Aloke Bera, Research Scientist

The main focus of my research is development of an automated high-throughput method for identifying anti-viral (flavivirus) drugs based on NS3 protein.

Bong-Suk Kim, Post-Doctoral Researcher

We are focusing on the structural proteins of West Nile (WN). WN is a prototype of the flavivirus family. We are performing some biochemical studies of WNV capsid protein (CP). WNC CP was expressed in E.coli and purified. We are working on nucleotide binding properties of this protein and we are concentrating on the study of core assembly in vitro.

Chinmay Patkar, Graduate student

Flaviviruses, a group of small, enveloped animal viruses containing positive sense, single-stranded genomic RNA, comprise of many significant human pathogens including West Nile virus, Dengue, Yellow fever, etc. The virus genome encodes for three structural proteins- capsid, membrane and envelope- that form the virus particle and seven non-structural proteins- NS1-NS5 - that are involved in transcription/replication of the genome and possibly packaging of the genome into particles.

It has been proposed that there exists a coupling between replication and packaging of the flavivirus genomic RNA. In order to understand the various aspects of replication and packaging separately, we have constructed replicons of yellow fever virus containing luciferase or GFP reporter genes but lacking the coding region for the structural proteins, hence unable to form virus particles. The replication of these replicons can be assayed for by analyzing reporter gene expression. Using trans-complementation assays, these replicons are being employed for studying the various cis-trans signals in the genome required for replication.

To facilitate genome packaging, the Yellow fever virus structural proteins are supplied in trans, using a Sindbis helper replicon, which can then package the replicon and release pseudo-infectious particles. Such a packaging system is being utilized for studying the elements on the capsid protein required for packaging of the genomic RNA and assembly of the virus particles. The involvement of non-structural proteins in assembly is also being investigated.

In collaboration with other labs in Purdue University, using in-silico analyses and structure-based drug design, an array of compounds is being created and medium and high-throughput screening of these compounds as inhibitors of alphaviruses and flaviviruses is in progress. High-throughput screening for inhibitors of flavivirus replication using compound libraries has also being undertaken.

Dagmar Sedlak, Virus production facilities and laboratories manager / Virologist

Dengue virus (member of Flaviviridae family) annually infects approximately 50 million people worldwide. To understand the molecular basis for the activity of neutralizing, non-neutralizing and cross-protective antibodies, we took a structural approach to investigate the role of antibodies in Dengue virus infection. The approach is to identify monoclonal antibodies that have defined biological properties against the virus. We produce the antibodies using hybridoma cells, then we further purify them and cleave off Fab fragments. We complex the fragments with virus, cryo-electron microscopy is carried out and then image reconstruction, which shows binding of monoclonal antibody to the surface of the virus. These and further studies are expected to help us understand the nature of antibody-virus interactions.

Elisa LaBauve, Graduate student

We are looking at the role of the flavivirus envelope (E) protein in host cell entry and viral assembly. We are conducting functional analysis of two conserved helices that lie within the stem region of the flavivirus E protein. This stem region links the E protein ectodomain to the transmembrane domain. Recent cryo-EM structural information has allowed for prediction of several positional changes that occur within these helices that are thought to mediate critical envelope protein interactions during various stages in the flavivirus life cycle. During the course of the life cycle, the envelope protein undergoes several conformational changes, starting with the immature prM-E heterodimer, proceeding to the mature E protein homodimer, and finally the formation of an E homotrimer during viral entry. We are looking at critical residues involved in these conformational changes.

Karla Combs, Graduate Student

We focus on flavivirus replication. We are working on developing a cell free system, which will allow us to further understand the viral life cycle without the hassle of cell culture. This system will allow us to look at the individual parts of the viral life cycle to determine at which stage mutations become deleterious. Also this system may be used to screen antiviral drugs. We are further studying NS3 helicase, which is a nonstructural protein of Yellow Fever , structure of which was solved by Jinhua Wu in J. Smith lab.. By using mutagenesis, we hope to determine what residues are important for helicase activity and possibly pathogenesis.

Mansoora Khaliq, Graduate Student

My interests are in determining the effects of specifically targeted organic compounds on the entry of flaviviruses.

Muriel Soler, Post-Doctoral Researcher

We are working on NS1 and NS2A nonstructural proteins of Yellow Fever Virus (Flavivirus). We don't know the structure of these 2 proteins but they seem to be implicated in replication and also packaging of the virus. Using transposon mutagenesis and a replicon system containing luciferase reporter gene, we tested the ability of the mutants to impair replication, packaging and their sensitivity to different temperatures. Map of NS1 and NS2A, highlighting more sensitive regions in terms of replication and packaging abilities can now be drawn.

Ranjan Sengupta, Graduate student

We are looking at specific events in the flavivirus, yellow fever virus life cycle with special emphasis on the contribution of the nonstructural protein NS3 in the virus life cycle. NS3, being a key multifunctional protein with defined domains, attracts speculations as how, when and where are the different functions carried out during an ongoing infection process. We are looking at the mutual cooperativity of these defined functional domains and trying to address the question of why would the virus have such strict conservation of these domains in one single protein within the flaviviridae family and beyond.

Xinqi Liu, Post-Doctoral Researcher

We also focus on crystallographic studies of some biologically important Flavivirus proteins. Currently we work on the NS1 protein. NS1 is a non-structural protein, which play an important role in the early stage of virus replication. NS1 is secreted extracellularly after synthesized. Secreted NS1 in the blood activate serious immunological reaction in the host immunity against the flavivirus infection.

Yu-hsuan Chang, Graduate student

My research interest focuses on the assembly process of flavivirus nucleocapsid core. Capsid proteins and the genomic RNA form the nucleocapsid core as the inner-most shell of the flavivirus. It has been found that capsid proteins are dimers in the solution, but the biological function is unclear. We are studying how the dimeric capsids are involved in assembly process by biochemical analysis and genetic approaches.