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Alok K. Upadhyay
Interdisciplinary Ph.D. Program
M.Sc. 1999, HP University, Shimla, India
B.Sc.1996, U.P. Collage, Varanasi, India
Thesis Advisor: Virendra N. Pandey, Ph.D.
Department of Biochemistry & Molecular Biology
Wednesday, December 2, 2009
11:00 a.m., MSB E-609b
Retroviruses containing a single stranded RNA genome have been of long-standing biological and pharmacological interest. They cause a variety of diseases including anemia, arthritis, and immunodeficiency states, as well as cancer (leukemia, sarcoma). The Human Immunodeficiency Virus-1 retrovirus, which causes immunodeficiency in humans (AIDS), has been the target of a massive crusade in modern biology. The viral enzyme, reverse transcriptase, is essential for HIV-1 replication. It is one of the major targets against which drugs have been effectively used to reduce the viral load in plasma. The viral enzyme is a heterodimeric protein (p66/p51); the smaller p51 subunit is derived from the larger p66 subunit by proteolytic removal of the C-terminal RNase H domain. Since the dimeric conformation of HIV-1 RT is absolutely essential for activity of the enzyme, it is imperative to decipher either the mechanism or the amino acid motifs that are essential for dimerization of the enzyme, then to translate this knowledge into designing and development of new drugs to inhibit the viral replication. To this end, I have characterized three motifs that are present in HIV-1 RT and investigated their implications in the dimerization, DNA binding, and polymerase functions of the enzyme. The first motif belonging to the ƒÒ7-ƒÒ8 loop, consists of S134, I135, N136 and N137 amino acid residues. I have generated subunit specific deletion mutant at these positions and shown that among these, amino acids residue S134, I135 and N136 in the p51 subunit (but not in p66) are absolutely essential for dimerization of the enzyme. The second motif I investigated is the connection subdomain of the enzyme. I have swapped a portion of the connection subdomain along with the RNase H domain between HIV-1 RT and MuLV RT and characterized the conformation and biochemical properties of the resulting chimeric enzymes. The chimeric HIV-1 RT carrying the RNase H domain from MuLV RT is not only a monomeric enzyme but has acquired some intrinsic properties of MuLV RT including metal use and fidelity. The third motif, the subject of present investigation, belongs to the ƒÒ1 and ƒÒ3 motif in the polymerase domain of both subunits. I have generated a total of six conservative and non conservative mutants at positions 24 (W24) and 61 (F61) and characterized them in detail. Although both these residues seem to have no role in dimerization process, they are crucial for binding and orientation of the duplex region of the template primer as well as positioning of +1 template overhang in the binary and ternary complexes. Together, these studies have defined not only the role of amino acids residues/motifs that are responsible for conferring dimeric formation to the enzyme but also their potential roles in DNA binding and the catalytic functions of the enzyme.