This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Khromykh, A. A.
Right arrow Articles by Westaway, E. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Khromykh, A. A.
Right arrow Articles by Westaway, E. G.

 Previous Article  |  Next Article 

Journal of Virology, December 1999, p. 10272-10280, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Efficient trans-Complementation of the Flavivirus Kunjin NS5 Protein but Not of the NS1 Protein Requires Its Coexpression with Other Components of the Viral Replicasedagger

Alexander A. Khromykh,1,* Petra L. Sedlak,1 Kimberley J. Guyatt,1 Roy A. Hall,2 and Edwin G. Westaway1

Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Queensland 4029,1 and Department of Microbiology, University of Queensland, St. Lucia, Brisbane, Queensland 4072,2 Australia

Received 6 July 1999/Accepted 8 September 1999

Successful trans-complementation of the defective Kunjin virus (KUN) RNA FLdGDD with a deletion of the RNA polymerase motif GDD in the NS5 gene by using a BHK cell line, repBHK, that continuously produced a functionally active KUN replication complex (RC) from replicon RNA was recently reported (A. A. Khromykh, M. T. Kenney, and E. G. Westaway, J. Virol. 72:7270-7279, 1998). In order to identify whether this complementation of FLdGDD RNA was provided by the wild-type NS5 protein alone or with the help of other nonstructural (NS) proteins also expressed in repBHK cells, we generated BHK cell lines stably producing the individual NS5 protein (SRns5BHK) or the NS1-NS5 polyprotein (SRns1-5BHK) by using a heterologous expression vector based on a modified noncytopathic Sindbis replicon. Western blot analysis with anti-NS5 antibodies showed that the level of production of NS5 was significantly higher in SRns5BHK cells than in SRns1-5BHK cells. Despite the higher level of expressed NS5, trans-complementation of FLdGDD RNA was much less efficient in SRns5BHK cells than in SRns1-5BHK cells and produced at least 100-fold less of the secreted complemented virus. In contrast, efficient complementation of KUN RNA with lethal cysteine-to-alanine mutations in the NS1 gene was achieved both in BHK cells producing the individual KUN NS1 protein from the Sindbis replicon vector and in repBHK cells, with both cell lines expressing similar amounts of NS1 protein. These results clearly demonstrate that flavivirus NS5 coexpressed with other components of the viral replicase possesses much higher functional (trans-complementing) activity than individually expressed NS5 and that efficient trans-complementation of mutated flavivirus NS1 and NS5 proteins occurs by different mechanisms. The results are interpreted and discussed in relation to our proposed model of formation of the flavivirus RC largely based on previous ultrastructural and biochemical analyses of KUN replication.

* Corresponding author. Mailing address: Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Herston Rd., Brisbane, Queensland 4029, Australia. Phone: (617) 3253-1568. Fax: (617) 3253-1401. E-mail: a.khromykh{at}mailbox.uq.edu.au.

dagger This is publication no. 96 from the Sir Albert Sakzewski Virus Research Centre.

Journal of Virology, December 1999, p. 10272-10280, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Ashour, J., Laurent-Rolle, M., Shi, P.-Y., Garcia-Sastre, A. (2009). NS5 of Dengue Virus Mediates STAT2 Binding and Degradation. J. Virol. 83: 5408-5418 [Abstract] [Full Text]  
  • Wilson, J. R., de Sessions, P. F., Leon, M. A., Scholle, F. (2008). West Nile Virus Nonstructural Protein 1 Inhibits TLR3 Signal Transduction. J. Virol. 82: 8262-8271 [Abstract] [Full Text]  
  • Hershkovitz, O., Zilka, A., Bar-Ilan, A., Abutbul, S., Davidson, A., Mazzon, M., Kummerer, B. M., Monsoengo, A., Jacobs, M., Porgador, A. (2008). Dengue Virus Replicon Expressing the Nonstructural Proteins Suffices To Enhance Membrane Expression of HLA Class I and Inhibit Lysis by Human NK Cells. J. Virol. 82: 7666-7676 [Abstract] [Full Text]  
  • Leung, J. Y., Pijlman, G. P., Kondratieva, N., Hyde, J., Mackenzie, J. M., Khromykh, A. A. (2008). Role of Nonstructural Protein NS2A in Flavivirus Assembly. J. Virol. 82: 4731-4741 [Abstract] [Full Text]  
  • Hoenen, A., Liu, W., Kochs, G., Khromykh, A. A., Mackenzie, J. M. (2007). West Nile virus-induced cytoplasmic membrane structures provide partial protection against the interferon-induced antiviral MxA protein. J. Gen. Virol. 88: 3013-3017 [Abstract] [Full Text]  
  • Malet, H., Egloff, M.-P., Selisko, B., Butcher, R. E., Wright, P. J., Roberts, M., Gruez, A., Sulzenbacher, G., Vonrhein, C., Bricogne, G., Mackenzie, J. M., Khromykh, A. A., Davidson, A. D., Canard, B. (2007). Crystal Structure of the RNA Polymerase Domain of the West Nile Virus Non-structural Protein 5. J. Biol. Chem. 282: 10678-10689 [Abstract] [Full Text]  
  • Chung, K. M., Liszewski, M. K., Nybakken, G., Davis, A. E., Townsend, R. R., Fremont, D. H., Atkinson, J. P., Diamond, M. S. (2006). From the Cover: West Nile virus nonstructural protein NS1 inhibits complement activation by binding the regulatory protein factor H. Proc. Natl. Acad. Sci. USA 103: 19111-19116 [Abstract] [Full Text]  
  • Pijlman, G. P., Kondratieva, N., Khromykh, A. A. (2006). Translation of the Flavivirus Kunjin NS3 Gene in cis but Not Its RNA Sequence or Secondary Structure Is Essential for Efficient RNA Packaging. J. Virol. 80: 11255-11264 [Abstract] [Full Text]  
  • Samuel, M. A., Diamond, M. S. (2006). Pathogenesis of West Nile Virus Infection: a Balance between Virulence, Innate and Adaptive Immunity, and Viral Evasion. J. Virol. 80: 9349-9360 [Full Text]  
  • Chung, K. M., Nybakken, G. E., Thompson, B. S., Engle, M. J., Marri, A., Fremont, D. H., Diamond, M. S. (2006). Antibodies against West Nile Virus Nonstructural Protein NS1 Prevent Lethal Infection through Fc {gamma} Receptor-Dependent and -Independent Mechanisms. J. Virol. 80: 1340-1351 [Abstract] [Full Text]  
  • Whitby, K., Pierson, T. C., Geiss, B., Lane, K., Engle, M., Zhou, Y., Doms, R. W., Diamond, M. S. (2005). Castanospermine, a Potent Inhibitor of Dengue Virus Infection In Vitro and In Vivo. J. Virol. 79: 8698-8706 [Abstract] [Full Text]  
  • Jones, M., Davidson, A., Hibbert, L., Gruenwald, P., Schlaak, J., Ball, S., Foster, G. R., Jacobs, M. (2005). Dengue Virus Inhibits Alpha Interferon Signaling by Reducing STAT2 Expression. J. Virol. 79: 5414-5420 [Abstract] [Full Text]  
  • Appel, N., Herian, U., Bartenschlager, R. (2005). Efficient Rescue of Hepatitis C Virus RNA Replication by trans-Complementation with Nonstructural Protein 5A. J. Virol. 79: 896-909 [Abstract] [Full Text]  
  • Liu, W. J., Chen, H. B., Wang, X. J., Huang, H., Khromykh, A. A. (2004). Analysis of Adaptive Mutations in Kunjin Virus Replicon RNA Reveals a Novel Role for the Flavivirus Nonstructural Protein NS2A in Inhibition of Beta Interferon Promoter-Driven Transcription. J. Virol. 78: 12225-12235 [Abstract] [Full Text]  
  • Evans, M. J., Rice, C. M., Goff, S. P. (2004). Genetic Interactions between Hepatitis C Virus Replicons. J. Virol. 78: 12085-12089 [Abstract] [Full Text]  
  • Ortego, J., Escors, D., Laude, H., Enjuanes, L. (2002). Generation of a Replication-Competent, Propagation-Deficient Virus Vector Based on the Transmissible Gastroenteritis Coronavirus Genome. J. Virol. 76: 11518-11529 [Abstract] [Full Text]  
  • Liu, W. J., Sedlak, P. L., Kondratieva, N., Khromykh, A. A. (2002). 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. J. Virol. 76: 10766-10775 [Abstract] [Full Text]  
  • Blitvich, B. J., Scanlon, D., Shiell, B. J., Mackenzie, J. S., Pham, K., Hall, R. A. (2001). Determination of the intramolecular disulfide bond arrangement and biochemical identification of the glycosylation sites of the nonstructural protein NS1 of Murray Valley encephalitis virus. J. Gen. Virol. 82: 2251-2256 [Abstract] [Full Text]  
  • Hurrelbrink, R. J., McMinn, P. C. (2001). Attenuation of Murray Valley Encephalitis Virus by Site-Directed Mutagenesis of the Hinge and Putative Receptor-Binding Regions of the Envelope Protein. J. Virol. 75: 7692-7702 [Abstract] [Full Text]  
  • Khromykh, A. A., Meka, H., Guyatt, K. J., Westaway, E. G. (2001). Essential Role of Cyclization Sequences in Flavivirus RNA Replication. J. Virol. 75: 6719-6728 [Abstract] [Full Text]  
  • Khromykh, A. A., Varnavski, A. N., Sedlak, P. L., Westaway, E. G. (2001). Coupling between Replication and Packaging of Flavivirus RNA: Evidence Derived from the Use of DNA-Based Full-Length cDNA Clones of Kunjin Virus. J. Virol. 75: 4633-4640 [Abstract] [Full Text]  
  • Khromykh, A. A., Sedlak, P. L., Westaway, E. G. (2000). cis- and trans-Acting Elements in Flavivirus RNA Replication. J. Virol. 74: 3253-3263 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»