TNPO3/CPSF6

TNPO3, transportin-SR2 or Tnp3 is a member of the karyopherin β superfamily of proteins, and works as a nuclear import receptor for serine-arginine-rich (SR) proteins, which are necessary for RNA splicing. It is an established fact that depletion of TNPO3 decreases the ability of wild type HIV-1 and other lentiviruses to infect cells [1-10]; however, the mechanism by which TNPO3 assists HIV-1 replication is under intense investigation with existing evidence to support a role for TNPO3 after reverse transcription but prior to integration [1-3,5,11].

We found that TNPO3 physically binds to in vitro-assembled HIV-1 CA-NC complexes, which recapitulate the surface of the viral core and are composed of capsid, nucleocapsid, and RNA (8). Our work revealed that the interaction between TNPO3 and the assembled HIV-1 CA-NC complexes bearing the capsid mutation N74D is diminished compared to that of the wt, suggesting that binding of TNPO3 to the HIV-1 core is specific. Depletion of TNPO3 inhibits HIV-1 viral replication after nuclear import but prior to proviral integration, which is consistent with a recent observation (22). TNPO3 thus represents a previously unappreciated class of HIV-dependency factor, which physically associates with the HIV-1 CA-NC complexes and is required for successful viral integration and genome site selection.

Our collective observations suggest that TNPO3 binds to the HIV-1 core in the cytoplasm and that this interaction is ultimately important for the virus’s integration into the host genome. Interestingly, depletion of TNPO3 has recently been shown to alter the pattern of HIV-1 site integration in the host genome (16). Therefore, one possibility is that TNPO3 is needed for the nuclear import of host factors that are required for viral integration. To test this hypothesis, we depleted TNPO3 and assessed the subcellular localization of the lens epithelium-derived growth factor (LEDGF/p75), a chromatin-associated host factor that is critical for HIV-1 integration (4). As shown in Fig. 2, the subcellular localization of LEDGF/p75 was not altered in TNPO3 K.D. cells compared to that in control cells (Fig. 2). In agreement, immunoﬂuorescence (IF) experiments also showed that the localization of LEDGF/p75 was not altered in TNPO3 K.D. cells compared to control cells. These experiments show that TNPO3 depletion does not affect the localization of the HIV-1 integration cofactor LEDGF/p75.

The fact that HIV-1-N74D is insensitive to TNPO3-depletion and overcomes the restriction imposed by a fragment derived from CPSF6 suggests a role for CPSF6 in the ability of TNPO3-depleted cells to block HIV-1 infection. The fragment derived from CPSF6 composed of residues 1-358 (CPSF6-358) localizes to the cytoplasm and potently restricts HIV-1 infection when overexpressed in different mammalian cells [12]. By contrast the full-length CPSF6 is a nuclear protein that when overexpressed in mammalian cells does not block lentiviral infection [12]. Our studies revealed that TNPO3-depleted cells are impaired in the integration process or exhibit a defect in the formation of 2-LTR circles. Because TNPO3-depleted cells inhibit HIV-1 infection in a CPSF6-dependent manner, we tested whether TNPO3-depleted cells inhibit HIV-1 infection by the mechanism used by CPSF6-358. For this purpose, we tested the ability of a cytosolic full-length CPSF6 to block HIV-1 infection. These results demonstrated that overexpression of a cytosolic full-length CPSF6 blocks HIV-1 infection at or before the nuclear import step. Overall these results suggested that inhibition of HIV-1 by TNPO3-depleted cells is CPSF6-dependent.

Valle-Casuso JC, Di Nunzio F,Yang Y, Reszka N, Lienlaf M, Arhel N, Perez P, Brass AL., Diaz-Griffero F (2012). TNPO3 is required for HIV-1 replication after nuclear import but prior to integration and binds the HIV-1 core. J. Virol. May;86(10):5931-6. DOI:10.1128/JVI.00451-12

Fricke T, Valle-Casuso JC, White TE, Brandariz-Nuñez A, Bosche WJ, Reszka N, Gorelick R, Diaz-Griffero F (2013). The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6. Retrovirology 10: 46. DOI: 10.1186/1742-4690-10-46

Figure 2. Subcellular localization of LEDGF and CPSF6 in TNPO3-depleted cells.

Figure 1. Binding of TNPO3 to HIV-1 capsid-nucleocapsid complexes.

Figure 3. Binding of endogenously expressed CPSF6 to in vitro assembled CA-NC complexes.

Figure 4. Cf2Th cells stably expressing the indicated CPSF6 variant or TRIM5αrh were challenged using similar amounts of HIV-1, and performed the fate of the capsid assay to separate pelletable from soluble cytosolic capsids 16 hours post-infection as described in Methods. Input, soluble and pellet fractions were analyzed by Western blotting using antibodies against HIV-1 CA p24. Similar results were obtained in three independent experiments and a representative experiments is shown.