Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor eIF-5A.

Eukaryotic initiation factor 5A(eIF-5A) is a cellular cofactor require d for the function of the human immunodeficiency virus type-1 (HIV-1) Rev trans-activator protein. The majority of a set of eIF-5A mutants did not support growth of yeast cells having an inactivated genomic copy of eIF-5A, indicating that the introduced mutation eliminated eIF-5A activity. Two nonfunctional mutants, eIF-5AM13 and eIF-5AM14, retained their binding capacity for the HIV-1 Rev response element:Rev complex. Both mutants were constitutively expressed in human T cells. When these T cells were infected with replication-competent HIV-1, virus replication was inhibited. The eIF-5AM13 and eIF5AM14 proteins blocked Rev trans-activation and Rev-mediated nuclear export.

The polypeptide chain of eukaryotic initiation factor 5A occurs in two distinct conformations in the absence of the hypusine modification.

Eukaryotic initiation factor 5A (eIF-5A) requires posttranslational modification of lysine at position 50 to hypusine for its biological activity. We have expressed an unmodified variant of eIF-5A in Escherichia coli and show that it has structural properties different from those of the native protein in terms of its near- and far-UV circular dichroism spectra and its equilibrium unfolding transition with guanidinium chloride. In contrast to the hypusine-modified protein, which unfolds in a two-state process, the complex unfolding transition of unmodified eIF-5A suggests that this variant occurs in two differently folded conformations, F1 and F2. Both conformations are populated under near-physiological conditions at a ration of 60 to 40, respectively. Equilibrium unfolding consists of parallel events: unfolding of F1 to one or several intermediate states (I), and unfolding of F2 to the unfolded state (U). Although the establishment of each of these individual equilibria is fast, the interconversion is slow at guanidinium chloride concentrations between 0 M and 3 M. Kinetic analysis reveals activation energies of 24.3 kcal mol-1 for the reaction of F1 and F2 and 24.1 kcal mol-1 for the reaction of F2 to F1. Both F1 and F2 possess well-defined secondary and tertiary structure. However, the tertiary structures of the two conformations differ as indicated by their distinct near-UV circular dichroism spectra. These differences may be restricted to the C-terminal part of the protein as 2-dimensional 1H-NMR spectra of unmodified eIF-5A reveal no doubled set of proton resonances for aromatic amino acid and histidine residues, of which almost all are located in the N-terminal region.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a new member of the human eIF-5A gene family.

Using an oligodeoxynucleotide generated by rapid PCR amplification of 5'-cDNA ends (5'-RACE) as a detection probe, we have isolated a new genomic clone encoding the human eukaryotic initiation factor 5A (eIF-5A). Sequence analysis revealed that the eIF-5A coding region is identical to the corresponding cDNA but interrupted by three introns. In a plasmid shuffle experiment we show functional replacement of the essential homologous gene in Saccharomyces cerevisiae by this human eIF-5A.

Activation of JAK3, but not JAK1, is critical to interleukin-4 (IL4) stimulated proliferation and requires a membrane-proximal region of IL4 receptor alpha.

The tyrosine kinases JAK1 and JAK3 have been shown to undergo tyrosine phosphorylation in response to interleukin-2 (IL), IL4, IL7, and IL9, cytokines which share the common IL2 receptor gamma-chain (IL2R gamma), and evidence has been found for a preferential coupling of JAK3 to IL2R gamma and JAK1 to IL2R beta. Here we show, using human premyeloid TF-1 cells, that IL4 stimulates JAK3 to a larger extent than JAK1, based upon three different evaluation criteria. These include a more vigorous tyrosine phosphorylation of JAK3 as measured by anti-phosphotyrosine immunoblotting, a more marked activation of JAK3 as determined by in vitro tyrosine kinase assays and a more manifest presence of JAK3 in activated IL4-receptor complexes. These observations suggest that IL4 receptor signal transduction does not depend on equimolar heterodimerization of JAK1 and JAK3 following IL4-induced heterodimerization of IL4R alpha and IL2R gamma. Indeed, when human IL4R alpha was stably expressed in mouse BA/F3 cells, robust IL4-induced proliferation and JAK3 activation occurred without detectable involvement of JAK1, JAK2, or TYK2. The present study suggests that JAK1 plays a subordinate role in IL4 receptor signaling, and that in certain cells exclusive JAK3 activation may mediate IL4-induced cell growth. Moreover, mutational analysis of human IL4R alpha showed that a membrane-proximal cytoplasmic region was critical for JAK3 activation, while the I4R motif was not, which is compatible with a role of JAK3 upstream of the recruitment of the insulin receptor substrate-1/4PS signaling proteins by IL4 receptors.

Distinct sequence motifs within the cytoplasmic domain of the human IL-4 receptor differentially regulate apoptosis inhibition and cell growth.

Hematopoietin receptors generally function as multimeric complexes composed of a unique ligand-binding chain and a second component often shared between several members of this receptor family. To better understand the signal transduction of the human IL-4 receptor (hIL4R), we analyzed the functionality of targeted mutations in two cytoplasmic regions of the ligand-binding hIL4R chain that we previously identified to be necessary for growth mediation in factor-dependent murine Ba/F3 cells. Here, we provide evidence that transient inhibition of apoptotic death of Ba/F3 cells and the competence to proliferate indefinitely depend on separated and distinct sequence motifs of the hIL4R. In particular, hIL4R constructs with a truncation of the recently described gp130 box1 from P242 to K264, or a deletion of the acidic region between S330 and S365, fail to stimulate growth or to mediate the inhibition of apoptosis. hIL4R bearing a point mutation within the gp130 box1 (P242S) is defective for growth stimulation but still signals the transient inhibition of apoptotic cell death and the induction of c-myc RNA. A third region required for IL4-mediated cell growth is localized between T462 and S476 and includes the sequence NPAY previously described to serve as interaction motif in signaling of epidermal growth factor and insulin receptors. Conversion of Y472 into F472 within the latter hIL4R motif affects the competence of stably transfected BA/F3 cells to proliferate indefinitely in the presence of hIL4. Sequences C-terminal of S476 are not essential for growth stimulation of BA/F3 transfectants.

The eukaryotic cofactor for the human immunodeficiency virus type 1 (HIV-1) rev protein, eIF-5A, maps to chromosome 17p12-p13: three eIF-5A pseudogenes map to 10q23.3, 17q25, and 19q13.2.

The eukaryotic initiation factor 5A (eIF-5A) has been identified as an essential cofactor for the HIV-1 transactivator protein Rev. Rev plays a key role in the complex regulation of HIV-1 gene expression and thereby in the generation of infectious virus particles. Expression of eIF-5A is vital for Rev function, and inhibition of this interaction leads to a block of the viral replication cycle. In humans, four different eIF-5A genes have been identified. One codes for the eIF-5A protein and the other three are pseudogenes. Using a panel of somatic rodent-human cell hybrids in combination with fluorescence in situ hybridization analysis, we show that the four genes map to three different chromosomes. The coding eIF-5A gene (EIF5A) maps to 17p12-p13, and the three pseudogenes EIF5AP1, EIF5AP2, and EIF5AP3 map to 10q23.3, 17q25, and 19q13.2, respectively. This is the first localization report for a eukaryotic cofactor for a regulatory HIV-1 protein.

The genomic structure encoding human initiation factor eIF-5A.

A genomic clone encoding the human eukaryotic initiation factor 5A (eIF-5A) was isolated and its entire nucleotide sequence was determined. The whole eIF-5A coding region is not interrupted by introns. The functional eIF-5A gene is highly homologous to the corresponding complementary DNA. One single 1.4-kb transcript thereof is expressed in human cell lines. Furthermore, we also isolated and sequenced two additional eIF-5A-related sequences which are, by expression and sequence analyses, identified as pseudogenes of the functional eIF-5A. The sequence homology between these pseudogenes and the functional eIF-5A is 71 and 87%.

Human interleukin-4 receptor signaling requires sequences contained within two cytoplasmic regions.

The signaling pathway used by the murine or human interleukin-4 receptor (hIL-4R) has not been elucidated so far. As an approach to mapping the cytoplasmic regions of the hIL-4R that are essential for mediating the biological response upon IL-4 binding we have cloned and expressed the wild-type hIL-4R and several cytoplasmic deletion mutants in the murine IL-3-dependent pro-B cell line BA/F3. Transfection of the wild-type hIL-4R conferred the ability on BA/F3 cells to proliferate in a dose-dependent way when treated with human IL-4. The analysis of six deletion mutants indicated that the signaling function of the hIL-4R depends on sequences within two discontinuous regions that are located between amino acid IIe233 and Ser365 and Thr462 and Ala580 of the intracytoplasmic domain. The deletion of either of these regions totally abrogated IL-4-inducible growth. The relevance of our data is discussed in relation to the results obtained from similar studies with other members of the hematopoietin receptor superfamily.