Walter A. Scott

Professor of Biochemistry and Molecular Biology

Ph.D. (1970) University of Wisconsin

Biochemistry of HIV-1,
Reverse Transcriptase

Tel: (305) 243-6359, Fax: (305) 243-3065

wscott@med.miami.edu

We are studying the replication machinery of human immunodeficiency virus (HIV). Replication is carried out by a virus-encoded protein, reverse transcriptase (RT). Since HIV RT is one of the simplest DNA polymerases, we hope to use this enzyme to investigate some of the most basic mechanisms of DNA synthesis such as nucleotide recognition, enzyme translocation, and internal conformational changes that occur during DNA synthesis.

RT is the target for many drugs that inhibit HIV replication. We have focused on a class of drugs including 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), and 2',3'-didehydro-3'-deoxythymidine (d4T) that are incorporated into growing DNA chains by RT and result in chain termination.  A novel activity of HIV RT gives this enzyme the ability to remove chain-terminating nucleotides from a blocked DNA chain after they have been incorporated.  Removal involves the transfer of the inhibitory nucleotide to an intracellular acceptor molecule.  The acceptor has not been identified unambiguously in infected cells; however, we have shown that a common intracellular molecule such as ATP could serve as acceptor.  The resulting product would be a dinucleoside polyphosphate, which has a unique structure and might be detectable in an infected cell extract. In the case of AZTMP-terminated DNA, the expected product would be AppppAZT. Therapy with AZT results in the selection of mutant viruses that are highly resistant to AZT. The AZT-resistance mutations occur in the RT coding region and the mutant enzyme has increased ability to carry out the unblocking reaction.

RT that is associated with a chain-terminated primer-template can either bind a nucleotide substrate for the unblocking reaction (Figure, left side), in which case the chain-terminating nucleotide is removed; or it can bind the natural dNTP substrate that matches the next position on the template to form a dead-end complex (Figure, right side). The dead-end complex cannot carry out either the DNA synthesis reaction or the unblocking reaction but it can be detected experimentally. The factors that determine which pathway the enzyme takes depend on translocation of the enzyme between two positions on the primer-template and we hope to explore the mechanism of translocation through the study of these reactions.

We are very interested in what determines the sensitivity of HIV to various nucleoside inhibitors, the mechanisms of mutations in the enzyme that cause resistance to these drugs, and how the drugs might be modified to reduce the chance of selecting resistance mutations and to make them more effective antiretroviral drugs.  We are also actively studying other steps in the HIV replication in hopes of finding new targets for drug development.  Future development of anti-HIV drugs will depend on basic research that advances our understanding of the HIV life cycle.

Representative Publications

1.    Meyer, P.R., Matsuura, S.E., Mian, A.M., So, A.G., and Scott, W.A.  (1999) A mechanism of AZT resistance: An increase in nucleotide-dependent primer unblocking by mutant HIV-1 reverse transcriptase. Mol. Cell 4: 35-43.

2.      Smith, A.J., Meyer, P.R., Asthana, D., Ashman, M.R., and Scott, W.A.  (2005)  Intracellular substrates for the primer-unblocking reaction by human immunodeficiency virus type 1 reverse transcriptase:  Detection and quantitation in extracts from quiescent- and activated-lymphocyte subpopulations.  Antimicrob. Agents Chemother.  49: 1761-1769.

3.      Smith, A.J., and Scott, W.A.  (2006) The influence of natural substrates and inhibitors on the nucleotide-dependent excision activity of HIV-1 reverse transcriptase in the infected cell.  Curr. Pharm. Design  12: 1827-1841.

4.      Meyer, P.R., Smith, A.J., Matsuura, S.E., and Scott, W.A.  (2006) Chain-terminating dinucleoside tetraphosphates are substrates for DNA polymerization by human immunodeficiency virus type 1 reverse transcriptase with increased activity against thymidine analogue-resistant mutants.  Antimicrob. Agents Chemother. 50:  3607-3614.

5.    Meyer, P.R., Rutvisuttinunt, W., Matsuura, S.E., So, A.G. and Scott, W.A.  (2007) Stable complexes formed by HIV-1 reverse transcriptase at distinct positions on the primer-template controlled by binding deoxynucleoside triphosphates or foscarnet.  J. Mol. Biol.  369: 41-54.

Honors and Professional Activities

Director, Human Retrovirus Laboratory for the IMPAACT Clinical Trials Program

Member, Virology Technical Advisory Committee for the Division of AIDS, NIH

Full member, NIH Review Panel, AIDS Discovery and Development of Therapeutics

Member, American Society for Biochemistry and Molecular Biology