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

Our research is focused on the structural E1 and E2 proteins of Sindbis virus (SINV). SINV is a prototype of the alphavirus family. The two envelope proteins, E1 and E2, form a heterodimer in the mature virus. We are studying the importance of the interactions of E1 and E2 proteins, since the disruption of those interaction could have an effect on folding, on transport of E1 and E2 to the surface of the membrane, or on fusion function. In addition, we are also 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.

Chanakha Navaratnarajah, Graduate student

The glycoprotein envelope of alphaviruses is made up of two proteins, E1 and E2. E1 is responsible for the fusion process with the host cell membrane and E2 is responsible for cell receptor binding. An atomic structure is available for the E1 glycoprotein, but attempts to crystallize E2 have so far been unsuccessful. We are studying E2 by two different methods: structural and genetic. The structural studies involve the expression, purification, crystallization and x-ray crystal structure solution of alphavirus E2 constructs. As part of our genetic studies of E2, we are looking at the effects of random in-frame insertions into E2 by transposon mutagenesis. The structural and genetic studies of E2 together will combine to give us new insight in to the structure and function of this glycoprotein.

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.

Eunmee Hong, Graduate student

We intend to elucidate the assembly pathway of the alphavirus nucleocapsid core and identify the role of Helix I region of the capsid protein in the assembly process using a variety of recombinant proteins and the in vitro core assembly system.

Joyce Jose, Post-doctoral researcher

We are studying the process of Alphavirus exit from infected cells, also known as budding. During this process, the nucleocapsid core interacts with the cytoplasmic domain of the E2 protein. A recent high resolution cryo-electron microscopy reconstruction has shown that there is a bridge of electron density connecting capsid proteins to the E2 glycoproteins. This density has been assigned to the cytoplasmic domain of E2. Although the cytoplasmic domain of E2 has 33 amino acids, it only takes 10 residues to reach the hydrophobic pocket. The position of the remaining 23 amino acids is unknown. We are attempting to define the molecular interactions between the nucleocapsid core and the cytoplasmic domain of E2 by using a combination of molecular genetics and biophysical methods.

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.

Mayuri, Graduate student

The four non-structural proteins in alphaviruses (nsP1,2,3 and 4) form a replicase complex, that plays a role in replication of the viral genome, in association with unknown host factors. We want to study the interactions between the non- structural proteins and their possible interactions with host factors, both of which are important in the establishment and stability of the replicase complex during the virus life cycle in the host cell.

nsP2 is a multi-functional non-structural protein which has helicase domain in the N-terminus, along with NTPase and ATPase activities associated with it and protease domain in the C-terminus, involved in the proteolytic processing of the polyproteins synthesized after the positive stranded genome is first released into the cytoplasm of the infected cell. Various temperature sensitive mutations mapped in the C-terminus have hinted at the role of nsP2 in regulating minus strand synthesis and 26S mRNA transcription, but an RNA binding region has not been characterized. Along with Janet Smith's lab we want to identify a possible RNA binding region in this protein using mutagenesis of residues with potential functional importance.

We are also working on developing a cell free system for translation and replication of the alphavirus genome. This is based on the 'life in a test tube' studies that have been done previously with poliovirus.

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.

Ranjit Warrier, Graduate student

Metastable macromolecular assembly systems can be used in the future for drug or nucleic acid delivery into cells.  In order to understand the assembly requirement for one such system, we are studying the assembly pathway of preformed nucleocapsid cores that accumulate in the cytoplasm of cells infected with Sindbis virus.  Each nucleocapsid core is assembled from one molecule of viral genomic RNA and 240 copies of the capsid protein.  These nucleocapsid cores interact with viral glycoproteins on the cellular membrane and bud out of the cell to form mature virus.  Upon infection of a new cell, the nucleocapsid core disassembles and releases the genomic RNA.  The initial events that lead to nucleocapsid formation are the capsid protein recognizing the genomic RNA specifically and capsid protein-capsid protein dimerization.  Amino acids 81-113 within the 264 amino acid Sindbis capsid protein have been implicated in these initial events. We are studying these initial events by using a combination of in vitro and in vivo characterization of mutant phenotypes and biophysical techniques such as analytical ultracentrifugation and electron microscopy.

Shailly Tomar, Graduate student

We are investigating the structural and functional aspects of alphavirus replicase proteins nsP1 and nsP4. nsP1 is a capping enzyme which has methyltransferase and guanylyltransferase activities and nsP4 is RNA dependent RNA polymerase. We use macromolecular crystallography in conjunction with biochemical and biophysical studies to understand the mechanisms of these enzymes involved in alphavirus genome replication and transription."

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.