Cooperativity among Rev-Associated Nuclear Export Signals Regulates HIV-1 Gene Expression and Is a Determinant of Virus Species Tropism

Abstract

Murine cells exhibit a profound block to HIV-1 virion production that was recently mapped to a species-specific structural attribute of the murine version of the chromosomal region maintenance 1 (mCRM1) nuclear export receptor and rescued by the expression of human CRM1 (hCRM1). In human cells, the HIV-1 Rev protein recruits hCRM1 to intron-containing viral mRNAs encoding the Rev response element (RRE), thereby facilitating viral late gene expression. Here we exploited murine 3T3 fibroblasts as a gain-of-function system to study hCRM1's species-specific role in regulating Rev's effector functions. We show that Rev is rapidly exported from the nucleus by mCRM1 despite only weak contributions to HIV-1's posttranscriptional stages. Indeed, Rev preferentially accumulates in the cytoplasm of murine 3T3 cells with or without hCRM1 expression, in contrast to human HeLa cells, where Rev exhibits striking en masse transitions between the nuclear and cytoplasmic compartments. Efforts to bias Rev's trafficking either into or out of the nucleus revealed that Rev encoding a second CRM1 binding domain (Rev-2xNES) or Rev-dependent viral gag-pol mRNAs bearing tandem RREs (GP-2xRRE), rescue virus particle production in murine cells even in the absence of hCRM1. Combined, these results suggest a model wherein Rev-associated nuclear export signals cooperate to regulate the number or quality of CRM1's interactions with viral Rev/RRE ribonucleoprotein complexes in the nucleus. This mechanism regulates CRM1-dependent viral gene expression and is a determinant of HIV-1's capacity to produce virions in nonhuman cell types. IMPORTANCECells derived from mice and other nonhuman species exhibit profound blocks to HIV-1 replication. Here we elucidate a block to HIV-1 gene expression attributable to the murine version of the CRM1 (mCRM1) nuclear export receptor. In human cells, hCRM1 regulates the nuclear export of viral intron-containing mRNAs through the activity of the viral Rev adapter protein that forms a multimeric complex on these mRNAs prior to recruiting hCRM1. We demonstrate that Rev-dependent gene expression is poor in murine cells despite the finding that, surprisingly, the bulk of Rev interacts efficiently with mCRM1 and is rapidly exported from the nucleus. Instead, we map the mCRM1 defect to the apparent inability of this factor to engage Rev multimers in the context of large viral Rev/RNA ribonucleoprotein complexes. These findings shed new light on HIV-1 gene regulation and could inform the development of novel antiviral strategies that target viral gene expression. T he nuclear pore complex (NPC) represents a key barrier to the replication of many viruses that infect eukaryotic cells (1-3). For retroviruses such as the primate lentivirus human immunodeficiency virus type 1 (HIV-1), the late, productive phases of the viral life cycle require efficient nuclear export of unspliced, and thus intron-containing, viral genomic RNAs (gRNAs) that (i) encode the group-specific antigen (Gag) and Gag-Pol polyproteins that form ...

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

“…1A) in trans with plasmids encoding fulllength Rev-minus infectious HIV-1 yellow fluorescent protein (YFP)-encoding reporter viruses (NL4-3/E-R-Rev-/YFP) and vesicular stomatitis virus G glycoprotein (VSV-G) for pseudotyping. As previously described (68), the expression of Rev-mChe or Rev-mChe-NES variants yielded similar levels of infectious virus production from human cells (Fig. 1B, compare lanes 3 and 6).…”

“…6A and B). Disruption to either nuclear import or export was measured by net per cell changes to bulk S-YFP levels in the nucleus versus the cytoplasm using a computational cell-segmentation strategy (68,76). The data are presented as a ratio of nuclear to cytoplasmic fluorescence (N/C ratio) normalized to the wild-type Rev-mChe condition (Fig.…”

“…Whether Rev's transient or long-term association with the nucleolus has any functional relevance remains undefined (22,68,97,98). However, that Rev-mChe bearing a C-terminal NES localizes poorly to the nucleolus and is less active than wild-type Rev at low levels of expression (Fig.…”

“…We previously showed that appending a second NES peptide to HIV-1 Rev or doubling the number of RRE sequences per viral RNA transcript was sufficient to overcome a profound block to HIV-1 in murine cells, attributable to a species-specific feature of the murine CRM1 ortholog (mCRM1) (68). This, combined with additional genetic studies from Hoffmann et al (69) and recent structural work by Booth et al, suggests that multiple NES domains are critical for export of the Rev/RRE complex and needed to recruit at least two molecules of CRM1 (70).…”

“…Alternatively, proteins such as Rev form oligomeric RNP complexes that present multiples of identical NES peptides to CRM1 or a CRM1 multimer (22,(62)(63)(64)(65)(66)(67). Recent structure-function studies from us and others (68,69), as well as elegant structural work from Frankel and coworkers (27,70), strongly suggest that Rev multimerization (consisting of 6 to 14 monomers on the RRE) is needed to form a multi-NES complex capable of recruiting at least two molecules of CRM1.…”