We found NS4 to have strong nucleolar localization, although it may shuttle between the nucleolus and cytoplasm and possibly carry out its biological functions in the latter. The nucleolus is a dynamic sub-nuclear structure that plays crucial roles in ribosome subunit biogenesis, the response to cellular stress and cell growth [51, 52] . Several examples of viral proteins targeting the nucleolus have been discovered in recent years [53] . The retroviral Rev and Rev-like proteins for example, shuttle between the nucleolus and cytoplasm, and function as post-transcriptional regulators of viral gene expression [54] [55] [56] [57] . One of the main functions of these proteins is to facilitate the export of unspliced viral mRNA (transcribed from the proviral DNA copy of the retroviral genome stably integrated in the cell genome) by simultaneously binding an RNA structure in the viral RNA and the karyopherin export factor Crm1 (chromosome region maintenance 1) [58] . Other RNA viruses (including those that replicate exclusively in the cytoplasm) have also been found to possess proteins that target the nucleoli. Examples include, among others, avian infectious bronchitis virus [59] , porcine reproductive and respiratory syndrome virus [60] , Newcastle disease virus [61] , Semliki forest virus [62] , dengue virus [63] , West Nile virus [64] , influenza virus [65] , avian reovirus [66] and encephalomyocarditis virus [67, 68] The reasons for the nucleolar targeting of many of these proteins have not always been entirely clear. 10 (TCID 50 /ml). In parallel, each virus preparation was also re-titrated by limiting dilution analysis to control that equal amounts of input virus was used in each experiment. This experiment was performed two times, each time in duplicate. doi:10.1371/journal.ppat.1002477.g008