Our Science – Hughes Website

Stephen H. Hughes, Ph.D.

HIV DRP Retroviral Replication Laboratory Laboratory Chief NCI-Frederick Building 539, Room 130A Frederick, MD 21702-1201 Phone:   301-846-1619 Fax:   301-846-6966 E-Mail:   hughes@ncifcrf.gov Link: Other Homepage

Biography

Dr. Stephen H. Hughes received his Ph.D. from Harvard University under the direction of Dr. Mario Capecchi and did postdoctoral research under the direction of Drs. J. Michael Bishop and Harold Varmus at the University of California, San Francisco. He was a Senior Staff Investigator at Cold Spring Harbor Laboratory until 1984, when he established the Gene Expression in Eukaryotes Section in the ABL-Basic Research Program at NCI-Frederick. Dr. Hughes became Deputy Director of the ABL-Basic Research Program in 1988 and Director of the Molecular Basis of Carcinogenesis Laboratory in 1995. In 1999, he joined the HIV Drug Resistance Program at the National Cancer Institute as Chief of the Retroviral Replication Laboratory. Dr. Hughes was appointed Director of the HIV Drug Resistance Program in 2006. He is a Co-organizer of the Annual Symposium on Antiviral Drug Resistance and has served as a Co-organizer of the Retroviruses and Viral Vectors Meetings at Cold Spring Harbor Laboratory and the Annual Meeting on Oncogenes. Dr. Hughes was named one of the most frequently cited AIDS researchers by Science Watch in 1996.

Research

HIV-1 Reverse Transcriptase, Retroviral Vector Design, and Molecular Mechanisms of Retroviral Replication

Our research can be divided into two interrelated parts: 1) the structure and function of HIV-1 reverse transcriptase (RT), and 2) retroviral replication and vector design. Our goals in the HIV-1 RT project are to gain a better understanding of the structure and function of HIV-1 RT and the mechanisms that underlie the resistance of HIV-1 to anti-RT drugs. We develop viral vectors to study retroviral replication and to investigate the effects of altering gene expression in cultured cells and in animal models. The two projects intersect directly through the use of HIV-1 vectors to study how mutations in RT and in the HIV-1 genome affect reverse transciption in cultured cells.

We study the structure and function of HIV-1 RT because it is an important target for anti-AIDS drugs (for example, AZT, 3TC, and ddI are RT inhibitors). We believe that the development of more effective anti-RT drugs will depend on a better understanding of not only wild-type HIV-1 RT but also the drug-resistant variants that arise in response to drug therapy. This project is part of a large collaboration, a critical part of which involves Dr. Edward Arnold and his colleagues, who have used X-ray crystallography to solve a number of different structures of HIV-1 RT (both wild type and mutant). Our biochemical and genetic experiments on HIV-1 RT are, in many cases, inspired by this structural analysis. In turn, the biochemical analyses have provided guidance for some of the crystallographic experiments. Recent structural and biochemical analyses have shed new light on the mechanisms of HIV-1 RT drug resistance. For example, resistance to the nucleoside analog 3TC appears to involve steric hindrance. Replacement of the methionine normally found at position 184 of RT, which is part of the polymerase active site, with either isoleucine or valine creates a steric gate. A beta-branched amino acid at position 184 blocks the appropriate binding of 3TCTP but still permits the incorporation of normal dNTPs.

In contrast, AZT resistance involves an enhanced excision of AZTMP after it has been incorporated into the growing DNA strand. The excision reaction is essentially the normal polymerization reaction run in reverse, except that the beta and gamma phosphates of ATP serve as the pyrophosphate donor. The mutations that confer resistance to AZT do not interact directly with AZTMP, but instead serve to enhance the ability of the mutant enzyme to bind ATP, which increases the rate of excision. The specificity of the excision mechanism for AZT is inherent in the structure of HIV-1 RT. Steric constraints involving the azido group cause the end of an AZTMP-terminated primer to preferentially reside in the nucleotide-binding site, which favors excision. Viruses that acquire additional mutations--for example, an insertion in the fingers subdomain--are able to excise a broader range of nucleoside analogs and are, as a consequence, broadly resistant to this class of drugs. We are working with Dr. Victor Marquez to develop novel nucleoside analogs that can effectively inhibit viruses that replicate using these excision-proficient HIV-1 RTs.

We have spent more than 15 years developing a series of retroviral vectors (the RCAS vectors) based on the avian sarcoma/leukosis virus (ASLV) family of retroviruses. Unlike most other retroviral vectors, the RCAS vectors are replication competent. Until recently, however, the RCAS vectors would efficiently infect only avian cells. In order for a retrovirus to infect a cell, a specific protein on the surface of the virus (the envelope or Env protein) must bind to its cognate receptor on the surface of the host cell. Mammalian cells lack the receptors recognized by the RCAS vectors. We solved this problem in two ways: by creating a version of RCAS vectors that uses an Env protein from a mammalian retrovirus and by modifying mammalian cells so they will express a receptor from avian cells. Both of these methods work well; each has particular advantages. One of the advantages of using the cloned avian receptor is that we can create transgenic mice expressing the receptor in a subset of their cells or tissues; in this way, we can control the expression of genes carried by the RCAS vectors in the mouse model.

We are also using HIV-1-based vectors in experiments designed to complement our structural and biochemical analysis of HIV-1 RT. Basically, we want to be able to understand how the data we (and others) obtain in structural and biochemical experiments relate to the actual process of reverse transcription that takes place in an uninfected cell.

The development of better vectors, and more effective AIDS treatments, rests ultimately on a better understanding of retroviruses and their life cycle. We are engaged in several projects designed to elucidate how retroviruses replicate. To give one example, the inability of avian retroviruses such as the RCAS vectors to replicate in mammalian cells raises an interesting question: How is replication blocked in these viruses?

Although the analyses are not yet complete, there is good evidence that unspliced and partially spliced viral RNAs are not appropriately handled in mammalian cells. By studying this problem, we hope to learn more about the rules (and machinery) that control the splicing and transport of viral RNAs, which could provide useful information about cellular and viral processes. In addition, we hope to obtain information that would be useful in the design and development of new RCAS vectors.

Our collaborators include Edward Arnold, Rutgers University; Mark Federspiel, Mayo Clinic; Amnon Hizi, Tel Aviv University; Victor Marquez, NCI; Michael Parniak, University of Pittsburgh; and Mamuka Kvaratskhelia, Ohio State University.

This page was last updated on 7/15/2008.