Research Projects

Acquistion of secretion proteins from Japanese Encephalitis Virus-infected cells

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus, a member of the family Flaviviridae, and causes serious viral encephalitis in humans. It is well known that upon viral infection host cell protein expression is induced leading to the production of cytoplasmic proteins and secretory inflammatory cytokines. There is growing evidence that virus particles associate and/or contain host proteins. These proteins may provide viruses with means to escape host immune defense or with mechanism for its release as well as cell entry. Until now, no global profile of secreted proteins in the cultured medium of JEV-infected cells has been measured. To this end, we characterize the effects of JEV infection on the profile of protein secretion of BHK-21 cells by developing a serum-free culture method in combination with LC-MS/MS. As the preliminary results were shown, five secreted proteins, including the molecular chaperones Hsp90, Hsp70, and GRP78, have been identified in the cultured medium from JEV-infected BHK-21 cells. The functional role of GRP78 in the JEV life cycle was further investigated as playing role in facilitating the JE virus infectivity. In this proposal, we will further characterize the global profile of secreted proteins from JEV-infected BHK-21 cells according to our established proteomic approaches; and dissect the functional role(s) of JEV-induced secreted molecular chaperones involving in the JEV life cycle. Finally, the possible mechanism of secretion of JE virus particle depends on co-assembly of molecular chaperones will be illustrated by the end of this project. Overall, the identification and functional analysis of secreted proteins from JEV-infected cells may reveal a role for host cell proteins in JEV pathogenesis.

Dissecting the Cellular Homeostasis Mechanisms Underlying Enterovirus Replication and Infection

Enteroviruses depend entirely on the host protein homeostasis machinery, composed of molecular chaperones and quality control (QC) components such as the ubiquitin-proteasome system, for viral protein production and function. Enterovirus replication poses several challenges to the cellular protein homeostasis machinery as the need to produce high amounts of protein in a very short time places a big burden for the host protein production and folding machineries. Furthermore, enteroviral proteins tend to be large, complex and multifunctional, and thus likely to require the assistance of molecular chaperones to fold. Indeed, we have shown that the Hsp90 isoform is essential for capsid folding and assembly for many, perhaps most, picornaviruses, including the enteroviruses and coxsakievirus. Since other aspects of enterovirus replication involve additional large multiprotein complexes, chaperones are likely to be broadly required for other aspects of the viral cycle. An important challenge to protein homeostasis in RNA virus arises from their very high mutation rates, which pose a big burden to viral protein stability and are likely to produce high levels of non-functional or destabilized proteins. These mutant proteins must be either maintained in a functional state or eliminated from the cell to prevent dominant negative effects on viral function. We hypothesize that these functions are carried out by chaperones, which can buffer metastable proteins, as well as by the ubiquitin-proteasome system, which targets misfolded proteins for degradation. To understand the molecular and cellular mechanisms by which cellular chaperone and quality control machineries control viral protein homeostasis, and allow the RNA virus to replicate we propose the following Aims: Aim 1: Define the chaperone components required for enterovirus replication. Aim 2: Define the role of the Quality control (QC) machinery in Enterovirus replication. Aim 3: Examine the plasticity and interplay of chaperone and QC pathways during viral infection.