Importin α3 Interacts with HIV-1 Integrase and Contributes to HIV-1 Nuclear Import and Replication

Abstract

HIV-1 employs the cellular nuclear import machinery to actively transport its preintegration complex (PIC) into the nucleus for integration of the viral DNA. Several viral karyophilic proteins and cellular import factors have been suggested to contribute to HIV-1 PIC nuclear import and replication. However, how HIV interacts with different cellular machineries to ensure efficient nuclear import of its preintegration complex in dividing and nondividing cells is still not fully understood. In this study, we have investigated different importin ␣ (Imp␣) family members for their impacts on HIV-1 replication, and we demonstrate that short hairpin RNA (shRNA)-mediated Imp␣3 knockdown (KD) significantly impaired HIV infection in HeLa cells, CD4؉ C8166 T cells, and primary macrophages. Moreover, quantitative real-time PCR analysis revealed that Imp␣3-KD resulted in significantly reduced levels of viral 2-long-terminal repeat (2-LTR) circles but had no effect on HIV reverse transcription. All of these data indicate an important role for Imp␣3 in HIV nuclear import. In an attempt to understand how Imp␣3 participates in HIV nuclear import and replication, we first demonstrated that the HIV-1 karyophilic protein integrase (IN) was able to interact with Imp␣3 both in a 293T cell expression system and in HIV-infected CD4 ؉ C8166 T cells. Deletion analysis suggested that a region (amino acids [aa] 250 to 270) in the C-terminal domain of IN is involved in this viral-cellular protein interaction. Overall, this study demonstrates for the first time that Imp␣3 is an HIV integrase-interacting cofactor that is required for efficient HIV-1 nuclear import and replication in both dividing and nondividing cells.HIV-1 replicates productively in nondividing cells, such as monocytes (49,61,74), macrophages (23,37,59,65,71), dendritic cells (47,64), and resting CD4 ϩ T lymphocytes (86), through its ability to undergo active nuclear import by hijacking the host nuclear import machinery. Moreover, active nuclear import is not only required for nondividing-cell infection but also plays a role in the infection of proliferating cells (35). This ability of HIV-1 to enter the nucleus at interphase may contribute significantly to the very high replication rate observed in infected individuals (30,70,73) and is one of the crucial steps in HIV-1 replication, which plays a leading role in the establishment of infection and AIDS pathogenesis.The viral double-stranded DNA (dsDNA), which associates with viral and cellular proteins, forms a high-molecular-mass nucleoprotein complex called the preintegration complex (PIC) in the cytosol of an infected cell (15,51). This large complex has to actively enter the nucleus through the intact nuclear membrane in order to be integrated. At the molecular level, the active nuclear import ability of HIV-1 is attributed to the karyophilic properties of viral PICs. It is known that several viral nucleophilic proteins, including integrase (IN), matrix (MA), and Vpr, are associated with this nucleoprotein complex and pla...

Transportin 3 and importin α are required for effective nuclear import of HIV-1 integrase in virus-infected cells

“…18 Recently, our observations regarding the interaction between the viral IN and importin α(3) in virus infected cells have been confirmed in independent studies using in-vitro pull-down assay and cell-based co-immunoprecipitation method in 293T cells and HIV-1 infected cells. 51 Furthermore, a short hairpin RNA (ShRNA) was employed to knockdown Impα3 in dividing C8166T, HeLa cells and non-dividing monocyte-derived macrophages (MDM). HIV-1 replication in Impα3 knockdown cells was significantly downregulated, (reviewed in ref.…”

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

“…In 2010, a potential role of importin-␣3 in HIV nuclear import was reported (11). Later the IN amino acids Arg 263 and Lys 264 were claimed to be involved in the binding to importin-␣3 (54).…”

“…Nuclear import in the human cell is orchestrated by a plethora of karyopherins, comprising both broad spectrum and highly specialized transporters. Several of these have been suggested to take part in HIV nuclear import, in particular importin-␣/␤ (8 -10), importin-␣3 (11), and importin-7 (12)(13)(14)(15). In 2008, transportin-SR2 (TRN-SR2, transportin-3, Tnpo3), encoded by the TNPO3 gene, was independently identified as a cellular cofactor of HIV in two genome-wide siRNA screens (16,17) and picked up as a specific binding partner of HIV IN (18).…”

Cellular and viral determinants of retroviral nuclear entry

“…Because of disagreement regarding possible interaction of importin ␣1 with IN in vitro (Woodward et al 2009;Ao et al 2010), the contributions of other members of the importin family was investigated in HeLa cells, CD4+ C8166 T cells, and primary macrophages by quantitative RT-PCR, as they have 50%-80% sequence similarities. The depletion of importin ␣3 decreases 2-LTR levels (Ao et al 2010).…”