Interaction of Transportin-SR2 with Ras-related Nuclear Protein (Ran) GTPase

Oliver Taltynov,Jonas Demeulemeester,Frauke Christ,Stéphanie De Houwer,Vicky G Tsirkone,Melanie Gerard,Stephen D Weeks,Sergei V Strelkov,Zeger Debyser

doi:10.1074/jbc.m113.484345

Oliver Taltynov, Jonas Demeulemeester + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m113.484345

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Abstract

The human immunodeficiency virus type 1 (HIV-1) and other lentiviruses are capable of infecting non-dividing cells and, therefore, need to be imported into the nucleus before integration into the host cell chromatin. Transportin-SR2 (TRN-SR2, Transportin-3, TNPO3) is a cellular karyopherin implicated in nuclear import of HIV-1. A model in which TRN-SR2 imports the viral preintegration complex into the nucleus is supported by direct interaction between TRN-SR2 and HIV-1 integrase (IN). Residues in the C-terminal domain of HIV-1 IN that mediate binding to TRN-SR2 were recently delineated. As for most nuclear import cargoes, the driving force behind HIV-1 preintegration complex import is likely a gradient of the GDP- and GTP-bound forms of Ran, a small GTPase. In this study we offer biochemical and structural characterization of the interaction between TRN-SR2 and Ran. By size exclusion chromatography we demonstrate stable complex formation of TRN-SR2 and RanGTP in solution. Consistent with the behavior of normal nuclear import cargoes, HIV-1 IN is released from the complex with TRN-SR2 by RanGTP. Although in concentrated solutions TRN-SR2 by itself was predominantly present as a dimer, the TRN-SR2-RanGTP complex was significantly more compact. Further analysis supported a model wherein one monomer of TRN-SR2 is bound to one monomer of RanGTP. Finally, we present a homology model of the TRN-SR2-RanGTP complex that is in excellent agreement with the experimental small angle x-ray scattering data.

Highlights

Transportin-SR2 (TRN-SR2) is a karyopherin implicated in nuclear import of the human immunodeficiency virus type 1 (HIV-1) preintegration complex
Functionality of the TRN-SR2-RanGTP Interaction—We first studied the direct interaction between TRN-SR2 and Ran loaded with either GDP or GTP (RanGDP or RanGTP, respectively)
According to one of the hypotheses, HIV nuclear import is mediated by the direct interaction between TRN-SR2 and HIV integrase present in the preintegration complex (PIC) [14]

Summary

Background

Transportin-SR2 (TRN-SR2) is a karyopherin implicated in nuclear import of the HIV-1 preintegration complex. Because CPSF6 binds TRN-SR2 and interacts with CA through its N-terminal domain, TRN-SR2 depletion may lead to cytoplasmic accumulation of CPSF6 that in turn may restrict HIV replication at the uncoating step [24, 35]. TRN-SR2 is a 923-amino acid protein consisting entirely of stacked HEAT repeats (two antiparallel ␣-helices connected by a small turn linker) [37] These create a curving structure with a high degree of flexibility, allowing binding to different types of cargo and regulatory proteins [38]. The complex of the importin and RanGTP moves back through the NPC to the cytoplasm where GTP is hydrolyzed, and the import factor is available for a new round of nuclear transport [39]. We present biochemical and structural biology studies on the interactions of TRN-SR2 with RanGTP

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION