The Requirement for Cellular Transportin 3 (TNPO3 or TRN-SR2) during Infection Maps to Human Immunodeficiency Virus Type 1 Capsid and Not Integrase

Recent genome-wide screens have highlighted an important role for transportin 3 in human immunodeficiency virus type 1 (HIV-1) infection and preintegration complex (PIC) nuclear import. Moreover, HIV-1 integrase interacted with recombinant transportin 3 protein under conditions whereby Moloney murine leukemia virus (MLV) integrase failed to do so, suggesting that integrase-transportin 3 interactions might underscore active retroviral PIC nuclear import. Here we correlate infectivity defects in transportin 3 knockdown cells with in vitro protein binding affinities for an expanded set of retroviruses that include simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), and Rous sarcoma virus (RSV) to critically address the role of integrase-transportin 3 interactions in viral infection. Lentiviruses, with the exception of FIV, display a requirement for transportin 3 in comparison to MLV and RSV, yielding an infection-based dependency ranking of SIV > HIV-1 > BIV and EIAV > MLV, RSV, and FIV. In vitro pulldown and surface plasmon resonance assays, in contrast, define a notably different integrase-transportin 3 binding hierarchy: FIV, HIV-1, and BIV > SIV and MLV > EIAV. Our results therefore fail to support a critical role for integrase binding in dictating transportin 3 dependency during retrovirus infection. In addition to integrase, capsid has been highlighted as a retroviral nuclear import determinant. Accordingly, MLV/HIV-1 chimera viruses pinpoint the genetic determinant of sensitization to transportin 3 knockdown to the HIV-1 capsid protein. We therefore conclude that capsid, not integrase, is the dominant viral factor that dictates transportin 3 dependency during HIV-1 infection.The early events in the retroviral life cycle, typified by reverse transcription and subsequent cDNA integration, occur within the context of high-molecular-weight nucleoprotein complexes that are derived from the core of the infecting virus particle (4, 20, 21, 36). This presents a particular challenge for the viruses, as the chromosomal targets of integration reside within the nucleus, which is separated from the cytoplasm by a double lipid bilayer. The nuclear membrane houses nuclear pore complexes that permit the passive diffusion of macromolecules with diameters of less than ∼9 nm (34). At 56 nm (36), the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC), derived from a prototypical retrovirus, grossly exceeds the diffusion limit. Different retroviruses have evolved different mechanisms to deal with the nuclear membrane barrier. The gammaretrovirus Moloney murine leukemia virus (MLV) relies on cell division for productive infection (35), apparently subverting the membrane when it dissolves during the M phase of the cell cycle (32, 42). HIV-1, in contrast, is actively transported during interphase (6), reflecting its ability to efficiently infect terminally differentiated and growth-arrested cells in the absence of cell division (31, 48).A number of HIV-1 PIC components, including matrix (MA), Vpr, integrase (IN), and the central DNA flap that forms during reverse transcription, have been implicated in nuclear import. However, this aspect of HIV-1 biology is relatively muddled, with multiple reports both supporting and refuting important roles for each of these components (see references 15, 19, and 51 for recent reviews). These apparent inconsistencies may be due to any number of reasons. HIV-1 might employ numerous overlapping mechanisms such that the alteration of one leads to minimal or partial phenotypes. Many viral mutations can exert pleiotropic affects, often complicating data interpretation. Moreover, the identification of the HIV-1 capsid (CA) protein as a dominant infectivity determinant in growth-arrested cells suggested that virus uncoating compared to active nuclear transport may be the rate-limiting step in this phase of the life cycle (51). IN also continues to attract particular interest, as it is the one protein component of the PIC that must remain associated with the reverse transcript after nuclear entry.Although numerous host cell factors have been implicated in HIV-1 nuclear transport, the field has yet to reach a consensus on the salient player(s). Various import proteins, including the importin α/importin β heterodimer (22, 23, 25), importin 7 (2, 22, 54), NUP153 (49), and transportin 3 (TNPO3 or TRN-SR2) (13), have been shown to interact with HIV-1 IN. TNPO3, a member of the karyopherin β protein family that imports serine/arginine-rich splicing factors (SR proteins) into the nucleus (27), has generated particular interest as of late. In addition to its identification as an HIV-1 IN binding protein (13), TNPO3 was identified in two independent genome-wide short interfering RNA (siRNA) screens for host factors required by HIV-1 for infection (5, 30). TNPO3 depletion yielded significant HIV-1 (5, 13, 30) and HIV-2 (13) but much less dramatic MLV (5, 13, 30) infection defects, indicating a potential role as a lentivirus-specific host cofactor. Moreover, the block to HIV-1 infection was pinpointed at PIC nuclear import (13). Furthermore, recombinant TNPO3 protein was shown to bind HIV-1 IN but not MLV IN in vitro, suggesting that the TNPO3-IN interaction might underlie lentivirus-specific PIC nuclear import (13).In this study we critically addressed the hypothesis that TNPO3 is indeed an IN-dependent cofactor of lentiviral infection. We correlated the infectivities of an expanded set of retroviral vectors in cells depleted for TNPO3 expression with relative IN-TNPO3 binding affinities. Our findings recapitulate the previously reported in vitro interaction between TNPO3 and HIV-1 IN (13). However, we find TNPO3 to be a relatively prolific IN binding protein, displaying affinity for the majority of tested IN proteins. Moreover, protein binding profiles did not correlate with the requirement for the host factor during virus infection. Furthermore, analyses of MLV/HIV-1 (MHIV) chimera viruses that harbor parts of MLV Gag and/or IN for the corresponding HIV-1 determinants highlight a role for CA in mediating the requirement for TNPO3 during HIV-1 infection.