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.

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.

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.

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.

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."