Zhou's paper is in press at the journal mBio. He developed highly inclined and laminated optical sheet (HILO) live-cell microscopy to track HIV-1 and host protein complexes. Here is an example of one of his movies, showing red virus particles moving along white microtubules in a cell:
Using live-cell imaging and virology techniques, Zhou showed that although CPSF6 is expressed predominantly in the nucleus of HeLa cells, it is also present in the cytoplasm of cells in apparent higher-order complexes near the nucleus. These CPSF6 complexes traffic on microtubules in uninfected cells.
Proper HIV-1 complex trafficking relies on CPSF6 binding to transportin 3 (TNPO3), which is necessary for nuclear localization. After HIV-1 infection, WT HIV-1 complexes traffic with CPSF6 complexes in a CA-dependent manner in HeLa cells:
Our collaborator, Dr. Peijun Zhang, expressed recombinant CPSF6 protein and showed that it oligomerizes in vitro. When added to CA tubular assemblies, CPSF6 disrupts them similar to what we showed in our previous study.
Surprisingly, loss of cyclophilin A (CypA) binding to HIV-1 capsid (i.e. with a small molecule like cyclosporine A (CsA) or a CA mutation) allows more CPSF6 binding to HIV-1 complexes in cells and in vitro, which corresponds to altered cytoplasmic trafficking and, thus, decreased infectivity in HeLa cells. Similar infectivity phenotypes were observed in CD4+ T cells. The graph below shows that when CypA binding to capsid is inhibited by CsA in cells, more CPSF6 accumulates around the viral particles in the cell cytoplasm:
With Emerson's help, Zhou showed that CypA is abundant throughout the cytoplasm but absent from the microtubule organizing center (MTOC) and nucleus of HeLa cells and CD4+ T cells. However, CypA is expressed in both the cytoplasm and nucleus of macrophages. In macrophages, HIV-1 trafficking and infectivity is altered by both lack of CypA binding or depletion of CPSF6.
In the model below, we propose that in HeLa and CD4+ T cells, CypA regulates premature engagement of the HIV-1 capsid by cytoplasmic CPSF6. If HIV-1 capsid binds to CPSF6 too early or too much, premature uncoating may occur.
In contrast, HIV-1 infection does not induce visible higher-order complexes with CPSF6 in the cytoplasm of macrophages. Higher nuclear CypA expression could in theory play a greater role in regulating capsid interactions and uncoating within the nucleus in this cell type.
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