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摘要
The large nucleoporin Nup358/RanBP2 forms eight filaments that project from the nuclear pore into the cytoplasm where they function as docking platforms for nucleocytoplasmic transport receptors. RNAi screens have implicated Nup358 in the HIV-1 life cycle. The 164 C-terminal amino acids of this 3,224 amino acid protein are a cyclophilin homology domain (Nup358Cyp), which has potential to bind the HIV-1 capsid and regulate viral progress to integration. Here we examined the virological role of Nup358 in conditional knockout mouse cells and in RNAi-depleted human CD4+ T cells. Cre-mediated gene knockout was toxic and diminished HIV-1 infectivity. However, cellular health and HIV-1 susceptibility were coordinately preserved if, prior to gene inactivation, a transposon was used to express all of Nup358 or only the N-terminal 1340 amino acids that contain three FG repeats and a Ran-binding domain. HIV-1, but not N74D capsid-mutant HIV-1, was markedly sensitive to TNPO3 depletion, but they infected 1-1340 segment-complemented Nup358 knockout cells equivalently. Human and mouse CypA both rescued HIV-1 in CypA gene -/- Jurkat cells and TRIM-Nup358Cyp fusions derived from each species were equally antiviral; each also inhibited both WT and N74D virus. In the human CD4+ T cell line SupT1, abrupt Nup358 depletion reduced viral replication but stable Nup358-depleted cells replicated HIV-1 normally. Thus, human CD4+ T cells can accommodate to loss of Nup358 and preserve HIV-1 susceptibility. Experiments with cylosporine, viruses with capsids that do not bind cyclophilins, and growth arrest did not uncover viral dependency on the C-terminal domains of Nup358. Our data reinforce the virological importance of TNPO3 and show that Nup358 supports nuclear transport functions important for cellular homeostasis and for HIV-1 nuclear import. However, the results do not suggest direct roles for the Nup358 cyclophilin or SUMO E3 ligase domains in engaging the HIV-1 capsid prior to nuclear translocation. Author Summary The purified cyclophilin homology domain (CHD) of Nup358/RanBP2 can interact with assembled HIV-1 capsids in vitro, which suggests that, in cells, the incoming virus core could be engaged functionally by the CHD prior to nucleopore traverse. Interpretations of Nup358 knockdowns have been complicated by toxicity due to globally altered cellular nucleocytoplasmic transport and by a lack of re-expression controls, which are difficult because of the protein's size. We present the first analyses of the HIV-1 life cycle in Nup358 knockout cells and in Nup358-depleted human CD4+ T cells, and the first detailed studies of domain requirements and re-expression controls. We find that the N-terminal portion containing three FG repeats rather than the C-terminal portion of Nup358 is sufficient to preserve HIV-1 infection susceptibility in dividing and non-dividing gene knockout cells. Mouse and human versions of CypA and Nup358Cyp are functionally equivalent and TNPO3 displays marked dependency factor activity in cells of both species. A human CD4+ T cell line can be stably deficient in Nup358 without loss of HIV-1 permissivity. The data support an important, though cyclophilin homology domain-independent, role for Nup358 in the HIV-1 life cycle and demonstrate conservation of HIV-1 early event pathways between human and mouse cells.
- 出版日期2014-2
