In addition, viral entry was also investigated using a recombinant HSV-1 (gL86) which expresses β-galactosidase upon entry into cells. In Rab27a-silenced cells, an important decrease in viral-associated GFP signal was observed 18 h p.i. (Figure 7B). Plaque assay showed a drastic reduction in plaque size of silenced shRNA-313 cells compared LY2874455 to control cells (Figure 7C). Moreover, the number of plaques also decreased, suggesting that Rab27a depletion could be affecting the viral egress. Moreover, cells were infected at a m.o.i. of 1 with K26GFP and then, processed for fluorescence activated cell sorter (FACS) analysis. The number of GFP-expressing cells and

their mean fluorescence were measured 24 hour after infection. As shown in Figure 7D, a significant decrease in these parameters was confirmed in Rab27a-silenced cells compared with non-target control shRNA-expressing and non-transfected cells. Histogram data have been expressed as percentage of maximum (% of max), in which NVP-BGJ398 mouse Y axis corresponds to the number of cells for each fluorescence intensity of the X axis, relative to the peak fraction of cells. To assess whether Rab27a is involved in the viral

cycle, we measured viral yield of infected cells. Viral titer of Rab27a-silenced infected cells also showed, within 24 h p.i., a significant decrease compared with non-target control shRNA-expressing and non-transfected cells (Figure 7E). This effect is not due to a differential entry capacity of virions into the cell, since kinetics of viral entry showed no difference among silenced and control cultures (data not shown). Altogether, these results suggest that Rab27a might

be required not only in viral egress, but also in viral production. Discussion Many details on the molecular mechanism utilized by HSV-1 to exploit the cellular trafficking machinery during morphogenesis are uncertain. In particular, several aspects regarding the process of the secondary Cisplatin solubility dmso envelopment and viral egress need further enlightenment. Final steps of viral assembly Sinomenine take place through secondary envelopment by budding into TGN-derived vesicles coated with viral glycoproteins and tegument proteins [10, 11, 36, 39–41]. Herein, we suggest the involvement of the Rab-GTPase Rab27a in this process. Various Rab GTPases have been involved in HSV-1 –as well as in other herpesviruses– envelopment [30–32]. In fact, Rab27a is required for assembly of HCMV [33]. Given the similarities among members of the herpesvirus family [10], we decided to analyze whether Rab27a plays any influential role in HSV-1 infection of oligodendrocytic cells. First of all, our results showed a significant level of expression of Rab27a in HOG cells, compared to HOM-2 and MeWo cell lines, which were used as positive controls.