Identification and analysis of truncated and elongated species of the flavivirus NS1 protein

The flavivirus non-structural glycoprotein NS1 is often detected in Western blots as a heterogeneous cluster of bands due to glycosylation variations, precursor-product relationships and/or alternative cleavage sites in the v iral polyprotein, In this study, we determined the basis of structural hete rogeneity of the NS1 protein of Murray Valley encephalitis virus (MVE) by g lycosylation analysis, pulse-chase experiments and terminal amino acid sequ encing. Inhibition of N-linked glycosylation by tunicamycin revealed that N S1 synthesised in MVE-infected C6/36 cells was derived from two polypeptide backbones of 39 kDa (NS1 degrees) and 47 kDa (NS1'). Pulse-chase experimen ts established that no precursor-product relationship existed between NS1 d egrees and NS1' and that both were stable end products. Terminal sequencing revealed that the N- and C-termini of NS1 degrees were located at amino ac id positions 714 and 1145 in the polyprotein respectively, consistent with the predicted sites based upon sequence homology with other flaviviruses. E xpression of the NS1 gene alone or in conjunction with NS2A by recombinant baculoviruses demonstrated that the production of NS1' was dependent on the presence of NS2A, indicating that the C-terminus of the larger protein was generated within NS2A. A smaller form (31 kDa) of NS1 (Delta NS1) was also identified in MVE-infected Vero cultures, and amino acid sequencing reveal ed a 129-residue truncation at the N-terminus of this protein. This corresp onds closely with the in-frame 121-codon deletion at the 5' end of the NS1 gene of defective MVE viral RNA (described by Lancaster et al. in 1998), su ggesting that Delta NS1 may be a translation product of defective viral RNA . (C) 1999 Elsevier Science B.V. All rights reserved.