Proteolytic processing of a Murray Valley encephalitis virus non-structural polyprotein segment containing the viral proteinase: accumulation of a NS3-4A precursor which requires mature NS3 for efficient processing.
The proteolytic processing of a non-structural polyprotein segment from the cytoplasmic domain of NS2A to the C terminus of NS5 of Murray Valley encephalitis (MVE) virus was examined, when expressed from cDNA via a vaccinia virus recombinant, in transiently transfected COS cells, or synthesized by cell-free translation. Cleavages mediated by the virus-encoded proteinase domain in NS3 at the junctions of NS2A-2B, NS2B-3 and NS4B-5 were catalysed efficiently. However, the cleavage at the NS3-4A junction, also mediated by the NS3 proteinase, was greatly delayed. Little or no NS3 was found, but an 85K precursor molecule accumulated; this was identified as NS3-4A. Termination codons were introduced by site-directed mutagenesis at the junctions of the NS3-4A, NS4A-4B and NS4B-5 genes to generate C-terminal truncations of the MVE virus polyprotein segment. In expression studies of these constructs the predicted NS3-mediated proteolytic cleavages were catalysed, except for that at the NS3-4A junction. In co-infections and co-transfections with constructs encoding the MVE virus nonstructural polyprotein region truncated at the C termini of NS3 or NS4A, efficient processing at the NS3-4A site was induced. Thus it appears that the MVE virus polyprotein is cleaved inefficiently in cis at the NS3-4A junction, whereas the site is processed efficiently in trans by mature NS3. The NS3-4A precursor is also seen in flavivirus-infected cells. Its function remains to be determined, but it could play a role in the replication of flavivirus, in view of the importance of polyprotein processing in the regulation of gene expression of positive-stranded RNA viruses, the modulation of processing at the NS3-4A site by NS3 or NS3-containing precursors described in the present study and the importance of NS3 as an integral part of the viral polymerase complex.
Lobigs
M
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Division of Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra.
D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D002478:Cells, Cultured; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D010450:Endopeptidases / Q000378:metabolism*; D005416:Flavivirus / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D020365:RNA Helicases; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D004277:DNA, Single-Stranded; D010450:Endopeptidases; D011498:Protein Precursors; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases