Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

The life cycle of enveloped viruses is a complex process relying on specific interactions with host factors that, in turn, represent potential targets for interfering with viral replication and pathogenesis. Although the molecular identity of cellular receptors involved in virion entry has been established for many viruses, few studies have investigated whether host proteins on intracellular compartments may function as receptors to facilitate viral trafficking and release from infected cells. In particular, viral-host interactions during dengue virus (DENV) egress are still poorly characterized and most cellular targets identified in high-throughput screens have not been mapped to the secretory pathway. DENV structural glycoproteins, pre-membrane (prM) and envelope (E), are sufficient to assemble native Recombinant Subviral Particles (RSPs) in the endoplasmic reticulum (ER). Newly formed RSPs mimic nascent virions and traffic through the secretory pathway, where they are processed into mature particles, before being released from cells. This study demonstrated that DENV requires host KDEL receptors (KDELRs), which cycle between the ER and Golgi apparatus to retrieve resident ER proteins, for vesicular transport from ER to Golgi. Depletion of KDELRs by siRNA reduced egress of both DENV progeny virions and RSPs produced in stable cell lines expressing prM and E. A direct interaction between KDELRs and DENV prM was demonstrated in either prME expressing or DENV infected cells by co-immunoprecipitation (co-IP) experiments. By immunoblotting with specific antibodies we first showed that KDELRs interacted with prM portion. By interfering with RSPs’ maturation, we then obtained conclusive evidence that interaction was restricted to the pr fragment which released from the mature RSPs after cleavage of prM by cellular protease furin. This finding was further confirmed by GST pull down assay, which mapped the interacting domain to the N-terminal 40 residues of the pr fragment, and by mutagenesis experiments, which showed that KDELRs interact with prM through three positively charged amino-terminal residues. Biochemical analysis and immunofluorescence microscopy indicated that mutations impairing KDELRs/prM binding did not affect RSPs formation, and translocation within the ER, whereas they strongly inhibited trafficking from ER to Golgi apparatus and, consequently, their release into the supernatant. Moreover, perturbation of KDELR cycle by siRNA-mediated depletion of class II Arfs, which accumulates KDELRs in the Golgi, phenocopied results obtained with both an interaction-deficient mutant and KDELR knockdowns. Finally, we compared the effect of KDELRs on all four DENV serotypes and found significant reductions of DENV1-3, but not DENV4, in keeping with the co-IP results that demonstrated, using RSP-producing cell lines, that only DENV1-3 prM proteins interacted with KDELRs. Of note, KDELRs depletion did not affect West Nile Virus progeny virus egress, suggesting that KDELRs might not be utilized by all Flavivirus. Taken together, several lines of evidence have been presented to indicate that the loss of interaction with KDELRs reduced DENV transport from ER to Golgi and, consequently, release from infected cells. These findings, therefore, have uncovered a novel function for KDELRs as an internal receptor required for DENV trafficking and identified a rate-limiting molecular step in the late stages of DENV lifecycle. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

Abstract not available

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: TNF-alpha; Type-I IFN; Dengue virus; differential display method. Includes bibliographical references (p. 64-74).

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Dengue is the most common mosquito-borne human disease, leading to 2.5 billion people at risk, 50-100 millions infections each year worldwide and among them, 500 000 severe dengue cases (dengue hemorrhagic fever, DHF/ dengue shock syndrome, DSS) plus more than 20 000 deaths. It can be caused by any of four dengue virus serotypes, which are antigenicly distinct and belong to the Flaviviridae family, genus Flavivirus. However, up till now there is no specific drug and vaccine against dengue. Understanding mechanisms developed by dengue virus to exploit host cells during all stages of the replication cycle is a first step towards the rationale design of anti-viral strategies. Very little is known about the late stages, which consist of assembly, budding and secretion of the virus. It is therefore very important to develop tools in order to study the egress of the virus. In this study, I investigated a stable cell line named Hela-prME that expresses serotype 1 dengue virus (DENV-1) prM and E native structural envelope proteins and constitutively produces dengue recombinant subviral particles (RSPs). Biochemical characterization of DENV-1 RSPs has validated that this cell line is a potential tool to study the dengue viral late-stage. Indeed, the maturation process observed with RSPs is similar to the pathway described for real virus (cleavage of prM fragment, homodimerization of E, acquisition of complex sugars). To better understand and depict the dengue virus late-stage secretion, I combined various electron microscopy (EM) techniques e.g. classical transmission electron microscopy (TEM), negative staining, immunogold labeling on cryo-ultrathin sections (Tokuyashu method) and tomography (ET) with such RSPs tool. The EM results obtained illustrate that electron dense particles and tubules labeled by antibodies directed against E and prM proteins were abundantly found in the lumen of endoplasmic reticulum (ER)-related cisternae of HeLa prME cells. Epositive particles were also found in other structures such as Golgi stacks and vesicles nearby as well as in aggregates with electron dense materials inside and surrounded by membrane. These particles are most likely corresponding to DENV-1 RSPs whereas the tubules may be other structures induced by assembly of prM and E proteins. This study has clearly shown that DENV-1 RSPs assemble in the ER and transport through the secretory pathway before being released. This work further validates the use of dengue RSPs and RSPs-producing cells as a model to study viral egress. / published_or_final_version / Pathology / Master / Master of Philosophy

No description available.

No description available.

No description available.

Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.