The export receptor Crm1 forms a dimer to promote nuclear export of HIV RNA.

David S Booth,Alan D Frankel,Yifan Cheng

doi:10.7554/elife.04121

David S Booth, Alan D Frankel + Show 1 more

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https://doi.org/10.7554/elife.04121

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Journal: eLife	Publication Date: Dec 8, 2014
Citations: 109	License type: CC BY 4.0

Affiliation: University of California, San Francisco

Abstract

The HIV Rev protein routes viral RNAs containing the Rev Response Element (RRE) through the Crm1 nuclear export pathway to the cytoplasm where viral proteins are expressed and genomic RNA is delivered to assembling virions. The RRE assembles a Rev oligomer that displays nuclear export sequences (NESs) for recognition by the Crm1-Ran(GTP) nuclear receptor complex. Here we provide the first view of an assembled HIV-host nuclear export complex using single-particle electron microscopy. Unexpectedly, Crm1 forms a dimer with an extensive interface that enhances association with Rev-RRE and poises NES binding sites to interact with a Rev oligomer. The interface between Crm1 monomers explains differences between Crm1 orthologs that alter nuclear export and determine cellular tropism for viral replication. The arrangement of the export complex identifies a novel binding surface to possibly target an HIV inhibitor and may point to a broader role for Crm1 dimerization in regulating host gene expression.

Highlights

Human immunodeficiency virus (HIV) replication depends on the coordinated expression of viral proteins from fully spliced, partially spliced, and unspliced forms of its RNA genome
In order to define the arrangement of HIV RNA export complexes for structural studies, we first determined the biochemical requirements for complex formation using recombinant Crm1, Ran, Rev tagged at the amino-terminus with a GB1 domain (GB1-Rev), and a 245 nt portion of Rev Response Element (RRE) RNA transcribed in vitro
The interaction between Crm1 and Rev requires a functional nuclear export sequences (NESs), as GB1-Rev bearing a dominant-negative M10 mutation in the NES, Leu78Asp and Glu79Leu (Malim et al, 1989), did not bind Crm1 under any condition. These results mirror in vivo and in vitro experiments showing that Rev uses an intact NES to bridge the interaction between the RRE and Crm1 utilizing Ran bound to GTP (RanGTP) (Fischer et al, 1995; Fornerod et al, 1997; Askjaer et al, 1998)

Summary

Introduction

Human immunodeficiency virus (HIV) replication depends on the coordinated expression of viral proteins from fully spliced, partially spliced, and unspliced forms of its RNA genome. Fully spliced RNAs encoding viral regulatory proteins initially reach the cytoplasm for translation (Feinberg et al, 1986). Later in the life cycle, the regulatory protein Rev directs the nuclear export of unspliced and partially spliced viral RNAs by engaging the Crm nuclear export pathway typically used to transport host protein cargoes and small RNAs (Sodroski et al, 1986; Emerman et al, 1989; Felber et al, 1989; Kohler and Hurt, 2007; McCloskey et al, 2012). The Rev nuclear export sequence (NES), located near the carboxy terminus, recruits Crm in cooperation with Ran bound to GTP (RanGTP) to elicit nuclear export of viral RNAs (Fischer et al, 1995)

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