Differential distribution of non-structural proteins of foot-and-mouth disease virus in BHK-21 cells

Differences in the kinetics of expression and cell distribution among FMDV non-structural proteins (NSPs) have been observed in BHK-21-infected cells. 3Dpol was the first protein detected by immunofluorescence (1.5 h p.i.), showing a perinuclear distribution. At 2–2.5 h p.i., 2B, 2C, 3B and 3C were detected, mostly exhibiting a punctuated, scattered pattern, while 3A and 3Dpol appeared concentrated at one side of the nucleus. This distribution was exhibited by all the NSPs from 3 h p.i., being 2C and, to a lesser extent, precursors 2BC and 3ABBB, the only proteins detected by Western blotting at that infection time. From 4 h p.i., all mature NSPs as well as precursors 2BC, 3ABBB, 3ABB, 3AB and 3CDpol were detected by this technique. In spite of their similar immunofluorescence patterns, 2C and 3A co-localized partially by confocal microscopy at 3.5 h p.i., and 3A, but not 2C, co-localized with the ER marker calreticulin, suggesting differences in the distribution of these proteins and/or their precursors as infection proceeded. Transient expression of 2C and 3AB resulted in punctuated fluorescence patterns similar to those found in early infected cells, while 3A showed a more diffuse distribution. A shift towards a fibrous pattern was noticed for 3ABB, while a major change was observed in cells expressing 3ABBB, which displayed a perinuclear fibrous distribution. Interestingly, when co-expressed with 3Dpol, the pattern observed for 3ABBB fluorescence was altered, resembling that exhibited by cells transfected with 3AB. Transient expression of 3Dpol showed a homogeneous cell distribution that included, as determined by confocal microscopy, the nucleus. This was confirmed by the detection of 3Dpol in nuclear fractions of transfected cells. 3Dpol and its precursor 3CDpol were also detected in nuclear fractions of infected cells, suggesting that these proteins can directly interact with the nucleus during FMDV infection.