Research in the Barklis Lab focuses on the assembly and replication of viruses, such as retroviruses, flaviviruses, and hantaviruses, using molecular genetic, biochemical and biophysical techniques. Molecular genetic and biochemical approaches have been used and continue to be employed to investigate viral protein interactions, RNA recognition and encapsidation, and cellular factors involved in virus replication and assembly.

To analyze virus particles, proteins and macromolecular complexes, a variety of biophysical methods also are utilized. For characterization of proteins and small oligomers we resort to sedimentation, crosslinking, dynamic light scattering, fluorescence polarization, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy methods. Virions and macromolecular complexes assembled in vitro from viral components are examined by transmission electron microscopy (EM) or atomic force microscopy (AFM). Typically viruses or virus assemblies formed in solution or on two-dimensional (2D) membrane sheets are imaged at high magnification, digitized and used to generate 7-25 angstrom resolution reconstructions using computer-based image alignment and averaging or electron diffraction approaches. Models and predictions from EM and AFM investigations are tested on virus particles and purified proteins using cell culture, nucleic acid binding, protein binding, mutagenesis and microscopy techniques.

In addition to elucidation of retrovirus and hantavirus replication and assembly pathways, the methods developed by Barklis Lab can be adapted to the analysis of subcellular complexes, interactions between proteins and nucleic acids, and to the elucidation of membrane receptor and channel protein structures. The lab also is using the knowledge gained from these experiments in long-term studies aimed at designing molecular machines and surfaces for nanotechnology applications. An immediate, practical goal is to identify structure-based compounds that can be employed as safe and specific antiviral agents. 

Figure shows four images, labeled A, B, C and D. A and C show show red markers (RNA). B and D show green markers (membrane phospholipids).
Binding site residues on the HIV-1 matrix protein for RNA (red) and for membrane phospholipids (green) were determined by nuclear magnetic spectrometry (NMR).