Functional similarity of HIV-I rev and HTLV-I rex proteins: identification of a new nucleolar-targeting signal in rev protein

We have tested the functional compatibility between rev protein of human immunodeficiency virus type I (HIV-I) and rex protein of human T-cell lymphotropic virus type I (HTLV-I). Each protein recognized the other's cis-acting sequence, albeit at reduced levels. Both proteins localize predominantly in the nucleolus. We have identified a new nucleolar-targeting signal in rev protein, which was homologous to that of rex protein. The sequence [35-RQARRNRRRRWRERQR-50] in rev protein, when fused to the amino-terminus of beta-galactosidase, directed the hybrid protein to the cell nucleolus. A deletion mutant which lacks several amino acid residues within the signal failed to function in the CAT assay system. These results demonstrate that the nucleolar targeting signals are essential for the functions of Rev and Rex.     

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.     

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.     

Different sites of interaction for Rev, Tev, and Rex proteins within the Rev-responsive element of human immunodeficiency virus type 1.

We have analyzed the action of the Rev and Tev proteins of human immunodeficiency virus type 1 (HIV-1) and of the Rex protein of human T-cell leukemia virus type I (HTLV-I) on a series of Rev-responsive element (RRE) mutants. The minimum continuous RRE region necessary and sufficient for Rev function was determined to be 204 nucleotides. Interestingly, this region was not sufficient for Tev or Rex function. These proteins require additional sequences, which may stabilize the structure of the RRE or may contain additional sequence-specific elements. Internal RRE deletions revealed that the targets for Rev and Rex can be separated, since mutants responding to Rev and not Rex and vice versa were identified. Tev was active on both types of mutants, suggesting that it has a more relaxed specificity than do both Rev and Rex proteins. Although Rev and Rex targets within the RRE appear to be distinct, the trans-dominant mutant RevBL prevents the RRE interaction with Rex. RevBL cannot inhibit the function of Rex on RRE deletions that lack the Rev-responsive portion. These results indicate the presence of distinct sites within the RRE for interaction with these proteins. The binding sites for the different proteins do not function independently and may interfere with one another. Mutations affecting the RRE may change the accessibility and binding characteristics of the different binding sites.     

Inhibition of human immunodeficiency virus type 1 Rev function by a Rev mutant which interferes with nuclear/nucleolar localization of Rev.

A nonfunctional mutant of human immunodeficiency virus type 1 Rev was created by deleting seven amino acid residues within the nucleolar targeting signal. This mutant Rev remained in the cytoplasm in expressed cells and strongly inhibited the function of Rev by interfering with the nuclear/nucleolar localization of coexpressed Rev. These findings strongly suggest the multimerization of Rev in the cytoplasm before migration to the nucleus/nucleolus, where wild-type Rev functions as a trans-regulator.     

Dominant-negative mutants are clustered in a domain of the human T-cell leukemia virus type I Rex protein: implications for trans dominance.

The 27-kDa Rex trans-acting protein appears to be essential for replication of human T-cell leukemia virus type I. Mutations introduced outside of the Rex RNA-binding domain-nucleolar localization signal display either wild-type activity or, conversely, yield dominant-negative proteins. We generated missense mutations in a particular domain of the Rex protein (amino acid residues 54 to 69) which is characterized by a cluster of dominant-negative mutants. Our results indicate that amino acids 57 to 67 are critically important for Rex function mediated through the RxRE cis-acting RNA sequence. Within this domain, only amino acids 61 to 63 could be mutated without loss of function. All other missense and deletion mutants yielded dominant-negative proteins. In vitro RNA-binding studies performed with glutathione S-transferase-Rex fusion proteins demonstrated that all of the mutant Rex proteins interacted specifically with RxRE RNA. Analysis of chimeric Rex-Rev proteins suggests that this Rex domain is important for oligomerization.     

Nuclear preservation and cytoplasmic degradation of human immunodeficiency virus type 1 Rev protein.

Rev, a major regulatory protein of human immunodeficiency virus type 1, has been demonstrated to shuttle between the nucleus and cytoplasm of infected cells. The fate of the Rev protein in living cells was evaluated by pulse-chase experiments using a transient Rev expression system. Sixteen hours after chasing with unlabelled amino acids, 45% of the labelled Rev was still present, which clearly indicates a long half-life of Rev in living cells. A Rev mutant which is deficient in the ability to migrate from the nucleus to the cytoplasm was degraded more slowly than the wild-type Rev protein. As well, another Rev mutant protein, which lacks a functional nucleolar targeting signal (NOS) and is unable to enter the cell nucleus, was rapidly degraded and undetectable 16 h after chasing. Nuclear-nucleolar targeting properties provided by a divergent NOS from a related retrovirus, which was used to substitute for the NOS of Rev, increased the intracellular half-life of this Rev mutant. Moreover, coexpression of an intracellular anti-Rev single-chain antibody (SFv), which has been shown to interfere with the nuclear translocation of Rev, accelerated the degradation of the wild-type Rev protein. Differential degradation of Rev in the nucleus and cytoplasm may play a critical role in determining and maintaining different stages of human immunodeficiency virus type 1 infection, in conjunction with the shuttling properties of the Rev protein.     

Structural and functional analysis of the human immunodeficiency virus type 2 Rev protein.

The Rev proteins of the human immunodeficiency virus (HIV) are necessary for expression of viral structural gene products. Site-directed mutations were made within the HIV-2 rev gene to identify functional domains. We observed that similar to HIV-1 Rev, the HIV-2 Rev protein was phosphorylated, albeit to a much lesser extent than was HIV-1 Rev. We also found that like HIV-1 Rev, HIV-2 Rev localized to the nucleus, with a marked accumulation in the nucleolus. Mutations within a stretch of basic residues prevented both nuclear and nucleolar localization. Furthermore, mutant Rev proteins able to localize in the nucleus but unable to localize in the nucleolus were nonfunctional.     

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.     

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.     

Binding of trans-dominant mutant Rev protein of human immunodeficiency virus type 1 to the cis-acting Rev-responsive element does not affect the fate of viral mRNA

The binding of Rev protein of human immunodeficiency virus type 1 (HIV-1) to the cis-acting Rev-responsive element (RRE) was compared to the binding of a trans-dominant Rev mutant. RevBL, which inhibits Rev function. Rev and RevBL expressed in bacteria were purified and shown to bind in vitro to the RRE with similar affinities. The study of the RRE mutants indicated that Rev and RevBL bind to the same target within the RRE in vitro and in vivo. In vivo experiments demonstrated that RevBL did not increase the steady-state levels of HIV-1 mRNA or protein. These experiments suggested that additional cellular factors interacting with Rev but not with RevBL are necessary for function. The Rex protein of human T-cell leukemia virus type I (HTLV-I) is similar to Rev and acts through a sequence named Rex-responsive element (RXRE) located in the long terminal repeat of HTLV-I. We examined the function of RevBL on a hybrid mRNA molecule containing both the RRE and RXRE. While RevBL prevented Rev function, it did not affect Rex function on the mRNA containing either the RXRE or both the RRE and RXRE. Therefore, binding of RevBL to the RRE had neither positive nor negative effects on the mRNA, since this mRNA could be efficiently utilized in the presence of a functional Rex-RXRE interaction. The results obtained in vivo and in vitro strongly suggest that RevBL inhibits Rev function by binding to the same site as Rev and preventing Rev binding and function.     

Functional mapping of the human immunodeficiency virus type 1 Rev RNA binding domain: new insights into the domain structure of Rev and Rex.

Expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the direct interaction of the viral trans-activator protein Rev with its cis-acting RNA sequence (Rev-response element [RRE]). A stretch of 14 amino acid residues of the 116-amino-acid Rev protein is sufficient to impose nucleolar localization onto a heterologous protein. Our results demonstrated that these same amino acid residues confer Rev-specific RRE binding to the heterologous human T-cell leukemia virus type I Rex protein. In addition, our results indicated that amino acids distinct from the nuclear localization signal are important for Rex-specific RRE RNA binding.     

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.