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.     

The Rex proteins of human T-cell leukemia virus type II differ by serine phosphorylation.

The Rex proteins of human T-cell leukemia virus types I and II (HTLV-I and HTLV-II) induce cytoplasmic expression of unspliced gag-pol mRNA and singly spliced env mRNA and are critical for virus replication. Two rex gene products, p27rex and p21rex of HTLV-I and p26rex and p24rex of HTLV-II, have been detected in HTLV-infected cells; however, the structural and biological relationship of the proteins has not been clearly elucidated. Endoproteinase digestion and phosphoamino acid analysis of HTLV-II Rex indicated that p24rex has the same amino acid backbone as p26rex and that the larger apparent molecular size of p26rex is attributable to serine phosphorylation.     

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.     

The open reading frame I (ORF I)/ORF II part of the human T-cell leukemia virus type I X region is dispensable for p40tax, p27rex, or envelope expression.

The X region of the human T-cell leukemia virus type I contains the second coding exon of the tax and rex regulatory proteins (open reading frame IV [ORF IV] and ORF III, respectively), as well as coding regions for more recently described proteins, p30II (or the tof protein) and p13II in ORF II and the putative rof protein and p12I in ORF I. Deletions and transcomplementation experiments showed that expression of the envelope, as well as that of the tax and rex proteins, was independent of the proteins encoded in the ORF I/ORF II region. Furthermore, p30II and p12I proteins could not replace the rex protein in a rex-dependent envelope or Gag protein expression system.     

Transcomplementation of simian immunodeficiency virus Rev with human T-cell leukemia virus type I Rex.

A molecular clone of the simian immunodeficiency virus SIVSMM isolate PBj14, lacking the ATG initiation codon for Rev protein (PBj-1.5), did not produce virus or large unspliced or singly spliced viral RNA upon transfection of HeLa cells. Low but significant levels of virus and large viral RNA production were observed upon transfection of PBj-1.5 into HeLa Rev cells expressing the rev gene of human immunodeficiency virus type 1. Furthermore, abundant virus and large viral RNA production occurred upon transfection of PBj-1.5 into HeLa Rex cells expressing the rex gene of human T-cell leukemia virus type I. Virus produced from HeLa Rex and HeLa Rev transfections was infectious, produced large amounts of virus, and was cytopathic for Rex-producing MT-4 cells. In contrast, no or only low levels of virus production were observed upon infection of H9 cells. These studies show that a defective SIV rev gene can be transcomplemented with human immunodeficiency virus type 1 Rev and with high efficiency by human T-cell leukemia virus type I Rex, and they suggest that rev-defective viruses could serve as a source for production of a live attenuated SIV vaccine.     

Biochemical and virological analysis of the 18-residue C-terminal tail of HIV-1 integrase

Background

Mouse mammary tumor virus (MMTV) encodes the Rem protein, an HIV Rev-like protein that enhances nuclear export of unspliced viral RNA in rodent cells. We have shown that Rem is expressed from a doubly spliced RNA, typical of complex retroviruses. Several recent reports indicate that MMTV can infect human cells, suggesting that MMTV might interact with human retroviruses, such as human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), and human endogenous retrovirus type K (HERV-K). In this report, we test whether the export/regulatory proteins of human complex retroviruses will increase expression from vectors containing the Rem-responsive element (RmRE).

Results

MMTV Rem, HIV Rev, and HTLV Rex proteins, but not HERV-K Rec, enhanced expression from an MMTV-based reporter plasmid in human T cells, and this activity was dependent on the RmRE. No RmRE-dependent reporter gene expression was detectable using Rev, Rex, or Rec in HC11 mouse mammary cells. Cell fractionation and RNA quantitation experiments suggested that the regulatory proteins did not affect RNA stability or nuclear export in the MMTV reporter system. Rem had no demonstrable activity on export elements from HIV, HTLV, or HERV-K. Similar to the Rem-specific activity in rodent cells, the RmRE-dependent functions of Rem, Rev, or Rex in human cells were inhibited by a dominant-negative truncated nucleoporin that acts in the Crm1 pathway of RNA and protein export.

Conclusion

These data argue that many retroviral regulatory proteins recognize similar complex RNA structures, which may depend on the presence of cell-type specific proteins. Retroviral protein activity on the RmRE appears to affect a post-export function of the reporter RNA. Our results provide additional evidence that MMTV is a complex retrovirus with the potential for viral interactions in human cells.     

Posttranscriptional regulation by the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins through a heterologous RNA binding site.

The human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins induce cytoplasmic expression of incompletely spliced viral mRNAs by binding to these mRNAs in the nucleus. Each protein binds a specific cis-acting element in its target RNAs. Both proteins also associated with nucleoli, but the significance of this association is uncertain because mutations that inactivate nucleolar localization signals in Rev or Rex also prevent RNA binding. Here we demonstrate that Rev and Rex can function when tethered to a heterologous RNA binding site by a bacteriophage protein. Under these conditions, cytoplasmic accumulation of unspliced RNA occurs without the viral response elements, mutations in the RNA binding domain of Rev do not inhibit function, and nucleolar localization can be shown to be unnecessary for the biological response.     

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.