Figure 5.

APOBEC3 proteins interact with human coronavirus (HCoV)-NL63 nucleoprotein (N). (a) 293T cell lysates enriched in active APOBEC3 proteins (A3C, A3F or A3H) or their catalytically inactive derivatives (mA3C, mA3F or mA3H) were incubated with purified HCoV-NL63 N (produced in Escherichia coli)³⁹ for 1 h at 37 °C. Subsequently, agarose spheres coated with anti-HA antibodies were added, and proteins were co-precipitated using spin columns. Samples were separated by electrophoresis and transferred onto PVDF membrane, and HCoV-NL63 N and HA-tagged APOBEC3 proteins were detected with specific antibodies. PC = positive control protein. (b) LLC-Mk2 cells were transfected with capped and polyadenylated mRNA encoding HCoV-NL63 N (Ctrl NL63 N), A3C (Ctrl A3C) or both transcripts (NL63 N + A3C), and incubated for 72 h at 37 °C. Cells were then fixed and immunostained for A3C (red), HCoV-NL63 N (green) and nuclei (blue). Co-localisation of A3C with HCoV-NL63-N was determined by confocal microscopy and marked with arrows. M, Manders’ coefficient (A3C overlapping with the virus). A representative image is shown.