Research and Professional Experience:
Postdoctoral Fellow, 2000-2005, Molecular Oncology, Washington University School of Medicine, St. Louis MO
HIV Molecular Virology
Human Immunodeficiency Virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). HIV infection is incurable, but treatment with a combination of anti-retroviral drugs can reduce virus replication to undetectable levels and forestall disease progression.
However drug resistance to the existing pool of anti-retroviral therapies continues to rise. Continued success in the repression of HIV replication in infected individuals will require the development of new inhibitors that are effective against drug-resistant strains of virus. Drugs that target novel areas of virus replication have the greatest probability to be effective against such viruses.
Our laboratory seeks to comprehend poorly understood aspects of HIV replication to identify new targets for anti-viral therapies. Several areas of HIV replication are potential targets. These include virus uncoating, preintegration complex (PIC) assembly and transport, viral transcriptional regulation, and virus assembly and release.
Currently we are focused on identifying novel cellular components of PICs as a first step to develop a new class of inhibitors. PICs are large viral DNA (vDNA) complexes formed during early HIV-1 infection of cells. They facilitate the transport of viral DNA into the nucleus of cells. Due to difficulties in producing and purifying PICs, their composition, assembly, and transport remain poorly defined. The viral proteins associated with the PIC do not account for the size of the complexes, suggesting they contain additional cellular components. Our long-term goal is to aid in the development of new HIV-1 inhibitors by determining how PICs assemble and mediate nuclear transport of vDNA in dividing and non-dividing cells.
Disparities exist in the literature concerning methods to both produce and purify preintegration complexes (PICs). We have analyzed several methods of PIC production and purification and developed a method that consistently produces PICs from several cell types using velocity gradient centrifugation. We have characterized the biochemical properties of these complexes and identified the location of PICs in gradients. Our protocol produces PICs that: 1) are biochemically similar to in vivo PICs; 2) are of sufficient quantity to analyze by mass spectrometry; and 3) are active for nuclear transport and integration in vitro.
In parallel studies in collaboration with Dr. Alan Engelman at the Dana Farber Cancer Institute we have developed a method to biotinylate HIV PIC proteins in vivo. Biotinylated proteins can be affinity purified with streptavidin to capture protein complexes and identify novel cellular proteins that interact with HIV.
Our mass spectrometry analysis is done in collaboration with Dr. Pawel Ciborowski, the Director of the Mass Spectrometry Core Facility at the University of Nebraska Medical Center, and Dr. Ron Cerny, the Director of the University of Nebraska at Lincoln Mass Spectrometry Facility. Both are members of the Nebraska Center for Virology.
Our MS analyses consistently identify viral proteins in every infected sample. We have also detected a number of cellular proteins previously demonstrated to interact with HIV-1 PICs and/or modulate HIV-1 replication. Several potential candidate proteins have been identified using both the targeted and affinity purification approaches. Four candidate proteins have been detected in virus particles. We are now working to characterize the role of these proteins in HIV replication and we continue to discover novel cellular components of PICs that play a critical role in retroviral replication.
Our laboratory is affiliated with the Nebraska Center for Virology a Center of Biomedical Research Excellence formed in the fall of 2000 under the National Institutes of Health Institutional Development Award program. The Center links the virology programs of Creighton University, the University of Nebraska at Lincoln, and the University of Nebraska Medical Center and conducts innovative research that addresses fundamental questions about the pathogenesis and replication of diverse viral agents that effect
human, animal, and plant health.