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.     

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.     

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.     

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.     

trans-dominant inhibition of human immunodeficiency virus type 1 Rev occurs through formation of inactive protein complexes.

The human immunodeficiency virus type 1 Rev protein controls expression of certain viral RNAs by binding to these RNAs in the nucleus. To investigate how dominant negative Rev mutants inhibit Rev function, we fused such mutants to hormone-dependent localization signals from the glucocorticoid receptor. Each was found to have fully potent inhibitory activity whether expressed in the nucleus or in the cytoplasm. Wild-type Rev colocalized with an inhibitory fusion protein, implying that the two proteins interact. The resulting complexes accumulated within nuclei in response to steroids but had no effect on expression of Rev-responsive mRNAs. A mutation known to block in vitro oligomerization of Rev abolished both complex formation and inhibitory activity of the mutant fusion proteins. Thus, trans-dominant inhibition of Rev does not require competition for nuclear substrates but may instead reflect the ability of a mutant to form nonfunctional complexes with the wild-type protein in vivo.     

Exchange of the basic domain of human immunodeficiency virus type 1 Rev for a polyarginine stretch expands the RNA binding specificity, and a minimal arginine cluster is required for optimal RRE RNA binding affinity, nuclear accumulation, and trans-activation

The Rev regulatory protein of human immunodeficiency virus (HIV) facilitates the nuclear export of unspliced and partially spliced HIV RNAs. Using a Rev:MS2 phage coat protein fusion that could be targeted to bind and activate the Rev-responsive element (RRE) RNA or heterologous MS2 phage operator RNA, we analyzed the role(s) of the arginine-rich RNA binding domain in RNA binding and transactivation. The arginine-rich domain could be functionally replaced by a stretch of nine arginines. However, polyarginine substitutions expanded the RNA binding specificity of the resultant mutant Rev protein. Polyarginine insertions in place of residues 24 to 60 that excised the RNA binding and oligomerization domains of Rev preserved the activation for MS2 RNA, but not for the RRE. A nine-arginine insertion outside of the natural context of the Rev nuclear localization signal domain was incompatible with activation of either RNA target. Insertions of fewer than eight arginines impaired RRE activation. Interrupted lysine clusters and disruption of the arginine stretch with lysine or neutral residues resulted in a similar phenotype. Some of these mutants with a null phenotype for RRE activated the heterologous MS2 RNA target. Under steady-state conditions, mutants that preserved the Rev response for RRE RNA localized to the nuclei; those with poor or no Rev response accumulated mostly in the cytoplasm. Many of the cytoplasmically resident derivatives became nuclear when leptomycin B (LMB) treatment inhibited nuclear export of nuclear export signal-containing proteins. Mutants that had a null activation potential for either RNA target were particularly resistant to LMB treatment. Abbreviated nuclear residence times and differences in RRE binding affinity may have compromised their activation potential for RRE. High-affinity binding to MS2 RNA through the intact coat protein was sufficient to overcome the short nuclear residence times and to facilitate MS2 activation by some derivatives.     

Comprehensive Mutational Analysis of the Moloney Murine Leukemia Virus Envelope Protein

The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the env gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues. The mutants with residual infectivity had insertions either in the amino-terminal signal sequence region, two disulfide-bonded loops in the receptor binding domain, discrete regions of the carboxy-terminal region of the surface subunit (SU), or the cytoplasmic tail. Insertions that allowed the mutants to reach the cell surface but not to mediate detectable infection were located within the amino-terminal sequence of the mature Env, within the SU carboxy-terminal region, near putative receptor binding residues, and throughout the fusion peptide. Independent analysis of select mutants in this group allowed more precise identification of the defect in Env function. Mapping of mutant phenotypes to a structural model of the receptor-binding domain provides insights into the protein's functional organization. The high-resolution functional map reported here will be valuable for the engineering of the Env protein for a variety of uses, including gene therapy.     

Genetic evidence for multiple nuclear functions of the herpes simplex virus ICP8 DNA-binding protein

We have isolated several mutant herpes simplex viruses, specifically mutated in the infected cell protein 8 (ICP8) gene, to define the functional domains of ICP8, the major viral DNA-binding protein. To facilitate the isolation of these mutants, we first isolated a mutant virus, HD-2, with the lacZ gene fused to the ICP8 gene so that an ICP8-beta-galactosidase fusion protein was expressed. This virus formed blue plaques on ICP8-expressing cell lines in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Mutated ICP8 gene plasmids cotransfected with HD-2 DNA yielded recombinant viruses with the mutant ICP8 gene incorporated into the viral genome. These recombinants were identified by formation of white plaques. Four classes of mutants were defined: (i) some expressed ICP8 that could bind to DNA but could not localize to the cell nucleus; (ii) some expressed ICP8 that did not bind to DNA but localized to the nucleus; (iii) some expressed ICP8 that neither bound to DNA nor localized to the nucleus; and (iv) one expressed ICP8 that localized to the cell nucleus and bound to DNA in vitro, but the mutant virus did not replicate its DNA. These classes of mutants provide genetic evidence that DNA binding and nuclear localization are distinct functions of ICP8 and that ICP8 has nuclear functions other than binding to DNA. Furthermore, the portion of ICP8 needed for a nuclear function(s) distinct from DNA binding is the part of ICP8 showing sequence similarity to that of the cellular protein cyclin or proliferating cell nuclear antigen.     

CRM1, an RNA transporter, is a major species-specific restriction factor of human T cell leukemia virus type 1 (HTLV-1) in rat cells

Rat ortholog of human CRM1 has been found to be responsible for the poor activity of viral Rex protein, which is essential for RNA export of human T cell leukemia virus type 1 (HTLV-1). Here, we examined the species-specific barrier of HTLV-1 by establishing rat cell lines, including both adherent and CD4(+) T cells, which express human CRM1 at physiological levels. We demonstrated that expression of human CRM1 in rat cells is not harmful to cell growth and is sufficient to restore the synthesis of the viral structural proteins, Gag and Env, at levels similar to those in human cells. Gag precursor proteins were efficiently processed to the mature forms in rat cells and released into the culture medium as sedimentable viral particles. An HTLV-1 pseudovirus infection system suggested that the released virus particles are fully infectious. Our newly developed reporter cell system revealed that Env proteins produced in rat cells are fully fusogenic, which is the basis for cell-cell HTLV-1 infection. Moreover, we show that the early steps in infection, from post-entry uncoating to integration into the host chromosomes, occur efficiently in rat cells. These results, in conjunction with reports describing efficient entry of HTLV-1 into rat cells, may indicate that HTLV-1 is unique in that its major species-specific barrier is determined by CRM1 at a viral RNA export step. These observations will enable us to construct a transgenic rat model expressing human CRM1 that is sensitive to HTLV-1 infection.     

Intersubunit disulfide isomerization controls membrane fusion of human T-cell leukemia virus Env

Human T-cell leukemia virus (HTLV-1) Env carries a typical disulfide isomerization motif, C(225)XXC, in the C-terminal domain SU. Here we have tested whether this motif is used for isomerization of the intersubunit disulfide of Env and whether this rearrangement is required for membrane fusion. We introduced the C225A and C228A mutations into Env and found that the former but not the latter mutant matured into covalently linked SU-TM complexes in transfected cells. Next, we constructed a secreted Env ectodomain and showed that it underwent incubation-dependent intersubunit disulfide isomerization on target cells. However, the rearrangement was blocked by the C225A mutation, suggesting that C(225) carried the isomerization-active thiol. Still, it was possible to reduce the intersubunit disulfide of the native C225A ectodomain mutant with dithiothreitol (DTT). The importance of the CXXC-mediated disulfide isomerization for infection was studied using murine leukemia virus vectors pseudotyped with wild-type or C225A HTLV-1 Env. We found that the mutant Env blocked infection, but this could be rescued with DTT. The fusion activity was tested in a fusion-from-within assay using a coculture of rat XC target and transfected BHK-21 effector cells. We found that the mutation blocked polykaryon formation, but this could be reversed with DTT. Similar DTT-reversible inhibition of infection and fusion was observed when a membrane-impermeable alkylator was present during the infection/fusion incubation. We conclude that the fusion activity of HTLV-1 Env is controlled by an SU CXXC-mediated isomerization of the intersubunit disulfide. Thus, this extends the applicability of the isomerization model from gammaretroviruses to deltaretroviruses.