This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplemental material
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
Google Scholar
Right arrow Articles by Pacini, L.
Right arrow Articles by Paonessa, G.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pacini, L.
Right arrow Articles by Paonessa, G.

 Previous Article  |  Next Article 

Journal of Virology, September 2009, p. 9079-9093, Vol. 83, No. 18
0022-538X/09/$08.00+0     doi:10.1128/JVI.00308-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Naturally Occurring Hepatitis C Virus Subgenomic Deletion Mutants Replicate Efficiently in Huh-7 Cells and Are trans-Packaged In Vitro To Generate Infectious Defective Particles{triangledown} ,{dagger}

Laura Pacini,1 Rita Graziani,1 Linda Bartholomew,1 Raffaele De Francesco,2 and Giacomo Paonessa1*

Istituto di Ricerche di Biologia Molecolare P. Angeletti, Merck Research Laboratories, Via Pontina km 30,600, I-00040 Pomezia (Roma), Italy,1 Istituto Nazionale Genetica Molecolare, Via Francesco Sforza 28, I-20122 Milano, Italy2

Received 12 February 2009/ Accepted 30 June 2009

Naturally occurring hepatitis C virus (HCV) subgenomic RNAs have been found in several HCV patients. These subgenomic deletion mutants, mostly lacking the genes encoding envelope glycoproteins, were found in both liver and serum, where their relatively high abundance suggests that they are capable of autonomous replication and can be packaged and secreted in viral particles, presumably harboring the envelope proteins from wild type virus coinfecting the same cell. We recapitulated some of these natural subgenomic deletions in the context of the isolate JFH-1 and confirmed these hypotheses in vitro. In Huh-7.5 cells, these deletion-containing genomes show robust replication and can be efficiently trans-packaged and infect naïve Huh-7.5 cells when cotransfected with the full-length wild-type J6/JFH genome. The genome structure of these natural subgenomic deletion mutants was dissected, and the maintenance of both core and NS2 regions was proven to be significant for efficient replication and trans-packaging. To further explore the requirements needed to achieve trans-complementation, we provided different combinations of structural proteins in trans. Optimal trans-complementation was obtained when fragments of the polyprotein encompassing core to p7 or E1 to NS2 were expressed. Finally, we generated a stable helper cell line, constitutively expressing the structural proteins from core to p7, which efficiently supports trans-complementation of a subgenomic deletion encompassing amino acids 284 to 732. This cell line can produce and be infected by defective particles, thus representing a powerful tool to investigate the life cycle and relevance of natural HCV subgenomic deletion mutants in vivo.

* Corresponding author. Mailing address: I.R.B.M. (Istituto di Ricerche di Biologia Molecolare) P. Angeletti, Via Pontina km 30,600, I-00040 Pomezia (Roma), Italy. Phone: 390691093223. Fax: 390691093654. E-mail: giacomo_paonessa{at}merck.com

{triangledown} Published ahead of print on 8 July 2009.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, September 2009, p. 9079-9093, Vol. 83, No. 18
0022-538X/09/$08.00+0     doi:10.1128/JVI.00308-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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