Eukaryotic cells control the export of RNA from the nucleus to ensure that properly processed RNAs enter the cytoplasm for gene expression. Regulated RNA export restricts the lifecycle of the pathogen human immunodeficiency virus (HIV) because the host cell prevents export of the unspliced, viral RNA genome that encodes proteins for and packages into new virions. The viral protein Rev circumvents this inhibition by oligomerizing on an intronic RNA structure called the Rev Response Element (RRE) and by recruiting the host nuclear export adaptors Crm1 and Ran. To export the RRE, the ternary complex formed between these components must favorably interact with the nuclear pore complex (NPC) to compensate for the large size and polyanionic charge that are physical barriers for the translocation of any large ribonuceloprotein complex (RNPs) through the NPC. We do not yet clearly understand how export adaptors facilitate the transport of large cargoes, like the Rev-RRE complex, since structures of NPC substrates are limited to small cargoes with single adaptors while functional evidence suggests that multiple export receptors act in concert to transport large RNPs. Likewise, we hypothesize the Rev-RRE complex organizes multiple Crm1 adaptors to facilitate export. To investigate how the ternary HIV export complex translocates through the NPC, we propose reconstituting the quaternary complex, determining its structure by cryo-electron microscopy, and using the structure to guide biochemical and cellular assays to understand how the number and position of adaptors affects export. Together these experiments will provide the physical foundation for further interrogating how the NPC coordinates multiple stages of RNA export.

Public Health Relevance

The progression of the human immunodeficiency virus (HIV) lifecycle depends on the viral protein Rev directing the nuclear export of the viral RNA through a Crm1-dependent pathway that circumvents the retention of unspliced RNA in the nucleus. Our goal is to understand how nuclear export adaptors assemble around the RNA cargo to facilitate its transport through the nuclear pore complex by solving the structure of the ternary export complex formed between Rev, a portion of the viral RNA, and export adaptors. These results will guide future work using the HIV export complex as a substrate for investigating the coordination of RNA export by the nuclear pore complex, potentially identifying specific nuclear transport proteins functioning as host cell cofactors that can be therapeutic targets.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31GM095350-02
Application #
8231263
Study Section
Special Emphasis Panel (ZRG1-IMST-D (29))
Program Officer
Hagan, Ann A
Project Start
2011-06-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
2
Fiscal Year
2012
Total Cost
$33,283
Indirect Cost
Name
University of California San Francisco
Department
Biochemistry
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143