The NIAMS Protein Expression Laboratory supports intramural NIH scientists in studying the structure and function of Human Immunodeficiency Virus (HIV) proteins. Most structural biology techniques, especially those for studying the three-dimensional structures of proteins, require large quantities of highly purified, monodisperse, and correctly folded proteins. The Protein Expression Laboratory responds to this need by analyzing and providing HIV proteins to NIH and collaborating scientists. Recent research includes the following.
The human immunodeficiency virus (HIV) consists of a number of proteins with regulatory and structural roles. In the Protein Expression Laboratory, HIV proteins important for the virus life cycle, and proteins that have anti-HIV activity, are expressed in bacteria using recombinant DNA methods (Wingfield et al., 1997). The proteins are purified and studied to establish their chemical and physical properties. Well-characterized proteins are made available to NIH investigators who study the molecular structure of these proteins. This structural information may provide impetus for targeted drug design and development.
The surface proteins of the HIV, and related simian immunodeficiency virus (SIV), consist of two non-covalently associated glycoproteins: gp120 and gp41. The gp120 mediates viral entry into the host cell by binding to receptors located on the host cell surface. This binding changes the shape of the transmembrane gp41, which facilitates or induces fusion between the viral and host membranes. Using both nuclear magnetic resonance spectroscopy and X-ray crystallography (Caffrey et al., 1998; Yang et al., 1999), the three-dimensional structure of the gp41 molecule was determined by scientists at the Protein Expression Laboratory and their collaborators. Based on structural and biochemical information, scientists at the Laboratory have suggested a mechanism for membrane fusion (Caffrey et al., 1999). Structural studies also provide an explanation for the in vivo formation of high molecular weight aggregates that may be responsible for HIV-associated neurological damage and dementia (Caffrey et al., 2000).
The HIV-1 protease consists of two identical subunits and is an important target for anti-HIV HIV-1 drugs. The virus gradually evolves to evade these drugs, lessening their efficacy. Based on the study of protease residues susceptible to the oxidative process, namely the sulfur-containing residues methionine and cysteine, it has been proposed that drugs which target the region between the subunits (interfacial) may provide an alternate site of intervention (Davis et al., 2000).
Related to the HIV-1 virus is the Hepatitis B Virus (HBV), the major worldwide cause of cancer. Liver cancer is one of the most common infectious diseases of humans. Although a vaccine has been developed, it is not universally available and, since the virus is vertically transmitted from mothers to infants, chronic HBV is often acquired in childhood. The HBV nucleocapsid plays an important structural and metabolic role in the life cycle of the virus. Studies of the molecular structure of the HBV nucleocapsid (Conway et al., 1997) indicate that drugs targeting the assembly of the capsid protein could prevent formation and the assembly of the virus (Zlotnick et al., 1999). Ongoing structural studies of the unassembled protein subunits are being carried out.
Scientific research at the Protein Expression Laboratory includes examination of microtubules that comprise one of the major cytoskeletal systems of the cells. These microtubules play an essential role in cytoplasmic organization and cell division. Investigators have shown that Rev, a key HIV-1 regulatory protein (Wingfield et al., 1991), binds strongly to microtubules. This results in the formation of large ring structures, the structural determination of which is being pursued (Watts, et al., 2000). The in vivo interaction between Rev and microtubules provides further insight into the role of Rev during HIV infection.
Current research also includes investigation into the catalytic mechanism of MAP3O, a plant protein obtained from melons using protein engineering methods that appears to have anti-HIV and anti-tumor actions. MAP 30 was originally isolated from the mature seeds of the bitter melon Momordica charantia, a medicinal plant found in China and Southeast Asia. Genomic DNA was isolated from the leaves of freshly grown bitter melon and was used to clone the MAP30 protein. A colleague in the project, Dr. Sylvia Lee-Huang, School of Medicine, New York University, performed the initial purification and cloning of this interesting protein. The structure of the protein in solution was solved recently by collaborators of the Laboratory (Wang et al., 2000).