Recruitment of the Crm1 Nuclear Export Factor Is Sufficient To Induce Cytoplasmic Expression of Incompletely Spliced Human Immunodeficiency Virus mRNAs

Abstract

Cytoplasmic expression of the incompletely spliced RNA transcripts that encode the late, structural proteins of human immunodeficiency virus type 1 (HIV-1) is dependent on the viral Rev regulatory protein. General agreement exists that Rev acts, at least in part, by recruiting the cellular Crm1 nuclear export factor to HIV-1 transcripts bearing the Rev response element RNA target, and thereby inducing their nuclear egress. However, several groups have argued that Crm1 recruitment may not be sufficient for Rev function. Thus, several additional candidate cofactors for Rev have been proposed, and Rev has also been suggested to also inhibit the nuclear splicing of HIV-1 transcripts and/or to directly enhance their cytoplasmic translation. To examine whether Crm1 recruitment is, instead, sufficient to activate the nuclear export of viral mRNAs, we targeted a leucine-rich Crm1 binding domain, derived from a heterologous protein that normally plays no role in RNA metabolism, to HIV-1 RNAs and showed that this tethered Crm1 binding domain is sufficient to induce the nuclear export and cytoplasmic translation of late HIV-1 mRNA species. More importantly, we show that direct tethering of the Crm1 nuclear export factor to target mRNAs, by fusion to a heterologous RNA binding domain, is in and of itself sufficient to induce the nuclear export and cytoplasmic expression of the unspliced HIV-1 mRNAs that encode the viral Gag proteins.Retroviral replication requires the cytoplasmic expression of both fully spliced and incompletely spliced forms of the initial, genome-length viral RNA transcript (reviewed in references 7 and 37). However, cells have evolved mechanisms to prevent the nuclear export of incompletely spliced cellular mRNAs, i.e., pre-mRNAs (4, 23). Retroviruses have therefore had to develop mechanisms that allow intron-containing viral transcripts to exit the nucleus in the face of this cellular proofreading mechanism. In several simple retroviruses, nuclear export of the genome-length transcript is mediated by cellular factors that are recruited to a cis-acting RNA target termed a constitutive transport element (CTE) (3,36,49). The cellular protein specific for the CTE found in simian type D viruses has been identified as Tap, a nuclear export factor that also plays a key role in mediating cellular mRNA export (7,15,19,43).In contrast to simple retroviruses, complex retroviruses encode a regulatory protein that is critical for the cytoplasmic expression of their incompletely spliced transcripts. In human immunodeficiency virus type 1 (HIV-1), this protein is termed Rev. Rev interacts with a cis-acting viral RNA target site, the Rev response element (RRE), and with Crm1, a host cell protein that is a member of the karyopherin or importin/exportin family of nucleocytoplasmic transport factors (12,14,29,31,35,42,50). Crm1 binds specifically to a short leucinerich motif found in the Rev protein that also functions as a nuclear export signal (NES) (2,11,30,48). NES binding by Crm1 requires a cellular cofactor, the GTP...

Inhibition of HIV-1 Replication by Cell-penetrating Peptides Binding Rev

“…Rev is also known to bind nucleoporins (29 -32), and recently, it has been shown that a kinesin-like protein binds to the NES of Rev and stimulates the activity of the protein (33). Although several cofactors are likely to intervene in the RNA export process mediated by Rev, hCRM1 and Ran have been shown to be sufficient to induce translocation to the cytoplasm of incompletely spliced HIV mRNAs (34,35).…”

“…Rev is also known to bind nucleoporins (29 -32), and recently, it has been shown that a kinesin-like protein binds to the NES of Rev and stimulates the activity of the protein (33). Although several cofactors are likely to intervene in the RNA export process mediated by Rev, hCRM1 and Ran have been shown to be sufficient to induce translocation to the cytoplasm of incompletely spliced HIV mRNAs (34,35).By considering that both Tat and Rev are small proteins that act through protein-protein interactions, we reasoned that it should be feasible to inhibit their function, which is absolutely necessary to viral replication, with peptide ligands competitively interfering with the associations in which they are engaged. Because it has the interest of reporting the interaction…”

HIV-1 Nef-associated Factor 1 Enhances Viral Production by Interacting with CRM1 to Promote Nuclear Export of Unspliced HIV-1 gag mRNA

“…The leucine-rich NES in bearing proteins, like HIV-1 Rev, mediates CRM1 recruitment for nuclear export (14,47,50). Based on the analysis of amino acid sequence, Naf1 was predicted to contain four putative NESs that interact with CRM1 (51).…”

“…These incompletely spliced HIV-1 RNAs contain a Rev response element (RRE), and the coordinate assembly of multiple Rev molecules on RRE recruits human protein complex containing CRM1 and Ran-GTP (GTP-binding nuclear protein Ran). Rev contains a leucine-rich nuclear export signal (NES) domain located near the carboxyl terminus, through which Rev interacts with CRM1, and the Rev-RRE-CRM1/Ran-GTP complex is then transported to the cytoplasm (6,(11)(12)(13)(14)(15)(16). Similarly, other retroviruses such as mouse mammary tumor virus and human T cell lymphotropic virus encode Rev-like regulatory proteins Rem or Rex, respectively, for hijacking the host CRM1 pathway to accomplish nuclear export of unspliced viral transcripts (17)(18)(19)(20)(21)(22).…”

hRIP, a cellular cofactor for Rev function, promotes release of HIV RNAs from the perinuclear region

“…Alternatively, cells were cotransfected with pgTAT and pCMV⌬CAN, a plasmid that expresses a truncated form of the nucleoporin CAN/Nup214 (⌬CAN). ⌬CAN blocks the ability of CRM1 to interact with the nuclear pore complex (NPC) and inhibits the nuclear export of Rev (Fornerod et al 1997;Bogerd et al 1998;Yi et al 2002). The intracellular distribution of tat RNA was visualized using a fluorochrome-labeled oligonucleotide probe complementary to stem loop IIB of the HIV-1 RRE.…”