Molecular dissection of dengue virus egress : involvement of host cellular factors-KDEL receptors

Abstract

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.