The Autophagy Receptor TAX1BP1 (T6BP) improves antigen presentation by MHC-II molecules.

CD4+ T lymphocytes play a major role in the establishment and maintenance of immunity. They are activated by antigenic peptides derived from extracellular or newly synthesized (endogenous) proteins presented by the MHC-II molecules. The pathways leading to endogenous MHC-II presentation remain poorly characterized. We demonstrate here that the autophagy receptor, T6BP, influences both autophagy-dependent and -independent endogenous presentation of HIV- and HCMV-derived peptides. By studying the immunopeptidome of MHC-II molecules, we show that T6BP affects both the quantity and quality of peptides presented. T6BP silencing induces the mislocalization of the MHC-II-loading compartments and rapid degradation of the invariant chain (CD74) without altering the expression and internalization kinetics of MHC-II molecules. Defining the interactome of T6BP, we identify calnexin as a T6BP partner. We show that the calnexin cytosolic tail is required for this interaction. Remarkably, calnexin silencing replicates the functional consequences of T6BP silencing: decreased CD4+ T cell activation and exacerbated CD74 degradation. Altogether, we unravel T6BP as a key player of the MHC-II-restricted endogenous presentation pathway, and we propose one potential mechanism of action.

The XPB Subunit of the TFIIH Complex Plays a Critical Role in HIV-1 Transcription and XPB Inhibition by Spironolactone Prevents HIV-1 Reactivation from Latency.

HIV transcription requires assembly of cellular transcription factors at the HIV-1promoter. The TFIIH general transcription factor facilitates transcription initiation by opening the DNA strands around the transcription start site and phosphorylating the C-terminal domain for RNA polymerase II (RNAPII) for activation. Spironolactone (SP), an FDA approved aldosterone antagonist, triggers the proteasomal degradation of the XPB subunit of TFIIH, and concurrently suppresses acute HIV infection in vitro Here we investigated SP as a possible block-and-lock agent for a functional cure aimed at the transcriptional silencing of the viral reservoir. The long-term activity of SP was investigated in primary and cell line models of HIV-1 latency and reactivation. We show that SP rapidly inhibits HIV-1 transcription by reducing RNAPII recruitment to the HIV-1 genome. shRNA knockdown of XPB confirmed XPB degradation as the mechanism of action. Unfortunately, long-term pre-treatment with SP does not result in epigenetic suppression of HIV upon SP treatment interruption, since virus rapidly rebounds when XPB reemerges; however, SP alone without ART maintains the transcriptional suppression. Importantly, SP inhibits HIV reactivation from latency in both cell line models and resting CD4+T cells isolated from aviremic infected individuals upon cell stimulation with latency reversing agents. Furthermore, long-term treatment with concentrations of SP that potently degrade XPB does not lead to global dysregulation of cellular mRNA expression. Overall, these results suggest that XPB plays a key role in HIV transcriptional regulation and XPB degradation by SP strengthens the potential of HIV transcriptional inhibitors in block-and-lock HIV cure approaches.IMPORTANCE Antiretroviral therapy (ART) effectively reduces an individual's HIV loads to below the detection limit, nevertheless rapid viral rebound immediately ensues upon treatment interruption. Furthermore, virally suppressed individuals experience chronic immune activation from ongoing low-level virus expression. Thus, the importance of identifying novel therapeutics to explore in block-and-lock HIV functional cure approaches, aimed at the transcriptional and epigenetic silencing of the viral reservoir to block reactivation from latency. We investigated the potential of repurposing the FDA-approved spironolactone (SP), as one such drug. SP treatment rapidly degrades a host transcription factor subunit, XPB, inhibiting HIV transcription and blocking reactivation from latency. Long-term SP treatment does not affect cellular viability, cell cycle progression or global cellular transcription. SP alone blocks HIV transcription in the absence of ART but does not delay rebound upon drug removal as XPB rapidly reemerges. This study highlights XPB as a novel drug target in block-and-lock therapeutic approaches.

Human T-Cell Lymphotropic Virus Type 1 Transactivator Tax Exploits the XPB Subunit of TFIIH during Viral Transcription.

Human T-cell lymphotropic virus type 1 (HTLV-1) Tax oncoprotein is required for viral gene expression. Tax transactivates the viral promoter by recruiting specific transcription factors but also by interfering with general transcription factors involved in the preinitiation step, such as TFIIA and TFIID. However, data are lacking regarding Tax interplay with TFIIH, which intervenes during the last step of preinitiation. We previously reported that XPB, the TFIIH subunit responsible for promoter opening and promoter escape, is required for Tat-induced human-immunodeficiency virus promoter transactivation. Here, we investigated whether XPB may also play a role in HTLV-1 transcription. We report that Tax and XPB directly interact in vitro and that endogenous XPB produced by HTLV-1-infected T cells binds to Tax and is recruited on proviral LTRs. In contrast, XPB recruitment at the LTR is not detected in Tax-negative HTLV-1-infected T cells and is strongly reduced when Tax-induced HTLV-1 LTR transactivation is blocked. XPB overexpression does not affect basal HTLV-1 promoter activation but enhances Tax-mediated transactivation in T cells. Conversely, downregulating XPB strongly reduces Tax-mediated transactivation. Importantly, spironolactone (SP)-mediated inhibition of LTR activation can be rescued by overexpressing XPB but not XPD, another TFIIH subunit. Furthermore, an XPB mutant defective for the ATPase activity responsible for promoter opening does not show rescue of the effect of SP. Finally, XPB downregulation reduces viability of Tax-positive but not Tax-negative HTLV-1-transformed T cell lines. These findings reveal that XPB is a novel cellular cofactor hijacked by Tax to facilitate HTLV-1 transcription.IMPORTANCE HTLV-1 is considered the most potent human oncovirus and is also responsible for severe inflammatory disorders. HTLV-1 transcription is undertaken by RNA polymerase II and is controlled by the viral oncoprotein Tax. Tax transactivates the viral promoter first via the recruitment of CREB and its cofactors to the long terminal repeat (LTR). However, how Tax controls subsequent steps of the transcription process remains unclear. In this study, we explore the link between Tax and the XPB subunit of TFIIH that governs, via its ATPase activity, the promoter-opening step of transcription. We demonstrate that XPB is a novel physical and functional partner of Tax, recruited on HTLV-1 LTR, and required for viral transcription. These findings extend the mechanism of Tax transactivation to the recruitment of TFIIH and reinforce the link between XPB and transactivator-induced viral transcription.

HIV-2/SIV viral protein X counteracts HUSH repressor complex.

To evade host immune defences, human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2) have evolved auxiliary proteins that target cell restriction factors. Viral protein X (Vpx) from the HIV-2/SIVsmm lineage enhances viral infection by antagonizing SAMHD1 (refs 1,2), but this antagonism is not sufficient to explain all Vpx phenotypes. Here, through a proteomic screen, we identified another Vpx target-HUSH (TASOR, MPP8 and periphilin)-a complex involved in position-effect variegation3. HUSH downregulation by Vpx is observed in primary cells and HIV-2-infected cells. Vpx binds HUSH and induces its proteasomal degradation through the recruitment of the DCAF1 ubiquitin ligase adaptor, independently from SAMHD1 antagonism. As a consequence, Vpx is able to reactivate HIV latent proviruses, unlike Vpx mutants, which are unable to induce HUSH degradation. Although antagonism of human HUSH is not conserved among all lentiviral lineages including HIV-1, it is a feature of viral protein R (Vpr) from simian immunodeficiency viruses (SIVs) of African green monkeys and from the divergent SIV of l'Hoest's monkey, arguing in favour of an ancient lentiviral species-specific vpx/vpr gene function. Altogether, our results suggest the HUSH complex as a restriction factor, active in primary CD4+ T cells and counteracted by Vpx, therefore providing a molecular link between intrinsic immunity and epigenetic control.

Specific Inhibition of HIV Infection by the Action of Spironolactone in T Cells.

Tat protein, the HIV transactivator, regulates transcription of the HIV genome by the host transcription machinery. Efficient inhibitors of HIV transcription that target Tat or the cellular cofactor NF-κB are well known. However, inhibition of HIV Tat-dependent transcription by targeting the general transcription and DNA repair factor II human (TFIIH) has not been reported. Here, we show that spironolactone (SP), an aldosterone antagonist approved for clinical use, inhibits HIV-1 and HIV-2 infection of permissive T cells by blocking viral Tat-dependent transcription from the long terminal repeat (LTR). We found that treatment of Jurkat and primary CD4+ T cells with SP induces degradation of the XPB cellular helicase, a component of the TFIIH complex, without affecting cellular mRNA levels, T cell viability, or T cell proliferation. We further demonstrate that the effect of SP on HIV infection is independent of its aldosterone antagonist function, since the structural analogue, eplerenone, does not induce XPB degradation and does not inhibit HIV infection. Rescue experiments showed that the SP-induced block of HIV infection relies, at least partially, on XPB degradation. In addition, we demonstrate that SP specifically inhibits Tat-dependent transcription, since basal transcription from the LTR is not affected. Our results demonstrate that SP is a specific inhibitor of HIV Tat-dependent transcription in T cells, which additionally suggests that XPB is a cofactor required for HIV infection. Targeting a cellular cofactor of HIV transcription constitutes an alternative strategy to inhibit HIV infection, together with the existing antiretroviral therapy. IMPORTANCE: Transcription from the HIV promoter is regulated by the combined activities of the host transcription machinery and the viral transactivator Tat protein. Here, we report that the drug spironolactone-an antagonist of aldosterone-blocks viral Tat-dependent transcription, thereby inhibiting both HIV-1 and HIV-2 infection of permissive T cells. This inhibition relies on the degradation of the cellular helicase XPB, a component of the TFIIH transcription factor complex. Consequently, XPB appears to be a novel HIV cofactor. Our discovery of the HIV-inhibitory activity of spironolactone opens the way for the development of novel anti-HIV strategies targeting a cellular cofactor without the limitations of antiretroviral therapy of drug resistance and high cost.

HIV-1 Vpr degrades the HLTF DNA translocase in T cells and macrophages.

Viruses often interfere with the DNA damage response to better replicate in their hosts. The human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) protein has been reported to modulate the activity of the DNA repair structure-specific endonuclease subunit (SLX4) complex and to promote cell cycle arrest. Vpr also interferes with the base-excision repair pathway by antagonizing the uracil DNA glycosylase (Ung2) enzyme. Using an unbiased quantitative proteomic screen, we report that Vpr down-regulates helicase-like transcription factor (HLTF), a DNA translocase involved in the repair of damaged replication forks. Vpr subverts the DDB1-cullin4-associated-factor 1 (DCAF1) adaptor of the Cul4A ubiquitin ligase to trigger proteasomal degradation of HLTF. This event takes place rapidly after Vpr delivery to cells, before and independently of Vpr-mediated G2 arrest. HLTF is degraded in lymphocytic cells and macrophages infected with Vpr-expressing HIV-1. Our results reveal a previously unidentified strategy for HIV-1 to antagonize DNA repair in host cells.

Evidence that HIV-1 restriction factor SAMHD1 facilitates differentiation of myeloid THP-1 cells.

BACKGROUND: SAMHD1 counteracts HIV-1 or HIV-2/SIVsmm that lacks Vpx by depleting the intracellular pool of nucleotides in myeloid cells and CD4+ quiescent T cells, thereby inhibiting the synthesis of retroviral DNA by reverse transcriptase. Depletion of nucleotides has been shown to underline the establishment of quiescence in certain cellular systems. These observations led us to investigate whether SAMHD1 could control the transition between proliferation and quiescence using the THP-1 cell model. FINDINGS: The entry of dividing THP-1 myeloid cells into a non-dividing differentiated state was monitored after addition of phorbol-12-myristate-13-acetate (PMA), an inducer of differentiation. Under PMA treatment, cells overexpressing SAMHD1 display stronger and faster adhesion to their support, compared to cells expressing a catalytically inactive form of SAMHD1, or cells depleted of SAMHD1, which appear less differentiated. After PMA removal, cells overexpressing SAMHD1 maintain low levels of cyclin A, in contrast to other cell lines. Interestingly, SAMHD1 overexpression slightly increases cell adhesion even in the absence of the differentiation inducer PMA. Finally, we found that levels of SAMHD1 are reduced in proliferating primary CD4+ T cells after T cell receptor activation, suggesting that SAMHD1 may also be involved in the transition from a quiescent state to a dividing state in primary T cells. CONCLUSIONS: Altogether, we provide evidence that SAMHD1 may facilitate some aspects of THP-1 cell differentiation. Restriction of HIV-1 by SAMHD1 may rely upon its ability to modify cell cycle parameters, in addition to the direct inhibition of reverse transcription.

Loss of infectivity of HIV-1 particles produced by mobile lymphocytes.

HIV-1 spreads by cell-free particles and through direct cell contacts. To discriminate between these two modes of dissemination, an assay in which the cells are cultured under shaking conditions impairing cell-to-cell transmission has been described. We addressed the impact of shaking on HIV-1 particle infectivity. Kinetics of HIV-1 infection in static or shaking conditions confirmed that HIV-1 replication is reduced in mobile lymphocyte T cells. Strikingly, the infectivity of viruses produced by mobile lymphocytes was dramatically reduced. In parallel, the amount of envelope protein present on these particles showed a continuous decrease over time. We conclude that inefficient HIV-1 replication in mobile lymphocytes in this experimental system is not only due to avoidance of viral cell-to-cell transfer but also to the loss of infectivity of the viral particles due to the alteration of the composition and functionality of the particles produced by these lymphocytes. It is important to take these observations into account when studying viral transmission under shaking conditions.

HIV-1 Vpr induces the degradation of ZIP and sZIP, adaptors of the NuRD chromatin remodeling complex, by hijacking DCAF1/VprBP.

The Vpr protein from type 1 and type 2 Human Immunodeficiency Viruses (HIV-1 and HIV-2) is thought to inactivate several host proteins through the hijacking of the DCAF1 adaptor of the Cul4A ubiquitin ligase. Here, we identified two transcriptional regulators, ZIP and sZIP, as Vpr-binding proteins degraded in the presence of Vpr. ZIP and sZIP have been shown to act through the recruitment of the NuRD chromatin remodeling complex. Strikingly, chromatin is the only cellular fraction where Vpr is present together with Cul4A ubiquitin ligase subunits. Components of the NuRD complex and exogenous ZIP and sZIP were also associated with this fraction. Several lines of evidence indicate that Vpr induces ZIP and sZIP degradation by hijacking DCAF1: (i) Vpr induced a drastic decrease of exogenously expressed ZIP and sZIP in a dose-dependent manner, (ii) this decrease relied on the proteasome activity, (iii) ZIP or sZIP degradation was impaired in the presence of a DCAF1-binding deficient Vpr mutant or when DCAF1 expression was silenced. Vpr-mediated ZIP and sZIP degradation did not correlate with the growth-related Vpr activities, namely G2 arrest and G2 arrest-independent cytotoxicity. Nonetheless, infection with HIV-1 viruses expressing Vpr led to the degradation of the two proteins. Altogether our results highlight the existence of two host transcription factors inactivated by Vpr. The role of Vpr-mediated ZIP and sZIP degradation in the HIV-1 replication cycle remains to be deciphered.

Interferon block to HIV-1 transduction in macrophages despite SAMHD1 degradation and high deoxynucleoside triphosphates supply.

BACKGROUND: Interferon-α (IFN-α) is an essential mediator of the antiviral response, which potently inhibits both early and late phases of HIV replication. The SAMHD1 deoxynucleoside triphosphate (dNTP) hydrolase represents the prototype of a new antiviral strategy we referred to as "nucleotide depletion". SAMHD1 depletes dNTP levels in myeloid cells below those required for optimal synthesis of HIV viral DNA. HIV-2 and its SIVsm and SIVmac close relatives encode a protein termed Vpx, which counteracts SAMHD1. The potentiality of IFN-α to cooperate with nucleotide depletion has been poorly investigated so far. Here we wondered whether IFN-α affects SAMHD1 expression, Vpx-induced SAMHD1 degradation, Vpx-mediated rescue of HIV-1 transduction and the dNTP supply in monocyte-derived macrophages (MDMs). RESULTS: IFN-α inhibited HIV-1 transduction in monocytes and in MDMs while SAMHD1 expression was not up-regulated. Vpx triggered SAMHD1 degradation in IFN-α treated cells, and weakly restored HIV-1 transduction from the IFN-α block. Vpx helper effect towards HIV-1 transduction was gradually inhibited with increasing doses of IFN-α. dNTP levels were not significantly affected in MDMs and CD4+ primary activated T lymphocytes by IFN-α and, in correlation with SAMHD1 degradation, restoration of dNTP levels by Vpx was efficient in MDMs treated with the cytokine. In contrast, IFN-α inhibited Vpx-mediated SAMHD1 degradation in THP-1 cells, where, accordingly, Vpx could not rescue HIV-1 transduction. CONCLUSION: Our results suggest that the early antiviral effect of IFN-α results from a mechanism independent of nucleotide depletion in MDMs. In addition, they indicate that the macrophage-like THP-1 cell line may provide a system to characterize an IFN-α-induced cell response that inhibits Vpx-mediated SAMHD1 degradation.

The scaffolding protein Dlg1 is a negative regulator of cell-free virus infectivity but not of cell-to-cell HIV-1 transmission in T cells.

BACKGROUND: Cell-to-cell virus transmission of Human immunodeficiency virus type-1 (HIV-1) is predominantly mediated by cellular structures such as the virological synapse (VS). The VS formed between an HIV-1-infected T cell and a target T cell shares features with the immunological synapse (IS). We have previously identified the human homologue of the Drosophila Discs Large (Dlg1) protein as a new cellular partner for the HIV-1 Gag protein and a negative regulator of HIV-1 infectivity. Dlg1, a scaffolding protein plays a key role in clustering protein complexes in the plasma membrane at cellular contacts. It is implicated in IS formation and T cell signaling, but its role in HIV-1 cell-to-cell transmission was not studied before. METHODOLOGY/PRINCIPAL FINDINGS: Kinetics of HIV-1 infection in Dlg1-depleted Jurkat T cells show that Dlg1 modulates the replication of HIV-1. Single-cycle infectivity tests show that this modulation does not take place during early steps of the HIV-1 life cycle. Immunofluorescence studies of Dlg1-depleted Jurkat T cells show that while Dlg1 depletion affects IS formation, it does not affect HIV-1-induced VS formation. Co-culture assays and quantitative cell-to-cell HIV-1 transfer analyses show that Dlg1 depletion does not modify transfer of HIV-1 material from infected to target T cells, or HIV-1 transmission leading to productive infection via cell contact. Dlg1 depletion results in increased virus yield and infectivity of the viral particles produced. Particles with increased infectivity present an increase in their cholesterol content and during the first hours of T cell infection these particles induce higher accumulation of total HIV-1 DNA. CONCLUSION: Despite its role in the IS formation, Dlg1 does not affect the VS and cell-to-cell spread of HIV-1, but plays a role in HIV-1 cell-free virus transmission. We propose that the effect of Dlg1 on HIV-1 infectivity is at the stage of virus entry.

Virus versus host cell translation love and hate stories.

Regulation of protein synthesis by viruses occurs at all levels of translation. Even prior to protein synthesis itself, the accessibility of the various open reading frames contained in the viral genome is precisely controlled. Eukaryotic viruses resort to a vast array of strategies to divert the translation machinery in their favor, in particular, at initiation of translation. These strategies are not only designed to circumvent strategies common to cell protein synthesis in eukaryotes, but as revealed more recently, they also aim at modifying or damaging cell factors, the virus having the capacity to multiply in the absence of these factors. In addition to unraveling mechanisms that may constitute new targets in view of controlling virus diseases, viruses constitute incomparably useful tools to gain in-depth knowledge on a multitude of cell pathways.

Human Discs Large is a new negative regulator of human immunodeficiency virus-1 infectivity.

Human immunodeficiency virus (HIV)-1 replication is positively or negatively regulated through multiple interactions with host cell proteins. We report here that human Discs Large (Dlg1), a scaffold protein recruited beneath the plasma membrane and involved in the assembly of multiprotein complexes, restricts HIV-1 infectivity. The endogenous Dlg1 and HIV-1 Gag polyprotein spontaneously interact in HIV-1-chronically infected T cells. Depleting endogenous Dlg1 in either adherent cells or T cells does not affect Gag maturation, production, or release, but it enhances the infectivity of progeny viruses five- to sixfold. Conversely, overexpression of Dlg1 reduces virus infectivity by approximately 80%. Higher virus infectivity upon Dlg1 depletion correlates with increased Env content in cells and virions, whereas the amount of virus-associated Gag or genomic RNA remains identical. Dlg1 knockdown is also associated with the redistribution and colocalization of Gag and Env toward CD63 and CD82 positive vesicle-like structures, including structures that seem to still be connected to the plasma membrane. This study identifies both a new negative regulator that targets the very late steps of the HIV-1 life cycle, and an assembly pathway that optimizes HIV-1 infectivity.

Implications of recombination for HIV diversity.

The human immunodeficiency virus (HIV) population is characterised by extensive genetic variability that results from high error and recombination rates of the reverse transcription process, and from the fast turnover of virions in HIV-infected individuals. Among the viral variants encountered at the global scale, recombinant forms are extremely abundant. Some of these recombinants (known as circulating recombinant forms) become fixed and undergo rapid expansion in the population. The reasons underlying their epidemiological success remain at present poorly understood and constitute a fascinating area for future research to improve our understanding of immune escape, pathogenicity and transmission. Recombinant viruses are generated during reverse transcription as a consequence of template switching between the two genetically different genomic RNAs present in a heterozygous virus. Recombination can thereby generate shortcuts in evolution by producing mosaic reverse transcription products of parental genomes. Therefore, in a single infectious cycle multiple mutations that are positively selected can be combined or, conversely, negatively selected mutations can be removed. Recombination is therefore involved in different aspects of HIV evolution, adaptation to its host, and escape from antiviral treatments.

Arabidopsis thaliana is a host of the legume nanovirus Faba bean necrotic yellows virus.

We report infection of Arabidopsis thaliana with the legume nanovirus Faba bean necrotic yellows virus (FBNYV) by its insect vector Aphis craccivora. Symptoms of FBNYV infection on A. thaliana include stunting and reduced apical dominance, and are rather mild, compared to the severe necrosis and early plant death induced by the virus in the natural host Vicia faba. An inoculation access period of 6h is sufficient to transmit FBNYV to A. thaliana. FBNYV is readily transmitted back from A. thaliana to V. faba, where it induces the characteristic severe disease symptoms. Hence, passage through A. thaliana does not affect FBNYV pathogenicity. FBNYV accumulates to the highest levels in roots and stems, compared to cauline and rosette leaves. In cauline leaves, the kinetics of virus accumulation correlates with the amount of master Rep protein accumulation.

The nanovirus-encoded Clink protein affects plant cell cycle regulation through interaction with the retinoblastoma-related protein.

Nanoviruses, multicomponent single-stranded DNA plant viruses, encode a unique cell cycle link protein, Clink, that interacts with retinoblastoma-related proteins (RBR). We have established transgenic Arabidopsis thaliana lines that conditionally express Clink or a Clink variant deficient in RBR binding. By controlled induction of Clink expression, we demonstrated the capacity of the Clink protein to alter RBR function in vivo. We showed that transcription of both S-phase-specific and G2/M-phase-specific genes was up-regulated depending on the RBR-binding proficiency of Clink. Concomitantly, ploidy levels increased in a substantial fraction of leaf cell nuclei. Also, leaf epidermis cells of transgenic plants producing Clink were smaller and more numerous, indicating additional cell divisions in this tissue. Furthermore, cytogenetic analyses following induction of Clink expression in mature leaves revealed the presence of metaphasic and anaphasic nuclei, clear evidence that Clink-mediated RBR inactivation is sufficient to induce quiescent cells to reenter cell cycle progression and, for at least a fraction of them, to pass through mitosis. Expression of Clink had no effect on genes transcribed by RNA polymerases I and III, suggesting that, in contrast to its mammalian homologue, A. thaliana RBR is not involved in the repression of polymerase I and polymerase III transcription. The results of these in vivo analyses firmly establish Clink as a member of the diverse class of multifunctional cell cycle modulator proteins encoded by small DNA viruses.

A functional histidine-tagged replication initiator protein: implications for the study of single-stranded DNA virus replication in planta.

Replication initiation of nanoviruses, plant viruses with a multipartite circular single-stranded DNA genome, is triggered by the master Rep (M-Rep) protein. To enable the study of interactions between M-Rep and viral or host factors involved in replication, we designed oligohistidine-tagged variants of the nanovirus Faba bean necrotic yellows virus (FBNYV) M-Rep protein that allow affinity purification of enzymatically active M-Rep from plant tissue. The tagged M-Rep protein was able to initiate replication of its cognate and other FBNYV DNAs in Nicotiana benthamiana leaf disks and plants. The replicon encoding the tagged M-Rep protein multiplied and moved systemically in FBNYV-infected Vicia faba plants and was transmitted by the aphid vector of the virus. Using the tagged M-Rep protein, we demonstrated the in planta interaction between wild-type M-Rep and its tagged counterpart. Such a tagged and fully functional replication initiator protein will have bearings on the isolation of protein complexes from plants.

A novel calmodulin-binding protein functions as a negative regulator of osmotic stress tolerance in Arabidopsis thaliana seedlings.

A clone for a novel Arabidopsisthaliana calmodulin (CaM)-binding protein of 25 kDa (AtCaMBP25) has been isolated by using a radiolabelled CaM probe to screen a cDNA expression library derived from A. thaliana cell suspension cultures challenged with osmotic stress. The deduced amino acid sequence of AtCaMBP25 contains putative nuclear localization sequences and shares significant degree of similarity with hypothetical plant proteins only. Fusion of the AtCaMBP25 coding sequence to reporter genes targets the hybrid protein to the nucleus. Bacterially expressed AtCaMBP25 binds, in a calcium-dependent manner, to a canonical CaM but not to a less conserved isoform of the calcium sensor. AtCaMBP25 is encoded by a single-copy gene, whose expression is induced in Arabidopsis seedlings exposed to dehydration, low temperature or high salinity. Transgenic plants overexpressing AtCaMBP25 exhibits an increased sensitivity to both ionic (NaCl) and non-ionic (mannitol) osmotic stress during seed germination and seedling growth. By contrast, transgenic lines expressing antisense AtCaMBP25 are significantly more tolerant to mannitol and NaCl stresses than the wild type. Thus, the AtCaMBP25 gene functions as a negative effector of osmotic stress tolerance and likely participates in stress signal transduction pathways.