were detected in bovine coronavirus (BCV)-infected cells and in cells transiently expressing the N protein. Filter binding was used to map N-binding sites within Drep, a BCV-defective genome that is replicated and packaged in the presence of helper virus. One high-efficiency N-binding site was identified between nucleotides 1441 and 1875 at the 3' end of the N ORF within Drep. For comparative purposes N-binding sites were also mapped for the mouse hepatitis coronavirus (MHV)-defective interfering (DI) RNA MIDI-C. Binding efficiencies similar to those for Drep were measured for RNA transcripts of a region encompassing the MHV packaging signal (nts 3949-4524), as well as a region at the 3' end of the MHV N ORF (nts 4837-5197) within MIDI-C. Binding to the full-length MIDI-C transcript (approximately 5500 nts) and to an approximately 1-kb transcript from the gene 1a region (nts 935-1986) of MIDI-C that excluded the packaging signal were both significantly higher than that measured for the smaller transcripts. This is the first identification of N-binding sequences for BCV. It is also the first report to demonstrate that N interacts in vitro with sequences other than the packaging signal and leader within the MHV genome. The data clearly demonstrate that N binds coronavirus RNAs more efficiently than nonviral RNAs. The results have implications with regard to the multifunctional role of N.

Animals↗

PubMed1998

Effects of mutations in the rubella virus E1 glycoprotein on E1-E2 interaction and membrane fusion activity.

Rubella virus (RV) virions contain two glycosylated membrane proteins, E1 and E2, that exist as a heterodimer and form the viral spike complexes on the virion surface. Formation of an E1-E2 heterodimer is required for transport of E1 out of the endoplasmic reticulum lumen to the Golgi apparatus and plasma membrane. To investigate the nature of the E1-E2 interaction, we have introduced mutations in the internal hydrophobic region (residues 81 to 109) of E1. Substitution of serine at Cys82 (mutant C82S) or deletion of this hydrophobic domain (mutant dt) of E1 resulted in a disruption of the E1 conformation that ultimately affected E1-E2 heterodimer formation and cell surface expression of both E1 and E2. Substitution of either aspartic acid at Gly93 (G93D) or glycine at Pro104 (P104G) was found to impair neither E1-E2 heterodimer formation nor the transport of E1 and E2 to the cell surface. Fusion of RV-infected cells is induced by a brief treatment at a pH below 6. 0. To test whether this internal hydrophobic domain is involved in the membrane fusion activity of RV, transformed BHK cell lines expressing either wild-type or mutant spike proteins were exposed to an acidic pH and polykaryon formation was measured. No fusion activity was observed in the C82S, dt, and G93D mutants; however, the wild type and the P104G mutant exhibited fusogenic activities, with greater than 60% and 20 to 40% of the cells being fused, respectively, at pH 4.8. These results suggest that it is likely that the region of E1 between amino acids 81 and 109 is involved in the membrane fusion activity of RV and that it may be important for the interaction of that protein with E2 to form the E1-E2 heterodimer.

Amino Acid Sequence↗

PubMed1988

RNA polymerases stall and/or prematurely terminate nearby both early and late promoters on polyomavirus DNA.

Levels of transcription within the E and L strands of the five major PstI fragments of polyomavirus (strain AT3) were measured by pulse-labeling RNA both in infected cells and in isolated nuclei or viral transcription complexes during the late phase of infection. Quantification was assured by hybridization to single-stranded DNAs in solution followed by collection of hybrids on nitrocellulose filters and ribonuclease treatment. The level of in vivo transcription in the region of the early (E strand) promoter was two- to threefold higher than that in all other E-strand regions, suggesting that most RNA polymerases prematurely terminate transcription shortly downstream from this promoter during the late phase. In vitro transcription levels in this region were five- to tenfold higher than in the remainder of the E strand, suggesting that many RNA polymerases 'stall' shortly after initiation in vivo but can be reactivated and continue transcription in vitro upon exposure to detergents and high salt solution. Some premature termination nearby the late (L strand) promoter was also detected by the same method. Strikingly, many RNA polymerases also stalled on the L strand in the region of the early promoter, some 5 x 10(3) bases downstream from the late promoter. Treatment of cells with dichlororibofuranosylbenzimidazole did not affect polymerases that stalled or terminated prematurely, but strongly reduced the presence of polymerases that normally transcribed throughout the entire E or L strand. Examination of the size of RNA chains produced during in vitro incubations showed that many polymerases stalled in vivo within 50 to 100 nucleotides downstream from the initiation sites on both DNA strands. The number of polymerases active in vitro at the E strand promoter was similar to the number of polymerases at the L strand promoter. However, in contrast to L-strand transcription, most of the polymerases that initiated at the E-strand promoter were incapable of extended transcription in vivo. These results suggest that large T antigen-mediated repression of E-strand transcription is not simply due to the exclusion of RNA polymerases from the early promoter. Stalling and/or premature termination by RNA polymerases shortly downstream from the early promoter appears to be a mechanism by which temporal regulation of polyomavirus gene expression can be effected.

Base Sequence↗

PubMed2005

Simplification therapy with once-daily didanosine, tenofovir and efavirenz in HIV-1-infected adults with viral suppression receiving a more complex antiretroviral regimen: final results of the EFADITE trial.

BACKGROUND: High pill burden and side effects often impact on the long-term success of highly active antiretroviral therapy (HAART), which has led clinicians to search for more convenient regimens. PATIENTS AND METHODS: A prospective, multicentre, open, comparative study in which HIV-1-infected patients on HAART and with plasma HIV-1 RNA <50 copies/ml for longer than 6 months were switched to tenofovir, didanosine and efavirenz (QD arm) or remained on the same treatment regimen (control arm). Patients with grade 4 toxicities or plasma HIV-1 RNA values repeatedly >1000 copies/ml discontinued the study. RESULTS: A total of 390 patients were included in the trial (309 in the QD arm and 81 in the control arm). The main baseline characteristics were well balanced between groups. In the QD arm, 41% of patients received high (standard) didanosine doses and 59% received reduced doses. At 12 months, plasma HIV-1 RNA <400 copies/ml was attained in 66% of QD patients and 73% of controls in the intent-to-treat (ITT) analysis (P=NS). However, the number of individuals with HIV-1 RNA <400 copies/ml in the QD arm was 56% versus 71% when comparing the use of high versus low didanosine doses (P=0.007). Treatment discontinuation occurred in 87 QD cases (28%) and 17 controls (21%). Twenty QD individuals (6.5%) and 2 controls (2.5%) discontinued because of virological failure (P=NS). The median CD4+ cell count change at 12 months was -26 and +27 cells/microl in QD patients and controls, respectively (P=0.001). In individuals who attained HIV-1 RNA <400 copies/ml, CD4+ cell changes were -25 and +15 cells/microl in QD patients and controls, respectively (P=0.001). Moreover, CD4+ cell declines in the QD arm were significantly greater in patients taking high versus low didanosine doses (-59 versus -15 cells/microl; P=0.04). The lipid profile improved significantly in the QD arm, particularly in patients who were on protease inhibitors prior to simplification. CONCLUSIONS: Simplification to didanosine-tenofovir-efavirenz provides a virological suppression rate at 12 months similar to that seen in patients who do not change therapy, as long as low didanosine doses are administered. Decreases in CD4+ cell levels in patients in the QD arm (especially decreases seen with high didanosine doses) and dyslipidaemias along with less convenient pill burden and schedules in controls were the main long-term concerns for each option.

Adenine↗

PubMed1987

Induction of H-2-specific antibodies by injections of syngeneic Sendai virus-coated cells.

The capacity of the B cell immunoglobulin receptor to recognize complexes of Sendai viral and H-2b antigens was investigated by studying the antibody response to injections of syngeneic Sendai virus-coated (SV+) spleen cells in C57BL/6 (B6) mice. Almost all mice produced alloreactive anti-H2 lymphocytotoxic antibodies. In contrast, such antibodies were found very exceptionally in mice injected with normal (SV-) cells or with Sendai virus (SV) only. The reaction pattern of the cytotoxic antibodies induced was variable and ranged from almost anti-private to widely cross-reactive serotypes. The results of reactions on H-2-congenic, -recombinant and -mutant mouse strains, and of capping and immunoprecipitation experiments showed that the cytotoxic antibodies were directed against H-2 class I molecules. The anti-H-2 antibodies exhibited enhanced binding for SV+ target cells, but absorption experiments showed that this was not the result of cross-reactions with cell surface Sendai viral determinants or with a molecular complex of H-2 plus SV. This conclusion was supported by the observation that syngeneic SV+ cells were not the predominant targets for the induced lymphocytotoxic antibodies. Our results do not support the existence of MHC-restricted antiviral antibodies, but show the induction of anti-class I H-2 alloantibodies by injections with syngeneic SV-coated cells. We present a model for regular induction of anti-H-2 antibodies without intentional alloimmunization.

Animals↗

PubMed2002

CD40, an extracellular receptor for binding and uptake of Hsp70-peptide complexes.

Tumor and viral antigens elicit a potent immune response by heat shock protein-dependent uptake of antigenic peptide with subsequent presentation by MHC I. Receptors on antigen-presenting cells that specifically bind and internalize a heat shock protein-peptide complex have not yet been identified. Here, we show that cells expressing CD40, a cell surface protein crucial for B cell function and autoimmunity, specifically bind and internalize human Hsp70 with bound peptide. Binding of Hsp70-peptide complex to the exoplasmic domain of CD40 is mediated by the NH(2)-terminal nucleotide-binding domain of Hsp70 in its ADP state. The Hsp70 cochaperone Hip, but not the bacterial Hsp70 homologue DnaK, competes formation of the Hsp70-CD40 complex. Binding of Hsp70-ADP to CD40 is strongly increased in the presence of Hsp70 peptide substrate, and induces signaling via p38. We suggest that CD40 is a cochaperone-like receptor mediating the uptake of exogenous Hsp70-peptide complexes by macrophages and dendritic cells.

Adenosine Triphosphatases↗

PubMed2004

Proteasome function in antigen presentation: immunoproteasome complexes, Peptide production, and interactions with viral proteins.

Proteasomes are the major nonlysosomal protein degradation machinery in eukaryotic cells and they are largely responsible for the processing of antigens for presentation by the MHC class I pathway. This review concentrates on recent developments in the area of antigen processing. Specialized proteasomes called immunoproteasomes and an 11S regulator of proteasomes (PA28) are induced by interferon-gamma, but it is not entirely clear why changes in proteasome structure are beneficial for antigen presentation. Different proteasome complexes have distinct subcellular distributions and subtle differences in cleavage specificity. Thus it is likely that the efficiency of production of MHC class I binding peptides varies in different locations. Immunoproteasome subunits are enriched at the ER where TAP transports peptides for association with newly synthesized MHC class I molecules. There is recent evidence to suggest that antigen presentation from viral expression vectors, or from peptides that are either delivered by bacterial toxins or derived from signal peptides, require proteasome activity for generation of the correct C-terminus of the epitope. The correct N-terminus may be generated by recently identified ER associated aminopeptidases. A number of viral protein interactions with proteasome subunits have been reported and such interactions may interfere with host anti-viral defenses and also contribute to mechanisms of cell transformation.

Animals↗

PubMed1993

The binding domain structure of retinoblastoma-binding proteins.

The retinoblastoma gene product (Rb), a cellular growth suppressor, complexes with viral and cellular proteins that contain a specific binding domain incorporating three invariant residues: Leu-X-Cys-X-Glu, where X denotes a nonconserved residue. Hydrophobic and electrostatic properties are strongly conserved in this segment even though the nonconserved amino acids vary considerably from one Rb-binding protein to another. In this report, we present a diagnostic computer pattern for a high-affinity Rb-binding domain featuring the three conserved residues as well as the conserved physico-chemical properties. Although the pattern encompasses only 10 residues (with only 4 of these explicitly defined), it exhibits 100% sensitivity and 99.95% specificity in database searches. This implies that a certain pattern of structural and physico-chemical properties encoded by this short sequence is sufficient to govern specific Rb binding. We also present evidence that the secondary structural conformation through this region is important for effective Rb binding.

Amino Acid Sequence↗

PubMed1999

Functional domains of template-activating factor-I as a protein phosphatase 2A inhibitor.

Template-Activating Factor-I (TAF-I) alpha and beta, chromatin remodeling factors, were identified as the stimulatory factor for replication of the adenovirus DNA complexed with viral basic core proteins. Recently, two cellular inhibitors for protein phosphatase 2A (PP2A) have been isolated. One of these inhibitors, designated IPP2A2, is a truncated version of TAF-Ibeta. Here, it is shown using recombinant TAF-I proteins that both TAF-Ialpha and beta have the PP2A inhibitor activity. The N-terminal region but not the C-terminal acidic region, the latter of which is essential for the chromatin remodeling activity, is shown to be required for the PP2A inhibitor activity. Roles of TAF-Ialpha- and beta-specific regions, the C-terminal acidic region, and other regions of TAF-I for the PP2A inhibitor activity are also discussed.

Animals↗

PubMed2000

Intranuclear binding by the HIV-1 regulatory protein VPR is dependent on cytosolic factors.

The regulatory protein Vpr of the human immunodeficiency virus HIV-1 performs multiple functions during the HIV replicative cycle. It is involved in the transport of the viral preintegration complex into the nucleus, and has the ability to interact with nuclear proteins such as transcription factors and cyclin-dependent kinases. In this study we examine for the first time the kinetics of intranuclear binding and accumulation at the nuclear envelope of fluorescently labelled full-length Vpr in vitro. We show that intranuclear binding is strongly dependent on the presence of cytosolic factors; in the absence of cytosol, Vpr associates predominantly with the nuclear envelope. Specific regulation of the interactions of Vpr with cytosolic factors, as well as with sites at the nuclear envelope and within the nucleus, is thus implicated, but conventional nuclear transport factors such as importin alpha/beta do not appear to be involved.

Animals↗

PubMed1999

Coiled-coil structure-mediated dimerization of template activating factor-I is critical for its chromatin remodeling activity.

Template activating factor-I (TAF-I)alpha and TAF-Ibeta have been identified as the host factors that activate DNA replication of the adenovirus genome complexed with viral basic core proteins (Ad core). TAF-I causes a structural change of the Ad core, thereby stimulating not only replication but also transcription from the Ad core DNA in vitro. TAF-I also activates transcription from the reconstituted chromatin consisting of DNA fragments and purified histones through chromatin remodeling. Although the carboxyl-terminal region, which is highly rich in acidic amino acids, is essential for the TAF-I activity, it remains unclear how other parts are involved in its activity. The native TAF-I isolated from HeLa cells exists as either hetero- or homo-oligomer. Here, we have demonstrated by cross-linking assays that most of TAF-I exists as a dimer. Analyses using deletion mutant TAF-I proteins revealed that the amino-terminal region of TAF-I common to both alpha and beta is essential for dimerization. This region is predicted to form a coiled-coil structure. Indeed, mutations disrupting this putative structure abolished the dimerization capability and reduced the TAF-I activity in the Ad core DNA replication assay. Furthermore, we found that TAF-I mutants lacking the acidic tail act in a dominant-negative manner in this assay. These observations strongly suggest that the dimerization of TAF-I is important for its activity.

Adenoviridae↗

PubMed1998

Arginine residues in the C-terminus of HIV-1 Vpr are important for nuclear localization and cell cycle arrest.

HIV-1 viral protein R (Vpr) is predominantly localized to the nucleus and plays an important role for viral preintegration complex import into the nucleus. In this study, we investigated the influence on subcellular localization of Arg residues in the C-terminus of Vpr. Consistent with previous studies, about 90% of the cells manifested diffuse nuclear staining in the Vpr-expressed cells. Besides diffuse nuclear staining, punctate perinuclear staining, and punctate cytoplasmic staining were also observed in the immunofluorescence studies. Deletion of the Ser-Arg-lle-Gly residues (amino acids 79-82; SRIG) had no effect on the Vpr localization. However, deletion of the Arg-Gln-Arg-Arg residues (amino acids 85-88; RQRR) resulted in a smooth perinuclear staining pattern. Substitution of five Arg residues with Asn (amino acids 80, 85, 87, 88, and 90; R-->N5) resulted in a diffuse cytoplasmic staining. Subcellular fractionation analyses support the immunofluorescence staining results. These findings indicate that the C-terminal Arg residues of HIV-1 Vpr play an important role for Vpr nuclear localization. All the Vpr mutants were appropriately expressed, exhibited no significant defect on the protein stability, and were incorporated efficiently into virus-like particles. Both SRIG and R-->N5 mutants lost their cell cycle arrest activities and the RQRR deletion only exhibited a low level of cell arrest activity. Therefore, the Arg residues in the HIV-1 Vpr C-terminus are important for Vpr nuclear localization and cell cycle arrest, but had no effect on protein stability or Vpr incorporation into virus-like particles.

Amino Acid Sequence↗

PubMed2000

Helix-destabilizing properties of the baculovirus single-stranded DNA-binding protein (LEF-3).

The helix-destabilizing properties of a single-stranded DNA-binding protein, LEF-3, of Bombyx mori nucleopolyhedrovirus (BmNPV) were studied. Partial duplexes of DNA containing single-stranded (ss) tails of different sizes and orientations were used as substrates for assay of the unwinding ability of LEF-3. Upon noncooperative binding to ssDNA, LEF-3 was capable of unwinding the duplexes with 5' ss tails. However, it did not cause melting of the duplexes containing 3' ss tails, even at oversaturation of ssDNA adjacent to the duplexes. Upon cooperative binding to long ss tails, LEF-3 also produced the polar melting effect; it unwound the duplexes with long 5' ss tails, but not those with long 3' ss tails. These data suggest that LEF-3 has a preferential direction for entry into duplex DNA, namely 5' to 3' with respect to the bound DNA strand. In agreement with its polarity, LEF-3 efficiently melted the primer-template complexes which serve as substrates for DNA polymerases. However, the formation of a complex with viral DNA polymerase before addition of LEF-3 protected the primer-templates from the destabilization effect of LEF-3. Although the destabilization effect of LEF-3 was highly sensitive to monovalent and divalent salts, the protein was capable of melting DNA duplexes in a polar manner at physiological conditions, i.e., 30 degrees C in 0.15 M NaCl. Therefore, the polar destabilization effect of LEF-3 seems to be physiologically important and may be connected, in particular, with the polar action of viral helicase holoenzyme during baculovirus replication.

Adenosine Triphosphate↗

PubMed2002

Subcellular localisation, protein interactions, and RNA binding of Potato mop-top virus triple gene block proteins.

Subcellular localisation, protein interactions, and RNA binding of the triple gene block proteins (TGBp) of Potato mop-top virus (PMTV) were studied. The 13-kDa (TGBp2) and 21-kDa (TGBp3) proteins with or without green fluorescent protein fused to their N-terminus, and the 51-kDa protein (TGBp1) were expressed individually from a recombinant Tobacco mosaic virus (TMV) vector. Fluorescent images and Western immunoblotting experiments of recombinant TMV-infected Nicotiana benthamiana cells suggested that TGBp2 and TGBp3 were associated with cellular endomembranes and that TGBp3 was associated with the cell wall, possibly located close to plasmodesmata. In Western blots, TGBp1 was detected in fractions containing the cell wall and those enriched for organelles and membranous structures. Self-interactions were demonstrated with all three proteins in yeast two-hybrid experiments, and a heterologous interaction was found between TGBp2 and TGBp3. No additional heterologous interactions were discovered between the different TGBp and none were detected in an in vitro binding assay. TGBp1 and TGBp2 but not TGBp3 were shown to bind ssRNA in a sequence nonspecific manner. The results support the model where TGBp2 and TGBp3 facilitate delivery and localisation of the ribonucleoprotein complex to the plasmodesmata. However, the process is facilitated by RNA-protein rather than protein:protein interactions between the TGBp1 in complex with viral RNA and membrane-localised TGBp2.

Blotting, Western↗