NAD+ pool depletion as a signal for the Rex regulon involved in Streptococcus agalactiae virulence.

In many Gram-positive bacteria, the redox-sensing transcriptional repressor Rex controls central carbon and energy metabolism by sensing the intra cellular balance between the reduced and oxidized forms of nicotinamide adenine dinucleotide; the NADH/NAD+ ratio. Here, we report high-resolution crystal structures and characterization of a Rex ortholog (Gbs1167) in the opportunistic pathogen, Streptococcus agalactiae, also known as group B streptococcus (GBS). We present structures of Rex bound to NAD+ and to a DNA operator which are the first structures of a Rex-family member from a pathogenic bacterium. The structures reveal the molecular basis of DNA binding and the conformation alterations between the free NAD+ complex and DNA-bound form of Rex. Transcriptomic analysis revealed that GBS Rex controls not only central metabolism, but also expression of the monocistronic rex gene as well as virulence gene expression. Rex enhances GBS virulence after disseminated infection in mice. Mechanistically, NAD+ stabilizes Rex as a repressor in the absence of NADH. However, GBS Rex is unique compared to Rex regulators previously characterized because of its sensing mechanism: we show that it primarily responds to NAD+ levels (or growth rate) rather than to the NADH/NAD+ ratio. These results indicate that Rex plays a key role in GBS pathogenicity by modulating virulence factor gene expression and carbon metabolism to harvest nutrients from the host.

Structural basis for NADH/NAD+ redox sensing by a Rex family repressor.

Nicotinamide adenine dinucleotides have emerged as key signals of the cellular redox state. Yet the structural basis for allosteric gene regulation by the ratio of reduced NADH to oxidized NAD(+) is poorly understood. A key sensor among Gram-positive bacteria, Rex represses alternative respiratory gene expression until a limited oxygen supply elevates the intracellular NADH:NAD(+) ratio. Here we investigate the molecular mechanism for NADH/NAD(+) sensing among Rex family members by determining structures of Thermus aquaticus Rex bound to (1) NAD(+), (2) DNA operator, and (3) without ligand. Comparison with the Rex/NADH complex reveals that NADH releases Rex from the DNA site following a 40 degrees closure between the dimeric subunits. Complementary site-directed mutagenesis experiments implicate highly conserved residues in NAD-responsive DNA-binding activity. These rare views of a redox sensor in action establish a means for slight differences in the nicotinamide charge, pucker, and orientation to signal the redox state of the cell.

Binding mode of the oxidized α-anomer of NAD+ to RSP, a Rex-family repressor.

The Rex-family repressors sense redox levels by alternative binding to NADH or NAD(+). RSP is the homologue of Rex in Thermoanaerobacter ethanolicus JW200(T) and regulates ethanol fermentation in this obligate anaerobe. The dimeric repressor binds to DNA by an open conformation. The crystal structure of RSP/α-NAD(+) complex shows a different set of ligand interactions mainly due to the unique configuration of the nicotinamide moiety. The positively charged ring is covered by the Tyr102 side chain and interacts with a sulfate ion adjacent to the N-terminus of helix α8. Consequently, the RSP dimer may be locked in a closed conformation that does not bind to DNA. However, α-NAD(+) does not show a higher affinity to RSP than ß-NAD(+). It has to be improved for possible use as an effector in modulating the repressor.

Phosphorylation regulates human T-cell leukemia virus type 1 Rex function.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) is a pathogenic complex deltaretrovirus, which is the causative agent of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to the structural and enzymatic viral gene products, HTLV-1 encodes the positive regulatory proteins Tax and Rex along with viral accessory proteins. Tax and Rex proteins orchestrate the timely expression of viral genes important in viral replication and cellular transformation. Rex is a nucleolar-localizing shuttling protein that acts post-transcriptionally by binding and facilitating the export of the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm. HTLV-1 Rex (Rex-1) is a phosphoprotein and general protein kinase inhibition correlates with reduced function. Therefore, it has been proposed that Rex-1 function may be regulated through site-specific phosphorylation. RESULTS: We conducted a phosphoryl mapping of Rex-1 over-expressed in transfected 293 T cells using a combination of affinity purification and liquid chromatography tandem mass spectrometry. We achieved 100% physical coverage of the Rex-1 polypeptide and identified five novel phosphorylation sites at Thr-22, Ser-36, Thr-37, Ser-97, and Ser-106. We also confirmed evidence of two previously identified residues, Ser-70 and Thr-174, but found no evidence of phosphorylation at Ser-177. The functional significance of these phosphorylation events was evaluated using a Rex reporter assay and site-directed mutational analysis. Our results indicate that phosphorylation at Ser-97 and Thr-174 is critical for Rex-1 function. CONCLUSION: We have mapped completely the site-specific phosphorylation of Rex-1 identifying a total of seven residues; Thr-22, Ser-36, Thr-37, Ser-70, Ser-97, Ser-106, and Thr-174. Overall, this work is the first to completely map the phosphorylation sites in Rex-1 and provides important insight into the regulation of Rex-1 function.

Characterization of the nuclear export signal of human T-cell lymphotropic virus type 1 Rex reveals that nuclear export is mediated by position-variable hydrophobic interactions.

We previously determined that amino acids 64 to 120 of human T-cell lymphotropic virus type 1 (HTLV-1) Rex can restore the function of an effector domain mutant of human immunodeficiency virus type 1 (HIV-1) Rev (T. J. Hope, B. L. Bond, D. McDonald, N. P. Klein, and T. G. Parslow, J. Virol. 65:6001-6007, 1991). In this report, we (i) identify and characterize a position-independent 17-amino-acid region of HTLV-1 Rex that fully complements HIV-1 Rev effector domain mutants and (ii) show that this 17-amino-acid region and specific hydrophobic substitutions can serve as nuclear export signals. Mutagenesis studies revealed that four leucines within the minimal region were essential for function. Alignment of the minimal Rex region with the HIV-1 Rev effector domain suggested that the position of some of the conserved leucines is flexible. We found two of the leucines could each occupy one of two positions within the context of the full-length HTLV-1 Rex protein and maintain function. The idea of flexibility within the Rex effector domain was confirmed and extended by identifying functional substitutions by screening a library of effector domain mutants in which the two regions of flexibility were randomized. Secondly, the functional roles of the minimal Rex effector domain and hydrophobic substitutions were independently confirmed by demonstrating that these effector domains could serve as nuclear export signals when conjugated with bovine serum albumin. Nuclear export of the wild-type Rex conjugates was temperature dependent and sensitive to wheat germ agglutinin and was blocked by a 20-fold excess of unlabeled conjugates. Together, these studies reveal that position-variable hydrophobic interactions within the HTLV-1 Rex effector domain mediate nuclear export function.

Importin beta can mediate the nuclear import of an arginine-rich nuclear localization signal in the absence of importin alpha.

The import of proteins into the nucleus is dependent on cis-acting targeting sequences, nuclear localization signals (NLSs), and members of the nuclear transport receptor (importin-beta-like) superfamily. The most extensively characterized import pathway, often termed the classical pathway, is utilized by many basic-type (lysine-rich) NLSs and requires an additional component, importin alpha, to serve as a bridge between the NLS and the import receptor importin beta. More recently, it has become clear that a variety of proteins enter the nucleus via alternative import receptors and that their NLSs bind directly to those receptors. By using the digitonin-permeabilized cell system for protein import in vitro, we have defined the import pathway for the Rex protein of human T-cell leukemia virus type 1. Interestingly, the arginine-rich NLS of Rex uses importin beta for import but does so by a mechanism that is importin alpha independent. Based on the ability of the Rex NLS to inhibit the import of the lysine-rich NLS of T antigen and of both NLSs to be inhibited by the domain of importin alpha that binds importin beta (the IBB domain), we infer that the Rex NLS interacts with importin beta directly. In addition, and in keeping with other receptor-mediated nuclear import pathways, Rex import is dependent on the integrity of the Ran GTPase cycle. Based on these results, we suggest that importin beta can mediate the nuclear import of arginine-rich NLSs directly, or lysine-rich NLSs through the action of importin alpha.

A multifunctional domain in human CRM1 (exportin 1) mediates RanBP3 binding and multimerization of human T-cell leukemia virus type 1 Rex protein.

Human CRM1 (hCRM1) functions in the Rex-mediated mRNA export of human T-cell leukemia virus type 1 (HTLV-1) as an export receptor and as an inducing factor for Rex multimerization on its cognate RNA. Although there are only 24 amino acid differences between hCRM1 and rat CRM1 (rCRM1), rCRM1 can hardly support Rex activity, suggesting a role for rCRM1 as a determinant restricting the host range of HTLV-1. Here, we used a series of mutants, which were generated by interchanging residues of these CRM1s, to examine the relationship of hCRM1 functions. The functions for Rex multimerization and binding to nuclear export signals are mapped to different amino acid residues, and these are separable, suggesting that CRM1 not only functions as an export receptor but also participates in the formation of the RNA export complex through higher-ordered interaction with Rex. The region for the interaction with RanBP3, comprising four residues (amino acids [aa] 411, 414, 474, and 481), and the region for Rex multimerization, including two residues (aa 411 and 414), form an overlapped domain. Our results provide the molecular basis underlying the species-specific ability of HTLV-1 to propagate in human cells.

X-ray structure of a Rex-family repressor/NADH complex insights into the mechanism of redox sensing.

The redox-sensing repressor Rex regulates transcription of respiratory genes in response to the intra cellular NADH/NAD(+) redox poise. As a step toward elucidating the molecular mechanism of NADH/NAD(+) sensing, the X-ray structure of Thermus aquaticus Rex (T-Rex) bound to effector NADH has been determined at 2.9 A resolution. The fold of the C-terminal domain of T-Rex is characteristic of NAD(H)-dependent enzymes, whereas the N-terminal domain is similar to a winged helix DNA binding motif. T-Rex dimerization is primarily mediated by "domain-swapped" alpha helices. Each NADH molecule binds to the C-terminal domain near the dimer interface. In contrast to NAD(H)-dependent enzymes, the nicotinamide is deeply buried within a hydrophobic pocket that appears to preclude substrate entry. We show that T-Rex binds to the Rex operator, and NADH but not NAD(+) inhibits T-Rex/DNA binding activity. A mechanism for redox sensing by Rex family members is proposed by analogy with domain closure of NAD(H)-dependent enzymes.

Anchoring an extended HTLV-1 Rex peptide within an RNA major groove containing junctional base triples.

BACKGROUND: The Rex protein of the human T cell leukemia virus type 1 (HTLV-1) belongs to a family of proteins that use arginine-rich motifs (ARMs) to recognize their RNA targets. Previously, an in vitro selected RNA aptamer sequence was identified that mediates mRNA transport in vivo when placed in the primary binding site on stem-loop IID of the Rex response element. We present the solution structure of the HTLV-1 arginine-rich Rex peptide bound to its RNA aptamer target determined by multidimensional heteronuclear NMR spectroscopy. RESULTS: The Rex peptide in a predominantly extended conformation threads through a channel formed by the shallow and widened RNA major groove and a looped out guanine. The RNA aptamer contains three stems separated by a pair of two-base bulges, and adopts an unanticipated fold in which both junctional sites are anchored through base triple formation. Binding specificity is associated with intermolecular hydrogen bonding between guanidinium groups of three non-adjacent arginines and the guanine base edges of three adjacent G.C pairs. CONCLUSIONS: The extended S-shaped conformation of the Rex peptide, together with previous demonstrations of a beta-hairpin conformation for the bovine immunodeficiency virus (BIV) Tat peptide and an alpha-helical conformation for the human immunodeficiency virus (HIV) Rev peptide in complex with their respective RNA targets, expands our understanding of the strategies employed by ARMs for adaptive recognition and highlights the importance of RNA tertiary structure in accommodating minimalist elements of protein secondary structure.

The human T-cell leukemia virus Rex protein.

A critical step in the life cycle of complex retroviruses, including HTLV-1 and HTLV-2 is the ability of these viruses to adopt a mechanism by which the genome-length unspliced mRNA as well as the partially spliced mRNAs are exported from the nucleus instead of being subjected to splicing or degradation. In HTLV, this is accomplished through the expression of the viral Rex, which recognizes a specific response element on the incompletely spliced mRNAs, stabilizes them, inhibits their splicing, and utilizes the CRM1-dependent cellular pathway for transporting them from the nucleus to the cytoplasm. Rex itself is regulated by phosphorylation, which implies that proper activation of the protein in response to certain cellular cues is an important tool for the virus to ensure that specific viral gene expression is allowed only when the host cell can provide the best conditions for virion production. Having such a critical role in HTLV life cycle, Rex is indispensable for efficient viral replication, infection and spread. Indeed, Rex is considered to regulate the switch between the latent and productive phases of the HTLV life cycle. Without a functional Rex, the virus would still produce regulatory and some accessory gene products; however, structural and enzymatic post-transcriptional gene expression would be severely repressed, essentially leading to non-productive viral replication. More detailed understanding of the exact molecular mechanism of action of Rex will thus allow for better design of therapeutic drugs against Rex function and ultimately HTLV replication. Herein we summarize the progress made towards understanding Rex function and its role in the HTLV life cycle.

[Specific arginine mediated RNA recognition]. / Oddzialywanie argininy z RNA.

In many biological systems substantial roles are played by interactions between amino acids and RNA. Among amino acids L-arginine seems to be particularly relevant, because the guanidinium group of arginine side chain can potentially form five hydrogen bonds with appropriately positioned acceptor groups of RNA. Extensive studies reveal that specific arginine recognition is achieved by many different RNAs over a broad range of binding affinities. Arginine is frequently found among amino acids in the nucleic acid-binding motifs in various proteins. For example, specific binding of the HIV-1 Tat protein to its RNA site (TAR) is mediated by a single arginine residue. Free arginine can be also bound by the guanosine site in the group I Tetrahymena ribosomal RNA intron catalytic centre, as well as by numerous RNA motifs, called arginine aptamers, which have been selected in vitro.

tax and rex Sequences of bovine leukaemia virus from globally diverse isolates: rex amino acid sequence more variable than tax.

Bovine leukaemia virus (BLV) is an important agricultural problem with high costs to the dairy industry. Here, we examine the variation of the tax and rex genes of BLV. The tax and rex genes share 420 bases and have overlapping reading frames. The tax gene encodes a protein that functions as a transactivator of the BLV promoter, is required for viral replication, acts on cellular promoters, and is responsible for oncogenesis. The rex facilitates the export of viral mRNAs from the nucleus and regulates transcription. We have sequenced five new isolates of the tax/rex gene. We examined the five new and three previously published tax/rex DNA and predicted amino acid sequences of BLV isolates from cattle in representative regions worldwide. The highest variation among nucleic acid sequences for tax and rex was 7% and 5%, respectively; among predicted amino acid sequences for Tax and Rex, 9% and 11%, respectively. Significantly more nucleotide changes resulted in predicted amino acid changes in the rex gene than in the tax gene (P < or = 0.0006). This variability is higher than previously reported for any region of the viral genome. This research may also have implications for the development of Tax-based vaccines.

Mutational analysis of the human T-cell leukemia virus type I trans-acting rex gene product.

Expression of the human T-cell leukemia virus type I (HTLV-I) rex gene is a prerequisite for the expression of the retroviral structural proteins. We have generated internal deletion mutants of this 27-kDa nucleolar trans-acting gene product to define functional domains in the Rex protein. The phenotype of the various mutant proteins was tested on the homologous HTLV-I rex response element sequence and the heterologous human immunodeficiency virus type 1 (HIV-1) rev response element sequence. Our results indicate that a region between amino acid residues 55 and 132 in the 189-amino-acid Rex protein is required for Rex-mediated trans activation on both retroviral response element sequences. In addition, substitution of the Rex nuclear localization signal by a sequence of the HIV-1 rev gene product targets the Rex protein to the correct subcellular compartment required for Rex function.

Functional domain structure of human T-cell leukemia virus type 2 rex.

The Rex protein of human T-cell leukemia virus (HTLV) acts posttranscriptionally to induce the cytoplasmic expression of the unspliced and incompletely spliced viral RNAs encoding the viral structural and enzymatic proteins and is therefore essential for efficient viral replication. Rex function requires nuclear import, RNA binding, multimerization, and nuclear export. In addition, it has been demonstrated that the phosphorylation status of HTLV-2 Rex (Rex-2) correlates with RNA binding and inhibition of splicing in vitro. Recent mutational analyses of Rex-2 revealed that the phosphorylation of serine residues 151 and 153 within a novel carboxy-terminal domain is critical for function in vivo. To further define the functional domain structure of Rex-2, we evaluated a panel of Rex-2 mutants for subcellular localization, RNA binding capacity, multimerization and trans-dominant properties, and the ability to shuttle between the nucleus and the cytoplasm. Rex-2 mutant S151A,S153A, which is defective in phosphorylation and function, showed diffuse cytoplasmic staining, whereas mutant S151D,S153D, previously shown to be functional and in a conformation corresponding to constitutive phosphorylation, displayed increased intense speckled staining in the nucleoli. In vivo RNA binding analyses indicated that mutant S151A,S153A failed to efficiently bind target RNA, while its phosphomimetic counterpart, S151D,S153D, bound twofold more RNA than wild-type Rex-2. Taken together, these findings provide direct evidence that the phosphorylation status of Rex-2 is linked to cellular trafficking and RNA binding capacity. Mutants with substitutions in either of the two putative multimerization domains or in the putative activation domain-nuclear export signal displayed a dominant negative phenotype as well as defects in multimerization and nucleocytoplasmic shuttling. Several carboxy-terminal mutants that displayed wild-type levels of phosphorylation and localized to the nucleolus were also partially impaired in shuttling. This is consistent with the hypothesis that the carboxy terminus of Rex-2 contains a novel domain that is required for efficient shuttling. This work thus provides a more detailed functional domain map of Rex-2 and further insight into its regulation of HTLV replication.

The activation domain of a hormone inducible HTLV-1 Rex protein determines colocalization with the nuclear pore.

Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) Rex is an essential regulatory protein that acts at the posttranscriptional level to promote expression of unspliced and singly spliced genes of the virus. Rex functions have been attributed to at least three separate domains of the protein determining nuclear/nucleolar accumulation and RNA binding (overlapping), multimerization, and nuclear export of Rex-responsive RNA. The steady-state intracellular localization of functional Rex molecules is mainly nucleolar. Fusions of wild-type Rex and the ligand binding domain of human estrogen receptor (ER) produced conditional molecules (ERRex and ERalaRex), which remained cytoplasmic in the absence of hormone and in response to hormone colocalized with the nuclear pore complex (NPC). These molecules induced in a hormone-dependent manner the expression of a Rex reporter plasmid and of the HTLV-1 Env protein and fusion of Env expressing cells. In contrast, activation domain mutants (ERRex delta and ERRexGly) translocated from the cytoplasm and acquired a diffuse nuclear localization. These mutants did not associate with the NPC and failed to show any of the expected Rex functions. Rex functions were perturbed by inactivating the RNA binding domain (mutant ERM2) or the oligomerization domain (mutant ERM7). However, these two mutant fusion proteins exhibited a hormone-dependent NPC colocalization. These observations provide in vivo evidence that intranuclear translocation of intact Rex to the NPC is dependent exclusively on a functional activation domain and is not influenced by binding to the target RNA.

Multiple arginine residues within the basic domain of HTLV-I Rex are required for specific RNA binding and function.

The Rex protein of the type I human T-cell leukemia virus (HTLV-I) is essential for viral replication, acting post-transcriptionally to enhance the expression of unspliced and singly spliced viral mRNAs that encode the Gag, Pol, and Env virion proteins. Rex function involves its direct interaction with a complex stem-loop structure termed the Rex RNA response element (RexRE), which is located within the 3' retroviral long terminal repeat. Binding of Rex to the RexRE involves a positively charged arginine-rich domain located near the N-terminus which also functions as a nuclear localization signal. Strikingly, substitution of all seven of the arginine residues present within this domain with positively charged lysine residues exerted no adverse effect on the nuclear targeting of Rex. However, these lysine substitutions completely abrogated both Rex binding to the RexRE and Rex function. Reversion of multiple substituted lysines to arginines at specific locations within this domain was required to restore both RexRE binding and biological function to the Rex protein. Thus, while the presence of positive charge alone in this domain appears sufficient for nuclear localization of Rex, multiple arginine residues at specific sites are essential for the full expression of RNA binding and functional activity of this retroviral trans-regulatory protein.

Rat CRM1 is responsible for the poor activity of human T-cell leukemia virus type 1 Rex protein in rat cells.

Rat models of human T-cell leukemia virus type 1 (HTLV-1)-related diseases such as adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis have been reported. However, these models do not completely reproduce human diseases partly because HTLV-1 replicates poorly in rats. We investigated here the possible reason for this. We found that the activity of Rex in rat cells is quite low compared to that in human cells. As Rex function depends largely on the CRM1 protein, whose human type (human CRM1 [hCRM1]) directly binds to Rex and exports it from the nucleus to the cytoplasm, we assessed whether rat CRM1 (rCRM1) could act as well as hCRM1 as a cofactor for Rex activity. We first cloned a cDNA encoding rCRM1 and found that both rCRM1 and hCRM1 could bind to and export Rex protein to the cytoplasm with similar efficiencies. However, unlike hCRM1, rCRM1 could hardly support Rex function because of its poor ability in inducing the Rex-Rex interaction required for RNA export into the cytoplasm. These observations suggest that the poor ability of rCRM1 to act as a cofactor for Rex function may be responsible for the poor replication of HTLV-1 in rats.

The genomic structure of a new simian T-lymphotropic virus, STLV-PH969, differs from that of human T-lymphotropic virus types I and II.

A new simian T-lymphotropic virus, STLV-PH969, was recently isolated from a wild-born Hamadryas baboon. Previous analysis had revealed that it differs sufficiently from the other HTLV/STLVs to be considered a new type, provisionally designated primate T-lymphotropic virus-L. Here we analyse a 3850 bp cDNA fragment spanning the 3' part of the STLV-PH969 genome. The fragment encodes three major proteins: Env, Tax and Rex. Sequence comparison and phylogenetic analysis indicate that in general STLV-PH969 tends to be more closely related to HTLV-II than to HTLV-I, although separate gene regions might have evolved under different constraints. Detailed comparison of the Env, Tax and Rex proteins among the HTLV-I, -II and STLV-PH969 prototypes reveals that the amino acid sequence of each protein shows a preferential conservation of functionally important domains. RNA-PCR on cytoplasmic messengers demonstrated splicing between a splice donor site immediately downstream of the env start codon, and two splice acceptor sites identified in the pX region. The predominant spliced messenger encodes both Tax and Rex. The other messenger potentially encodes a new viral protein from the proximal part of the pX region that is similar in amino acid composition to p12I and p10xI of HTLV-I and HTLV-II respectively. This genomic organization of the proximal pX region of STLV-PH969 is different from that found in HTLV-I and HTLV-II. Therefore, the distinct classification of this virus can be justified, not only in terms of sequence divergence but also in terms of its different genomic structure.