A short form of the tick‐borne encephalitis virus NS3 protein

Abstract

Using monoclonal untibodics to the tick-borne encephalitis virus (TBE) nonstructural protein NS3 two forms of this protein were rcvc&d in TBE-infcctcd mammalian cells: a full-length rorm (69 kDa) and a short form (49 kDa) which has not been observed before and was tilled NW. Rceombinant plasmids were constructed and various fragmcnu of the TBE NS3 gent were cxpresscd in rabbit rcticulocytc lysate. By analyzing immune precipitates of '5S-labclcd translation products. we could monitor and localize internal clcuvage of NS3, due to which the NS3' protein was gcneratcd.

cis - and trans -Acting Elements in Flavivirus RNA Replication

“…Although a helper NS2B-full-length NS3 protease complex is obviously produced in trans from the replicon RNA in repBHK cells, it is possible that translation in cis of an NS2B-full-length NS3 complex is required for proper cleavage of core protein translated upstream from the same mRNA and for subsequent release of secreted mature virions. The NS3 deletions in the helicase domain also removed the conserved dibasic cleavage site QRR2GR (in the helicase motif VI) which generates the truncated NS3Ј product of ϳ460 residues shown to be present in tick-borne encephalitis virus-and dengue virus-infected cells (2,12,36,42). Since the function of this truncated NS3Ј protein has not been established, NS3Ј may play some role in virus assembly and/or secretion when produced from the same RNA molecule.…”

Complementation Analysis of the Flavivirus Kunjin NS3 and NS5 Proteins Defines the Minimal Regions Essential for Formation of a Replication Complex and Shows a Requirement of NS3 in cis for Virus Assembly

“…One of the suggested explanations for the lack of virus assembly and/or release was possible involvement of C-terminally truncated NS3 product, NS3Ј or p50, resulting from an alternative cleavage at QRR2GR site (Fig. 4B) and shown to be present in tick-borne encephalitis virus-and dengue virusinfected cells (1,30,35) and in KUN-infected cells (W. J. Liu and A. A. Khromykh, unpublished results) in virus assembly.…”

“…In one of the genomic length constructs with deletions (a derivative of repdNS1.1/3.8, Fig. 4) we retained the proposed cleavage site in the helicase domain QRR2GR to allow the release of truncated NS3Ј protein (1,30,35) in order to examine whether NS3Ј may have a function in virus assembly. However, despite demonstrable complementation of RNA replication in repBHK cells, no secreted or intracellular virus particles were detected in this experiment, thus demonstrating that allowing production and release of NS3Ј did not rescue the inability of NS3-deleted RNA to assemble into virions.…”

“…The protease activity resides in the first 167 to 180 amino acids (5,6,10,11,40), while the C-terminal region commencing from codons 160 to 170 contains motifs for nucleoside triphosphatase, RNA helicase, and RNA-stimulated triphosphatase (4,9,12,27,38,39). C-terminally truncated NS3 product (NS3Ј or p50), resulting from an alternative cleavage (QRR2GR [arrow marks cleavage site]) have been detected in tick-borne encephalitis virus-and dengue virus-infected cells (1,30,35), but the function of this truncated protein is not known.Our previous studies demonstrated that replication of the defective KUN genomic RNA with NS5 protein truncated to retain only the first 397 amino acids was efficiently complemented by the helper RC produced from the KUN replicon (subgenomic) RNA in repBHK cells, while genomic RNA with a further truncation leaving only the first 227 amino acids in NS5 was complemented very inefficiently (20). Amino acid sequence comparisons of the N-terminal region of NS5 proteins from different flaviviruses revealed the presence of three small (10 to 20 amino acids) highly conserved domains, which we named a (KUN amino acids 141 to 151), b (KUN amino…”

“…The protease activity resides in the first 167 to 180 amino acids (5,6,10,11,40), while the C-terminal region commencing from codons 160 to 170 contains motifs for nucleoside triphosphatase, RNA helicase, and RNA-stimulated triphosphatase (4,9,12,27,38,39). C-terminally truncated NS3 product (NS3Ј or p50), resulting from an alternative cleavage (QRR2GR [arrow marks cleavage site]) have been detected in tick-borne encephalitis virus-and dengue virus-infected cells (1,30,35), but the function of this truncated protein is not known.…”