Anti-Rex aptamers as mimics of the Rex-binding element

RNA molecules that bind tightly and specifically to a Rex fusion protein have been isolated from a conformationally constrained pool of random sequence RNAs. The anti-Rex aptamers effectively mimic several features of the wild-type Rex-binding element (XBE). The highest-affinity aptamers effectively compete with the wild-type XBE for binding to the RNA-binding domain of Rex, an arginine-rich motif (ARM), but do not bind to the functionally analogous Rev protein or its ARM. However, characteristic sequence and structural motifs found in some of the anti-Rex aptamers may provide insights into how the Rex protein can interact with other viral RNAs, such as the Rev-responsive element. The anti-Rex aptamers can functionally substitute for the XBE in vivo, a result which supports a previously proposed model for mRNA transport in which the viral genome serves as a platform for assembling a nucleoprotein complex that can co-opt the cellular transport apparatus. Overall, these studies suggest that anti-Rex aptamers may serve as RNA decoys of the Rex protein.     

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.     

Multimer Formation Is Not Essential for Nuclear Export of Human T-Cell Leukemia Virus Type 1 Rex trans-Activator Protein

The Rex trans-regulatory protein of human T-cell leukemia virus type 1 (HTLV-1) is required for the nuclear export of incompletely spliced and unspliced viral mRNAs and is therefore essential for virus replication. Rex is a nuclear phosphoprotein that directly binds to its cis-acting Rex response element RNA target sequence and constantly shuttles between the nucleus and cytoplasm. Moreover, Rex induces nuclear accumulation of unspliced viral RNA. Three protein domains which mediate nuclear import-RNA binding, nuclear export, and Rex oligomerization have been mapped within the 189-amino-acid Rex polypeptide. Here we identified a different region in the carboxy-terminal half of Rex which is also required for biological activity. In inactive mutants with mutations that map within this region, as well as in mutants that are deficient in Rex-specific multimerization, Rex trans activation could be reconstituted by fusion to a heterologous leucine zipper dimerization interface. The intracellular trafficking capabilities of wild-type and mutant Rex proteins reveal that biologically inactive and multimerization-deficient Rex mutants are still efficiently translocated from the nucleus to the cytoplasm. This observation indicates that multimerization is essential for Rex function but is not required for nuclear export. Finally, we are able to provide an improved model of the HTLV-1 Rex domain structure.     

Phosphorylation of two serine residues regulates human T-cell leukemia virus type 2 Rex function

The function of the human T-cell leukemia virus (HTLV) Rex phosphoprotein is to increase the level of the viral structural and enzymatic gene products expressed from the incompletely spliced viral RNAs containing the Rex-responsive element. The phosphorylation of HTLV type 2 Rex (Rex-2), predominantly on serine residues, correlates with an altered conformation, as detected by a gel mobility shift, and is required for specific binding to its viral RNA target sequence. Thus, the phosphorylation state of Rex in the infected cell may be a switch that determines whether the virus exists in a latent or a productive state. A mutational analysis of Rex-2 that focused on serine and threonine residues was performed to identify regions or domains within Rex-2 important for function, with a specific emphasis on identifying Rex-2 phosphorylation mutants. We identified mutations near the carboxy terminus that disrupted a novel region or domain and abrogated Rex-2 function. Mutant M17 (with S151A and S153A mutations) displayed reduced phosphorylation that correlated with reduced function. Replacement of both serine residues 151 and 153 with phosphomimetic aspartic acid restored Rex-2 function and locked Rex-2 in a phosphorylated active conformation. A mutant containing threonine residues at positions 151 and 153 displayed a phenotype indistinguishable from that of wild-type Rex. Furthermore, this same mutant showed increased threonine phosphorylation and decreased serine phosphorylation, providing conclusive evidence that one or both of these residues are phosphorylated in vivo. Our results provide the first direct evidence that the phosphorylation of Rex-2 is important for function. Further understanding of HTLV Rex phosphorylation will provide insight into the regulatory control of HTLV replication and ultimately the pathobiology of HTLV.     

Efficient transactivation of the minute virus of mice P38 promoter requires upstream binding of NS1.

The P38 promoter of the autonomous parvovirus minute virus of mice is strongly transactivated by the nonstructural protein NS1, a sequence-specific DNA-binding protein. In the context of the complete viral genome, the only unique cis-acting signals required for P38 transactivation by NS1 are the proximal Sp1 site and the TATA element. In the absence of additional upstream sequences, a dependence upon the NS1 binding site within the transactivation response region is observed. Addition of synthetic NS1 binding sites to transactivation response region deletion mutants can restore the ability of NS1 to transactivate P38, and NS1 transactivation has been directly correlated to its ability to bind upstream of the P38 promoter.     

Transcriptional regulation of central carbon and energy metabolism in bacteria by redox-responsive repressor Rex

Redox-sensing repressor Rex was previously implicated in the control of anaerobic respiration in response to the cellular NADH/NAD(+) levels in gram-positive bacteria. We utilized the comparative genomics approach to infer candidate Rex-binding DNA motifs and assess the Rex regulon content in 119 genomes from 11 taxonomic groups. Both DNA-binding and NAD-sensing domains are broadly conserved in Rex orthologs identified in the phyla Firmicutes, Thermotogales, Actinobacteria, Chloroflexi, Deinococcus-Thermus, and Proteobacteria. The identified DNA-binding motifs showed significant conservation in these species, with the only exception detected in Clostridia, where the Rex motif deviates in two positions from the generalized consensus, TTGTGAANNNNTTCACAA. Comparative analysis of candidate Rex sites revealed remarkable variations in functional repertoires of candidate Rex-regulated genes in various microorganisms. Most of the reconstructed regulatory interactions are lineage specific, suggesting frequent events of gain and loss of regulator binding sites in the evolution of Rex regulons. We identified more than 50 novel Rex-regulated operons encoding functions that are essential for resumption of the NADH:NAD(+) balance. The novel functional role of Rex in the control of the central carbon metabolism and hydrogen production genes was validated by in vitro DNA binding assays using the TM0169 protein in the hydrogen-producing bacterium Thermotoga maritima.     

Identification of a motif in the C terminus of herpes simplex virus regulatory protein ICP4 that contributes to activation of transcription.

Expression of most viral genes during productive infection by herpes simplex virus is regulated by the viral protein ICP4 (also called IE175 or Vmw175). The N-terminal portion of ICP4 contains well-defined transactivation, DNA binding, and dimerization domains that contribute to promoter regulation. The C-terminal half of ICP4 contributes to the activity of ICP4, but the functional motifs have not been well mapped. To localize functional motifs in the C-terminal half of ICP4, we have compared the relative specific activities of ICP4 variants in transient-transfection assays. Deletion of the C-terminal 56 residues reduces the specific activity more than 10-fold. Mutational analysis identified three consecutive residues (1252 to 1254) that are conserved in ICP4 orthologs and are essential for full activity, especially in the context of ICP4 variants with a deletion in the N-terminal transactivation domain. Recombinant viruses that encode variants of ICP4 with mutations in the N-terminal transactivation domain and/or the extreme C terminus were constructed. The phenotypes of these recombinant viruses support the hypothesis that efficient promoter activation by ICP4 requires motifs at both the N and C termini. The data suggest that the C terminus of ICP4 functions not as an independent transactivation domain but as an enhancer of the ICP4 N-terminal transactivation domain. The data provide further support for the hypothesis that some ICP4 motifs required for promoter activation are not required for promoter repression and suggest that ICP4 utilizes different cellular factors for activation or repression of viral promoters.     

Avian retroviral RNA element promotes unspliced RNA accumulation in the cytoplasm.

All retroviruses need mechanisms for nucleocytoplasmic export of their unspliced RNA and for maintenance of this RNA in the cytoplasm, where it is either translated to produce Gag and Pol proteins or packaged into viral particles. The complex retroviruses encode Rev or Rex regulatory proteins, which interact with cis-acting viral sequences to promote cytoplasmic expression of incompletely spliced viral RNAs. Since the simple retroviruses do not encode regulatory proteins, we proposed that they might contain cis-acting sequences that could interact with cellular Rev-like proteins. To test this possibility, we initially looked for a cis-acting sequence in avian retroviruses that could substitute for Rev and the Rev response element in human immunodeficiency virus type 1 expression constructs. A cis-acting element in the 3' untranslated region of Rous sarcoma virus (RSV) RNA was found to promote Rev-independent expression of human immunodeficiency virus type 1 Gag proteins. This element was mapped between RSV nucleotides 8770 and 8925 and includes one copy of the direct repeat (DR) sequences flanking the RSV src gene; similar activity was observed for the upstream DR. To address the function of this element in RSV, both copies of the DR sequence were deleted. Subsequently, each DR sequence was inserted separately back into this deleted construct. While the viral construct lacking both DR sequences failed to replicate, constructs containing either the upstream or downstream DR replicated well. In the absence of both DRs, Gag protein levels were severely diminished and cytoplasmic levels of unspliced viral RNA were significantly reduced; replacement of either DR sequence led to normal levels of Gag protein and cytoplasmic unspliced RNA.     

Human T-cell leukemia virus (HTLV) type II Rex protein binds specifically to RNA sequences of the HTLV long terminal repeat but poorly to the human immunodeficiency virus type 1 Rev-responsive element.

The human T-cell leukemia viruses (HTLVs) encode a trans-regulatory protein, Rex, which differentially regulates viral gene expression by controlling the cytoplasmic accumulation of viral mRNAs. Because of insufficient amounts of purified protein, biochemical characterization of Rex activity has not previously been performed. Here, utilizing the baculovirus expression system, we purified HTLV type II (HTLV-II) Rex from the cytoplasmic fraction of recombinant baculovirus-infected insect cells by heparin-agarose chromatography. We directly demonstrated that Rex specifically bound HTLV-II 5' long terminal repeat RNA in both gel mobility shift and immunobinding assays. Sequences sufficient for Rex binding were localized to the R-U5 region of the HTLV-II 5' long terminal repeat and correlate with the region required for Rex function. The human immunodeficiency virus type 1 (HIV-1), has an analogous regulatory protein, Rev, which directly binds to and mediates its action through the Rev-responsive element located within the HIV-1 env gene. We demonstrated that HTLV-II Rex rescued an HIV-1JR-CSF Rev-deficient mutant, although inefficiently. This result is consistent with a weak binding activity to the HIV-1 Rev-responsive element under conditions in which it efficiently bound the HTLV-II long terminal repeat RNA.     

Phosphorylation regulates RNA binding by the human T-cell leukemia virus Rex protein.

The Rex protein of human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) regulates the expression of the viral structural genes and is critical for viral replication. Rex acts by specifically binding to RNAs containing sequences of the R region of the 5' long terminal repeat. Two forms of Rex detected in HTLV-II-infected cells, p26rex and p24rex, differ in the extent of serine phosphorylation. Two-dimensional phosphopeptide analysis indicates that p26rex is extensively phosphorylated at multiple sites. Using a sensitive immunobinding assay, we show that the phosphorylation state of Rex determines the efficiency of binding of Rex to HTLV-II target RNAs. Thus, the phosphorylation state of Rex in the infected cell may be a switch that determines whether virus exists in a latent or productive state. These studies also suggest that phosphorylation of RNA-binding regulatory proteins is a more general mechanism of gene regulation.     

Separation of primary structural components conferring autoregulation, transactivation, and DNA-binding properties to the herpes simplex virus transcriptional regulatory protein ICP4

A truncated ICP4 peptide which contains the amino-terminal 774 amino acids of the 1,298-amino-acid polypeptide is proficient for DNA binding, autoregulation, and transactivation of some viral genes (N. A. DeLuca and P. A. Schaffer, J. Virol. 62:732-743, 1988) and hence exhibits many of the properties characteristic of intact ICP4. To define the primary sequence important for the activities inherent in the amino-terminal half of the ICP4 molecule, insertional and deletion mutagenesis of the sequences encoding these residues were conducted. The DNA-binding activity of the molecule as assayed by the association with a consensus binding site was sensitive to insertional mutagenesis in two closely linked regions of the molecule. One region between amino acids 445 and 487 is critical for DNA binding and may contain a helix-turn-helix motif. The second region between amino acids 263 and 338 reduces the binding activity to a consensus binding site. When analyzed in the viral background, the DNA-binding activity of a peptide containing an insertion at amino acid 338 to a consensus binding site was reduced while the association with an alternative sequence was eliminated, suggesting a possible mechanism by which ICP4 may recognize a broader range of sequence elements. Mutations which eliminated DNA binding also eliminated or reduced both transactivation and autoregulation, supporting the requirement for DNA binding for these activities. Peptides that retained the deduced DNA-binding domain but lacked amino acids 143 through 210 retained the ability to associate with the consensus site and autoregulatory activity but were deficient for transactivation, demonstrating that the structural requirements for transactivation are greater than those required for autoregulation.     

Effects of translation initiation factor eIF-5A on the functioning of human T-cell leukemia virus type I Rex and human immunodeficiency virus Rev inhibited trans dominantly by a Rex mutant deficient in RNA binding.

The viral transactivator proteins Rex and Rev are necessary for the expression of structural proteins of human T-cell leukemia virus type I and human immunodeficiency virus type 1, respectively. Although the interaction of Rex/Rev with a cellular cofactor(s) has been thought to be required for Rex/Rev action, there is no suitable system to search for the cofactor(s) in mammalian cells. We found that a Rex mutant, TAgRex, which contains a simian virus 40 nuclear localization signal in place of the N-terminal 19 amino acids of Rex, could dominantly inhibit wild-type Rex/Rev functions. The inhibition did not require either Rev response element/Rex response element binding or the oligomerization ability of the mutant, but it did require a region around amino acid 90 of the Rex protein, suggesting that TAgRex sequestered the cellular cofactor. Complementation with the eukaryotic translation initiation factor 5A (eIF-5A) in this system could restore the impaired Rex function. These results indicate that eIF-5A is the cofactor indispensable for Rex function. Additionally, by using a two-hybrid system, the homo-oligomer formation of Rex was found to be mediated by the region around amino acid 90 in addition to Tyr-64 and Trp-65 of Rex protein. Thus, eIF-5A may play a part in the formation of the Rex homo-oligomer.     

Functional analysis of human T-cell leukemia virus type I rex-response element: direct RNA binding of Rex protein correlates with in vivo activity.

The human T-cell leukemia virus type I rex gene product plays a critical role in the expression of the retroviral structural proteins Gag and Env from incompletely spliced mRNAs. Rex protein acts through a cis element (rex-response element [RxRE]) which is located in the U3/R region of the 3' long terminal repeat and is present on all human T-cell leukemia virus type I-specific mRNAs. Two domains of the predicted secondary structure of the RxRE are crucially important for Rex action in vivo as measured by two assay systems. In vitro studies using highly purified recombinant Rex protein revealed a specific and direct interaction with radiolabeled RxRE sequences. The correlation between our in vivo results and the direct binding of Rex protein to mutant and wild-type RxRE sequences supports both the existence of the predicted secondary structure and the importance of this direct interaction with the cis-acting RNA sequence for Rex function in vivo.