The Cargo-Binding Domain of Transportin 3 Is Required for Lentivirus Nuclear Import

Abstract

Lentiviruses, unlike the gammaretroviruses, are able to infect nondividing cells by transiting through nuclear pores to access the host genomic DNA. Several nuclear import and nuclear pore components have been implicated as playing a role in nuclear import, including transportin 3 (TNPO3), a member of the importin-␤ family of nuclear import proteins. We demonstrated that TNPO3 was required by several lentiviruses, with simian immunodeficiency virus mac239 (SIVmac239) and equine infectious anemia virus (EIAV) the most dependent and human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) the least. Analysis of HIV-1/SIVmac239 chimeric viruses showed that dependence on TNPO3 mapped to the SIVmac239 capsid. Mutation of a single amino acid, A76V in the SIVmac239 capsid, rendered the virus TNPO3 independent and resistant to mCPSF6-358, a truncated splicing factor that prevents HIV-1 nuclear import. Using a complementation assay based on 293T cells that express a TNPO3-targeted short hairpin RNA (shRNA), we showed that the Drosophila TNPO3 homologue can substitute for its human counterpart and that it mapped a key functional domain of TNPO3 to the carboxy-terminal cargo-binding domain. Within the cargo-binding domain, two hydrophobic motifs were required for TNPO3-dependent infection. The mutated TNPO3 proteins maintained their ability to localize to the nucleus, suggesting that their inability to restore lentivirus infection resulted from an inability to bind to a host or viral cargo protein.

Interaction of the HIV-1 Intasome with Transportin 3 Protein (TNPO3 or TRN-SR2)

“…However, it remains to be seen whether this TNPO3 segment engages HIV-1 IN or another cargo in infected cells. Another TNPO3 region mapped by the published study implicated the C-terminal 18 amino acids (56). The truncated protein (␦18) as well as the F918A/ F922A mutant exhibited reduced activities.…”

“…A previous study mapped several TNPO3 amino acid sites as being important for lentiviral replication (56). The most significant effect on simian immunodeficiency virus nuclear import was observed when the L767A/L768A substitutions were introduced in TNPO3.…”

The HIV-1 Integrase Mutant R263A/K264A Is 2-fold Defective for TRN-SR2 Binding and Viral Nuclear Import

“…IN catalytic site mutations or the addition of integrase inhibitors during HIV infection reproducibly result in abortive integration and a steep increase in two-LTR circles (34), whereas a decrease in two-LTR circles is generally accepted to reflect a block in nuclear import (35). In line, a reduction in the number of two-LTR circles was measured after RNAi-mediated TRN-SR2 depletion (18,20,21,36), suggesting a nuclear import defect. In a direct HIV nuclear import assay using IN-eGFP-labeled PICs (18,37), we demonstrated a reduction of nuclear versus cytoplasmic PICs after TRN-SR2 knockdown (18), underscoring a role for TRN-SR2 in HIV nuclear import.…”

Identification of Residues in the C-terminal Domain of HIV-1 Integrase That Mediate Binding to the Transportin-SR2 Protein

“…Although the exact role of TRN-SR2 in HIV replication is still under debate, there is a consensus that even moderate knockdown of this cofactor results in a strong inhibition of HIV replication at an early replication step prior to integration. Because TRN-SR2 belongs to the importin-␤ family of nuclear import factors (46 -49), it is not far-fetched to imagine a direct or indirect role in the nuclear import of the viral PIC (7,19,21). Recently, the hypothesis was formulated that TRN-SR2 also acts as a nuclear export factor for tRNA and HIV capsid (27), but this model awaits independent confirmation.…”

“…Using RNAi technology, the crucial role of TRN-SR2 in HIV replication was demonstrated (18 -20). A specific reduction in two long terminal repeat (LTR) circles and integration was measured by quantitative PCR (Q-PCR) (19,21,22). Direct evidence for TRN-SR2-mediated nuclear import of HIV was obtained by using fluorescently labeled PICs (19).…”