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-β 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.

Fig. 1.

TNPO3 knockdown inhibits HIV-1 and SIVmac239 nuclear import. (A) HeLa cells were transfected with nontarget control (NTC), TNPO3, Nup153, or mock siRNA and then infected with HIV-1 and SIVmac239 reporter virus. (B) HeLa cells were transfected with NTC and two different TNPO3 siRNAs (TNPO3.9 and TNPO3.12) or were mock transfected. The cells were then infected with HIV-1 or SIVmac239 reporter virus. AZT was added to one sample to control for intravirion reverse transcripts. DNA was harvested from infected cells at 2, 10, 24, and 48 h postinfection, and viral late-reverse transcription (RT) products and 2-LTR circles were measured by qRT-PCR. Each point represents the mean of triplicate samples, with the error bars representing the standard deviations.

Fig. 2.

TNPO3 knockdown in dividing and nondividing cells blocks infection with multiple lentiviruses. (A) Empty-vector-transduced 293T cells and 293T cells that stably express a TNPO3-targeted shRNA were infected with MLV-GFP, FIV-GFP, EIAV-RFP, SIVmac-HSA, and HIV-1-HSA reporter virus at the indicted MOI. (B) Empty-vector-transduced HeLa cells and HeLa cells that stably express a TNPO3-targeted shRNA were infected with MLV-GFP, FIV-GFP, EIAV-RFP, SIVmac-HSA, and HIV-1-HSA reporter virus at an MOI of 0.3. (C) PMA-differentiated wild-type and stable TNPO3 knockdown THP-1 cells were infected with SIVmac239 or HIV-1 reporter virus. (D) Control and TNPO3 knockdown 293T cells were infected with wild-type HIV-1, wild-type SIVmac239, SIVmac239, SIVmac with HIV-1 capsid and p2 (SIV-HCAp2), or HIV-1 with SIVmac239 capsid (HIV-SCA) reporter virus. (E) Control and TNPO3 knockdown 293T cells were infected with HIV-SCA reporter virus containing a wild-type SIVmac239 CA or an SIVmac239 CA with an A76V mutation. (F) Control and mCPSF6-358-expressing HeLa cells treated with DMSO or 2 μg/ml aphidicolin overnight were infected with HIV-SCA reporter virus containing a wild-type SIVmac239 CA or an SIVmac239 CA with an A76V mutation. These experiments were done in triplicate, and the error bars indicate the standard deviations of the mean.

Fig. 3.

The carboxy-terminal cargo-binding domain of TNPO3 is required for SIVmac239 infection. (A) The shRNA-target site in TNPO3 (red) was mutated (black) to generate an shRNA-resistant TNPO3 expression vector. (B) TNPO3 knockdown 293T cells were transfected with expression vector encoding shRNA-resistant TNPO3, importin-13 (Imp13), and empty vector. The transfected cells and wild-type 293T cells (cntrl cells) were then infected with SIVmac239 reporter virus. (C) TNPO3 knockdown 293T cells were transfected with wild-type, ΔCargo, or TNPO3 with the carboxy-terminal 18 amino acids deleted (ΔC18).

Fig. 4.

Drosophila TNPO3 facilitates SIVmac239 infection. (A) The amino acid sequence of Drosophila TNPO3 was aligned with human TNPO3 using ClustalW. Identical amino acids are shaded dark gray, and conservative changes are shaded light gray. (B) TNPO3 knockdown cells were complemented with wild-type human TNPO3, ΔC18, and drTNPO3 and then challenged with SIVmac239 reporter virus.

Fig. 5.

F918 and F922 in the cargo-binding domain of TNPO3 are required to facilitate SIVmac239 infection. (A) Specific amino acid residues in the carboxy-terminal 18 amino acids of TNPO3 were mutated to alanine (asterisks). (B) TNPO3 knockdown cells were complemented with wild-type, ΔCargo, or mutated TNPO3 expression vector and then infected with SIVmac239 reporter virus. (C) TNPO3 knockdown cells were complemented with wild-type, ΔCargo, F918A, F922A, or double F918A/F922A point mutated TNPO3 expression vector and then infected with SIVmac239 reporter virus. (D) Mutant TNPO3 proteins were visualized by immunoblot analysis.

Fig. 6.

A dileucine motif in the cargo-binding domain of TNPO3 is required to facilitate SIVmac239 infection. (A) Specific amino acid residues in the cargo-binding domain were mutated to alanine (asterisks). Individual loop mutants are boxed in black. (B) TNPO3 knockdown cells were complemented with a wild-type, ΔCargo, or mutated TNPO3 expression vector. (C) TNPO3 knockdown cells were complemented with a wild-type, ΔCargo, and TNPO3 expression vector containing single or multiple point mutations of amino acid residues L767, L766, R769, and S770. (D) The mutated TNPO3 proteins were visualized by immunoblot analysis.

Fig. 7.

The cargo-binding domain is required for TNPO3-dependent infection by several lentiviruses. TNPO3 knockdown cells were complemented with shRNA-resistant wild-type, ΔCargo, F918A/F922A, and TNPO3 LL767-8AA expression vector or with the empty vector. The cells were then infected with SIVmac239, HIV-1, or EIAV reporter virus.

Fig. 8.

Nonfunctional mutated TNPO3 proteins localize to the nucleus. (A) TNPO3 knockdown cells were complemented with a wild-type TNPO3-GFP, mutated TNPO3-GFP, or GFP expression vector. The cells were gated for GFP fluorescence, and the percentages of infected GFP⁻ and GFP⁺ cells were determined. (B) HeLa cells transfected with GFP-tagged shRNA-resistant full-length or mutant TNPO3 expression vectors were imaged by confocal microscopy. GFP fusion proteins are shown in green. The nucleus was counterstained with Hoechst (blue) and the plasma membrane with wheat germ agglutinin (red).