1887

Abstract

Mature protein C of tick-borne encephalitis virus (TBEV) is cleaved from the polyprotein precursor by the viral NS2B/3 protease (NS2B/3). We showed previously that replacement of the NS2B/3 cleavage site at the C terminus of protein C by the foot-and-mouth disease virus (FMDV) 2A StopGo sequence leads to the production of infectious virions. Here, we show that infectious virions can also be produced from a TBEV mutant bearing an inactivated 2A sequence through the expression of the FMDV 3C protease (3C) either or (from a TBEV replicon). Cleavage at the C terminus of protein C depended on the catalytic activity of 3C as well as on the presence of an optimized 3C cleavage site. Passage of the TBEV mutants bearing a 3C cleavage site either in the absence of 3C or in the presence of a catalytically inactive 3C led to the appearance of revertants in which protein C cleavage by NS2B/3 had been regained. In three different revertants, a cleavage site for NS2B/3, namely RR*C, was now present, leading to an elongated protein C. Furthermore, two revertants acquired additional mutations in the C terminus of protein C, eliminating two basic residues. Although these latter mutants showed wild-type levels of early RNA synthesis, their foci were smaller and an accumulation of protein C in the cytoplasm was observed. These findings suggest a role of the positive charge of the C terminus of protein C for budding of the nucleocapsid and further support the notion that TBEV protein C is a multifunctional protein.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.038398-0
2012-03-01
2019-12-08
Loading full text...

Full text loading...

/deliver/fulltext/jgv/93/3/504.html?itemId=/content/journal/jgv/10.1099/vir.0.038398-0&mimeType=html&fmt=ahah

References

  1. Amberg S. M., Rice C. M.. ( 1999;). Mutagenesis of the NS2B-NS3-mediated cleavage site in the flavivirus capsid protein demonstrates a requirement for coordinated processing. . J Virol 73:, 8083–8094.[PubMed]
    [Google Scholar]
  2. Atkins J. F., Wills N. M., Loughran G., Wu C. Y., Parsawar K., Ryan M. D., Wang C. H., Nelson C. C.. ( 2007;). A case for “StopGo”: reprogramming translation to augment codon meaning of GGN by promoting unconventional termination (Stop) after addition of glycine and then allowing continued translation (Go). . RNA 13:, 803–810. [CrossRef][PubMed]
    [Google Scholar]
  3. Birtley J. R., Knox S. R., Jaulent A. M., Brick P., Leatherbarrow R. J., Curry S.. ( 2005;). Crystal structure of foot-and-mouth disease virus 3C protease. New insights into catalytic mechanism and cleavage specificity. . J Biol Chem 280:, 11520–11527. [CrossRef][PubMed]
    [Google Scholar]
  4. Chappell K. J., Nall T. A., Stoermer M. J., Fang N. X., Tyndall J. D., Fairlie D. P., Young P. R.. ( 2005;). Site-directed mutagenesis and kinetic studies of the West Nile Virus NS3 protease identify key enzyme-substrate interactions. . J Biol Chem 280:, 2896–2903. [CrossRef][PubMed]
    [Google Scholar]
  5. Curry S., Roqué-Rosell N., Zunszain P. A., Leatherbarrow R. J.. ( 2007;). Foot-and-mouth disease virus 3C protease: recent structural and functional insights into an antiviral target. . Int J Biochem Cell Biol 39:, 1–6. [CrossRef][PubMed]
    [Google Scholar]
  6. Dokland T., Walsh M., Mackenzie J. M., Khromykh A. A., Ee K. H., Wang S.. ( 2004;). West Nile virus core protein; tetramer structure and ribbon formation. . Structure 12:, 1157–1163. [CrossRef][PubMed]
    [Google Scholar]
  7. Donnelly M. L., Luke G., Mehrotra A., Li X., Hughes L. E., Gani D., Ryan M. D.. ( 2001;). Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. . J Gen Virol 82:, 1013–1025.[PubMed]
    [Google Scholar]
  8. Filocamo G., Pacini L., Migliaccio G.. ( 1997;). Chimeric Sindbis viruses dependent on the NS3 protease of hepatitis C virus. . J Virol 71:, 1417–1427.[PubMed]
    [Google Scholar]
  9. Gehrke R., Heinz F. X., Davis N. L., Mandl C. W.. ( 2005;). Heterologous gene expression by infectious and replicon vectors derived from tick-borne encephalitis virus and direct comparison of this flavivirus system with an alphavirus replicon. . J Gen Virol 86:, 1045–1053. [CrossRef][PubMed]
    [Google Scholar]
  10. Hahm B., Back S. H., Lee T. G., Wimmer E., Jang S. K.. ( 1996;). Generation of a novel poliovirus with a requirement of hepatitis C virus protease NS3 activity. . Virology 226:, 318–326. [CrossRef][PubMed]
    [Google Scholar]
  11. Kofler R. M., Heinz F. X., Mandl C. W.. ( 2002;). Capsid protein C of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence. . J Virol 76:, 3534–3543. [CrossRef][PubMed]
    [Google Scholar]
  12. Kroschewski H., Allison S. L., Heinz F. X., Mandl C. W.. ( 2003;). Role of heparan sulfate for attachment and entry of tick-borne encephalitis virus. . Virology 308:, 92–100. [CrossRef][PubMed]
    [Google Scholar]
  13. Lai V. C., Zhong W., Skelton A., Ingravallo P., Vassilev V., Donis R. O., Hong Z., Lau J. Y.. ( 2000;). Generation and characterization of a hepatitis C virus NS3 protease-dependent bovine viral diarrhea virus. . J Virol 74:, 6339–6347. [CrossRef][PubMed]
    [Google Scholar]
  14. Lee E., Stocks C. E., Amberg S. M., Rice C. M., Lobigs M.. ( 2000;). Mutagenesis of the signal sequence of yellow fever virus prM protein: enhancement of signalase cleavage In vitro is lethal for virus production. . J Virol 74:, 24–32. [CrossRef][PubMed]
    [Google Scholar]
  15. Lindenbach B. D., Thiel H. J., Rice C. M.. ( 2007;). Flaviviridae: the viruses and their replication. . In Fields Virology, pp. 1101–1152. Edited by Knipe D. M., Howley P. M... Philadelphia:: Lippincott Williams & Wilkins;.
    [Google Scholar]
  16. Lobigs M.. ( 1993;). Flavivirus premembrane protein cleavage and spike heterodimer secretion require the function of the viral proteinase NS3. . Proc Natl Acad Sci U S A 90:, 6218–6222. [CrossRef][PubMed]
    [Google Scholar]
  17. Lobigs M., Lee E.. ( 2004;). Inefficient signalase cleavage promotes efficient nucleocapsid incorporation into budding flavivirus membranes. . J Virol 78:, 178–186. [CrossRef][PubMed]
    [Google Scholar]
  18. Ma L., Jones C. T., Groesch T. D., Kuhn R. J., Post C. B.. ( 2004;). Solution structure of dengue virus capsid protein reveals another fold. . Proc Natl Acad Sci U S A 101:, 3414–3419. [CrossRef][PubMed]
    [Google Scholar]
  19. Mandl C. W.. ( 2005;). Steps of the tick-borne encephalitis virus replication cycle that affect neuropathogenesis. . Virus Res 111:, 161–174. [CrossRef][PubMed]
    [Google Scholar]
  20. Mandl C. W., Ecker M., Holzmann H., Kunz C., Heinz F. X.. ( 1997;). Infectious cDNA clones of tick-borne encephalitis virus European subtype prototypic strain Neudoerfl and high virulence strain Hypr. . J Gen Virol 78:, 1049–1057.[PubMed]
    [Google Scholar]
  21. Orlinger K. K., Hoenninger V. M., Kofler R. M., Mandl C. W.. ( 2006;). Construction and mutagenesis of an artificial bicistronic tick-borne encephalitis virus genome reveals an essential function of the second transmembrane region of protein e in flavivirus assembly. . J Virol 80:, 12197–12208. [CrossRef][PubMed]
    [Google Scholar]
  22. Schrauf S., Schlick P., Skern T., Mandl C. W.. ( 2008;). Functional analysis of potential carboxy-terminal cleavage sites of tick-borne encephalitis virus capsid protein. . J Virol 82:, 2218–2229. [CrossRef][PubMed]
    [Google Scholar]
  23. Schrauf S., Mandl C. W., Bell-Sakyi L., Skern T.. ( 2009;). Extension of flavivirus protein C differentially affects early RNA synthesis and growth in mammalian and arthropod host cells. . J Virol 83:, 11201–11210. [CrossRef][PubMed]
    [Google Scholar]
  24. Stocks C. E., Lobigs M.. ( 1998;). Signal peptidase cleavage at the flavivirus C-prM junction: dependence on the viral NS2B-3 protease for efficient processing requires determinants in C, the signal peptide, and prM. . J Virol 72:, 2141–2149.[PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.038398-0
Loading
/content/journal/jgv/10.1099/vir.0.038398-0
Loading

Data & Media loading...

Supplements

Supplementary Table 

PDF

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error