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. 2015 Nov 20;11(11):e1005274. doi: 10.1371/journal.ppat.1005274

Fig 2. Hsc70 was redistributed from the cytoplasm to both the periphery and within KSHV-induced RTCs.

Fig 2

(A) Unreactivated TREx BCBL1-RTA cells exhibited endogenous Hsc70 protein equally distributed between the cytoplasm and the nucleus (i). The same Hsc70 location was seen at 12 h reactivation prior to RTC formation, when RTA was diffuse in the nucleus (ii). At 20 h reactivation viral RTA was assembled into incipient RTCs. Hsc70 was not detected in the cytoplasm and instead numerous nuclear foci that were positioned predominantly adjacent to RTCs were seen (iii). At later reactivation times very large Hsc70 foci were completely recruited within RTCs. Hsc70 cytoplasmic depletion is indicated (iv arrows). Nucleus highlighted in yellow shows a typical KSHV fully developed-RTC with cellular chromatin marginalised to the nuclear periphery. (B) Unreactivated (-) or reactivated for 26 h (+) TREx BCBL1-RTA cells were fractionated into whole cell (WC), cytoplasmic (C) and nuclear (N) fractions. Equal amounts of total protein from each fraction were analysed by Western blotting. Nuclear fractions were characterised by enrichment of lamin B1 and absence of cytoplasmic GAPDH, while cytoplasmic fractions showed the inverse profile. A small decrease in cytoplasmic Hsc70 which correlated with a small increase in nuclear Hsc70 was detected in reactivated cells, further supporting that Hsc70 was redistributed from the cytoplasm to the nucleus. (C) TREx BCBL1-RTA cells remained unreactivated (i) or reactivated for 24 h (ii-iii’) followed by triple-labelling with antibodies specific for RTA and Hsc70 and Click-iT EdU Alexa Fluor 647, the latter allowed visualization of newly synthesized DNA. During early reactivation, a proportion of Hsc70 protein was adjacent to small viral RTCs. Arrow points to a small KSHV RTC filled with viral DNA (ii). Hsc70 also partially co-localised with viral DNA (ii) and RTA (ii’) in small RTCs. During later reactivation times, Hsc70 completely moved within fully-developed RTCs strongly co-localising with viral DNA (iii) and RTA (iii’).