- Jul 2015
Fig. 1 IAV requires the Ub-binding function of HDAC6 for capsid uncoating.
In this figure the authors showed the results obtained when they investigated HDAC6 role in IAV entry.
Panel A: The top part shows a cartoon of IAV entry that consists of multiple steps. The authors studied what step HDAC6 was critical during the virus host cell entry. Bottom part shows quantitative analysis graphs, each corresponding to the above step of IAV entry under investigation.
Panel A (endocytosis): The authors measured the amount of HA-stained spots per cell in wild type MEFs (WT MEF) compared to HADC5 Knock-out MEFs (HDAC6 KO). The third bar represents WT MEFs that were treated with dynasore (Dyn) in order to stop the endocytosis process. Depletion of HDAC6 did not influence the endocytosis step.
Panel A (HA-Acidification): In this step HDAC6 KO MEFS did not show any difference with respect to the wild type cells. It was concluded that HDAC6 does not influence this step of the virus infection.
Panel A (Fusion): fusion of capsid to endosome membrane is not affected by the lack of HDAC6 in knock-out MEF cells as shown in graph.
Panel A (Uncoating): Measuring the amount of cells with dispersed M1, the authors noticed that cells lacking HDAC6 dispersed less M1. This observation led to the conclusion that HDAC6 was fundamental for the uncoating process.
Panel A (vRNP import): Indeed, As vRNP import represents the step following uncoating, HDAC6 KO MEFs showed less NP-positive nuclei because the previous uncoating step was reduced.
In analyzing HA Acidification, Fusion, Uncoating and vRNP import, the authors used BafA1, which is a drug that is able to block uncoating, as a control for their experiment.
Panel B is a graphical representation of the 2 deacetylase catalytic domains of HDAC6 and the zinc-finger ubiquitin binding domain that is close to the C-terminus of the enzyme.
Panel C HDAC6 (WTr) (light gray bar) was a line of HDAC6 KO MEFs able to express again HDAC6. HDAC6 (HDm) (blue bar) was a line of HDAC6 KO MEFs expressing HDAC6 mutated in its deacetylase domain. HDAC6 (ZnFm) (purple bar) was a line of HDAC6 KO MEFs expressing HDAC6 with a mutation in its zinc-finger domain. The authors evaluated the effect of these mutations on the uncoating capacity of IAV. Depletion of deacetylase activity of HDAC6 did not influence the uncoating. Instead, loss of zinc-finger ubiquitin binding domain was detrimental for the uncoating.
The two asterisks on top of the purple bar indicate that this result is statistically significant, that is a result that is caused by something other than mere random chance.
Panel D: life in technicolor!!! These experiments were performed using the confocal microscopy technique. Fluorescent dye molecules bind to specific parts of the cell, so that only those parts are visualized. Fluorescent dyes are chemical compounds that re-emit light upon light excitation (light absorption).<br> The following video, created by Northwest Missouri State University, explains the basics of confocal microscopy. https://www.youtube.com/watch?v=jUAvneBhDcQ
The authors observed that after 2.5 hours from infection, M1 is dispersed inside WT MEFs, indicating that uncoating has occurred. Instead, M1 is confined inside vacuoles when cells lack HDAC6 and the mutant cells in which HDAC6 lacks the Zinc-finger ubiquitin binding region, meaning that the uncoating hasn't occurred.