The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6

Abstract

BackgroundExpression of the cellular karyopherin TNPO3/transportin-SR2/Tnp3 is necessary for HIV-1 infection. Depletion of TNPO3 expression in mammalian cells inhibits HIV-1 infection after reverse transcription but prior to integration.ResultsThis work explores the role of cleavage and polyadenylation specificity factor subunit 6 (CPSF6) in the ability of TNPO3-depleted cells to inhibit HIV-1 infection. Our findings showed that depletion of TNPO3 expression inhibits HIV-1 infection, while the simultaneous depletion of TNPO3 and CPSF6 expression rescues HIV-1 infection. Several experiments to understand the rescue of infectivity by CPSF6 were performed. Our experiments revealed that the HIV-1 capsid binding ability of the endogenously expressed CPSF6 from TNPO3-depleted cells does not change when compared to CPSF6 from wild type cells. In agreement with our previous results, depletion of TNPO3 did not change the nuclear localization of CPSF6. Studies on the formation of 2-LRT circles during HIV-1 infection revealed that TNPO3-depleted cells are impaired in the integration process or exhibit a defect in the formation of 2-LTR circles. To understand whether the cytosolic fraction of CPSF6 is responsible for the inhibition of HIV-1 in TNPO3-depleted cells, 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 the nuclear import step. Fate of the capsid assays revealed that cytosolic expression of CPSF6 enhances stability of the HIV-1 core during infection.ConclusionsThese results suggested that inhibition of HIV-1 by TNPO3-depleted cells requires CPSF6.

Nuclear import of APOBEC3F-labeled HIV-1 preintegration complexes

“…The decrease in WT A3F-YFP particles upon Nup153 knockdown was similar to the decrease in 2-LTR circles (1.7-vs. 2.3-fold decrease, respectively). There was no decrease in the number of nuclear A3F-YFP particles upon TNPO3 depletion in cells infected with WT or D110E virus, consistent with reports indicating that TNPO3 depletion acts on HIV-1 at a step after entry of the PIC into the nucleus (12,15,17,18). The decrease in 2-LTR circles (2.7-fold decrease) upon TNPO3 depletion, but not nuclear import of A3F-YFP particles, indicates that TNPO3 may affect the formation and/or stability of 2-LTR circles, which is consistent with a previous report (15).…”

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

“…Although we and others have shown that TRN-SR2 directly interacts with HIV IN (18,19,27,38,39), the mechanism of action has been questioned (19,23,36,40,41). Especially reports on a set of HIV capsid mutations (e.g.…”

“…CPSF6 is a cellular protein involved in splicing and polyadenylation of pre-mRNA that carries an RS domain at its C terminus. Because CPSF6 binds HIV CA (40), it has been suggested that TRN-SR2 depletion might result in cytoplasmic accumulation of CPSF6, which in turn might perturb viral uncoating through CPSF6-CA binding, leading to an indirect block in nuclear import and restricted HIV replication (36,40,41). Still, the cytoplasmic accumulation of CPSF6 after TRN-SR2 knockdown is not always observed (36,41).…”

“…Because CPSF6 binds HIV CA (40), it has been suggested that TRN-SR2 depletion might result in cytoplasmic accumulation of CPSF6, which in turn might perturb viral uncoating through CPSF6-CA binding, leading to an indirect block in nuclear import and restricted HIV replication (36,40,41). Still, the cytoplasmic accumulation of CPSF6 after TRN-SR2 knockdown is not always observed (36,41). In any case, the fact that a virus defective for interaction with CPSF6 (CA N74D ) remains sensitive to TRN-SR2 knockdown in a multiple-round replication experiment (25) suggests that cytoplasmic accumulation of CPSF6 is not the sole cause for the HIV replication deficit upon TRN-SR2 depletion.…”

BI-2 destabilizes HIV-1 cores during infection and Prevents Binding of CPSF6 to the HIV-1 Capsid

“…Expression of CPSF6 is required for the HIV-1 infection phenotype observed in human TNPO3-depleted cells [17][18][19]. We and others have previously demonstrated that the small-molecule HIV-1-inhibitor PF74 prevents the binding of CPSF6 to HIV-1 capsid [6,17].…”