HIV-1 capsid is involved in post-nuclear entry steps

Abstract

BackgroundHIV-1 capsid influences viral uncoating and nuclear import. Some capsid is detected in the nucleus but it is unclear if it has any function. We reported that the antibiotic Coumermycin-A1 (C-A1) inhibits HIV-1 integration and that a capsid mutation confers resistance to C-A1, suggesting that capsid might affect post-nuclear entry steps.ResultsHere we report that C-A1 inhibits HIV-1 integration in a capsid-dependent way. Using molecular docking, we identify an extended binding pocket delimited by two adjacent capsid monomers where C-A1 is predicted to bind. Isothermal titration calorimetry confirmed that C-A1 binds to hexameric capsid. Cyclosporine washout assays in Jurkat CD4+ T cells expressing engineered human TRIMCyp showed that C-A1 causes faster and greater escape from TRIMCyp restriction. Sub-cellular fractionation showed that small amounts of capsid accumulated in the nuclei of infected cells and C-A1 reduced the nuclear capsid. A105S and N74D capsid mutant viruses did not accumulate capsid in the nucleus, irrespective of C-A1 treatment. Depletion of Nup153, a nucleoporin located at the nuclear side of the nuclear pore that binds to HIV-1 capsid, made the virus less susceptible to TRIMCyp restriction, suggesting that Nup153 may help maintain some integrity of the viral core in the nucleus. Furthermore C-A1 increased binding of CPSF6, a nuclear protein, to capsid.ConclusionsOur results indicate that capsid is involved in post-nuclear entry steps preceding integration.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0262-0) contains supplementary material, which is available to authorized users.

Capsid-Dependent Host Factors in HIV-1 Infection

“…Although specific interactions between CA and nucleoporins such as NUP358 and NUP153 may be thought to favor the nuclear pore model, partially uncoated capsids should still engage these factors. Recent research indicates that some CA remains associated with the PIC following nuclear import [60, 72, 74, 78, 108], which is consistent with both the nuclear pore and cytoplasmic models of uncoating. Uncoating is exquisitely fine-tuned through intrinsic core stability and by host factors that can directly interact with CA and either stabilize or destabilize viral capsids [12, 110].…”

“…Thus, although CPSF6 is not an essential viral cofactor, HIV-1 prefers this interaction when push comes to shove. The N74D change delayed uncoating [77] and N74D and N74A mutants displayed fewer CA signals in the nucleus than WT HIV-1 [60, 74, 78]. Interestingly, the N74D mutant as well as other CPSF6-binding deficient mutants render HIV-1 almost completely insensitive to depletion of cofactors implicated in nuclear entry including TNPO3, NUP358, and NUP153 [9, 65, 66, 79].…”

“…NUP153 is required for PIC nuclear entry as shown by decreases in 2-LTR circle DNA [52, 58, 79] and nuclear PICs [74, 105], although its role likely extends beyond transport to include integration and integration targeting [10, 78, 79, 111]. The genetic determinant of NUP153 dependency maps to HIV-1 CA [79].…”

Localized Phosphorylation of a Kinesin-1 Adaptor by a Capsid-Associated Kinase Regulates HIV-1 Motility and Uncoating

“…Given the size of the viral core, the capsid (CA) shell has to disassemble or “uncoat” to allow the genome to pass through nuclear pores (Campbell and Hope, 2015). Although some CA can be detected in the nucleus and functions in processes after nuclear entry (Chen et al, 2016; Hulme et al, 2015; Koh et al, 2013; Peng et al, 2014; Schaller et al, 2011; Stultz et al, 2017), whether reverse transcription and uncoating occur during transport along MTs in the cytoplasm or at nuclear pores remains a matter of debate (Arhel et al, 2007; Fassati and Goff, 2001; Le Sage et al, 2014; McDonald et al, 2002; Miller et al, 1997; Rasaiyaah et al, 2013; Zhou et al, 2011). However, recent live cell imaging supports the notion that uncoating begins in the cytoplasm (Francis et al, 2016).…”

The roles of the nuclear pore complex in cellular dysfunction, aging and disease

“…Nup214, a cytoplasmic ring nucleoporin with known roles in nucleocytoplasmic transport directly interacts with and acts as the functional import receptor of adenovirus 2 nucleocapsids [95]. The nuclear basket nucleoporin Nup153, which plays direct roles in importin alpha/beta mediated protein import [96], binds to the HIV capsid and knockdown of this nucleoporin reduces capsid entry [97]. In a proteomic analysis of HCV capsid interacting proteins, Nup98 was identified as critical for HCV entry into the nucleus and the subsequent propagation of the virus [98].…”