Differences in membrane association and sub-cellular distribution between NS2-3 and NS3 of bovine viral diarrhoea virus

The sub-cellular location and mechanism of membrane association of NS3 and NS2-3 polypeptides of bovine viral diarrhoea virus (BVDV) have been examined. Both NS3 and NS2-3 proteins were detected in post-nuclear membrane fractions but not in cytosolic fractions of BVDV infected cells; a proportion of NS3, but not NS2-3, could be dissociated from the membranes with 800 mM KCl or at pH 11. Following extraction with 1% Triton X-114, NS3 was predominantly present in the aqueous phase, but NS2-3 was only recovered in the detergent phase. Confocal microscopy showed that in BVDV infected cells, NS3 and/or NS2-3 co-localise with the endoplasmic reticulum (ER) protein, ERP60, but not Golgi or lysosomal proteins. Sub-cellular fractionation analysis demonstrated that NS2-3 was almost exclusively associated with the rough ER membrane but a significant proportion of NS3 was present in the smooth ER membrane fractions in addition to the rough ER membrane. These differences in the distribution of NS2-3 and NS3 on ER membranes in cells infected with cytopathogenic (CP) strains of BVDV were also observed using confocal microscopy and antibodies that are specific to either NS2 or NS3. This distinct distribution of NS3 and NS2-3 on the ER membrane has revealed a further difference between CP and non-cytopathogenic (NCP) strains of BVDV.