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Filippini, Sara (2008) Production of HCV infectious viral particles through
trans-complementation of gpE1/gpE2 and characterization of early events that follow HCV binding to target cells.
[Ph.D. thesis]

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Abstract (english)

Trans-complementation of gpE1/gpE2 allows recovery of infectious Hepatitis C virus

Although observed for other members of the Flaviviridae including bovine diarrea virus and Kunjin virus, trans-complementation of replication-defective replicons was never observed for hepatitis C virus (HCV). This suggested an isolated and independent nature of the HCV replication complexes, with little or not exchange of factors between them. In particular, HCV non structural proteins seem to remain associated to their respective complexes and may not be able to access other complexes.

In the present work we demonstrated for the first time for HCV that when the structural proteins E1 and E2 are provided in trans by the use of complementing cell lines constitutively expressing them it is possible to achieve production of infectious viral particles.

Using a 293T cells retroviral system we created three packaging cell lines constitutively expressing the two surface proteins E1 and E2 from genotype 2a (S6.1/E1E2:2a), GT 1a (S6.1/E1E2:1a), and GT 1b (S6.1/E1E2:1b).
When viral backbone RNA lacking the E1E2 genes (JFH?E1E2) deriving from the JFH-1 isolate (GT 2a) was transfected into S6.1/E1E2:2a cells, HCV infectious particles were released, indicating that for successful virus assembly, budding and release the structural glycoproteins E1 and E2 can be provided in trans. These results were consistent with what we observed in S6.1 naïve cells transfected with the full length RNA genome of JFH-1, use as positive control. The release of infectious viral particles, though, could not be detected in S6.1/E1E2:1a and S6.1/E1E2:1b transfected with JFH?E1E2.
We can hypotize that in the case of heterologous trans-complementation, genetic incompatibility between JFH-1 backbone and the structural proteins of GT 1a and GT 1b could have totally or partially affected the production of viral particles in our system, although these is only a speculative hypothesis that need to be verified in other experiments of homologous and heterologous trans-complementation, characterizing the relationships among HCV proteins and other viral factors

Characterization of early events that follow HCV binding to target cells

As we have previously seen, by transfecting permissive cells with the mRNA of the HCV isolate JFH-1 it is possible to produce infectious viral particles that might be used for experiments on the viral life cycle, as binding, attachment and entry. One of the main advantages of the cell culture-derived HCV (HCVcc) in fact is probably the mechanisms of interaction of these viral particles with the host cell receptors that closely mimic what happens in a natural infection event.
Besides the two main HCV receptors identified so far, CD81 and SR-BI, many cellular factors act in concert to mediate HCV binding and entry into hepatocytes. Recently Evans et al. (2007) identified a tight junction protein that is highly expressed in liver cells, Claudin-1 (CLDN-1), which seems to act late in the entry process, after virus binding and interaction with the HCV co-receptor CD81.
In the present study we demonstrated that there is a strict connection among CD81 and CLND-1 during the early events of viral attachment and entry. In fact engagement of CD81 by the use of the recombinant protein E2, the E1E2 heterodimer complex or anti-CD81 antibody led to a translocation of this receptor, normally present on the whole cellular surface, to the areas of cell-cell junctions, where CLDN-1 dwells.
The use of chemical inhibitors of actin polymerization, such as Latrunculin A and Jasplakinolide, proved the involvement of the actin cytoskeleton in CD81 translocation. Time course experiments with LatA demonstrated that an intact and functional cytoskeleton is required at a very early stage in the relocalization process, beside having also a relevant physiological importance on viral internalization. In fact, pretreatment of cells with these specific actin inhibitors as well as silencing the expression of some proteins of the Rho GTPases family, such as Rho, Rac ad Cdc42, which normally modulate the actin rearrangement, greatly reduced HCVcc infectivity.

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EPrint type:Ph.D. thesis
Tutor:Montecucco, Cesare
Supervisor:Crotta, Stefania
Ph.D. course:Ciclo 20 > Scuole per il 20simo ciclo > BIOSCIENZE > BIOLOGIA CELLULARE
Data di deposito della tesi:2008
Anno di Pubblicazione:2008
Key Words:HCV trans-complementation, CD81 relocalization
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/11 Biologia molecolare
Struttura di riferimento:Dipartimenti > pre 2012 - Dipartimento di Scienze Biomediche Sperimentali
Codice ID:639
Depositato il:25 Sep 2008
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