Our Science – Pavlakis Website
George N. Pavlakis, M.D., Ph.D.
Analysis of HIV-1 Expression, Protein Function, and Pathogenic Mechanisms of AIDS
The Human Retrovirus Section studies the molecular biology and pathogenic mechanisms of HIV-1. We are interested in new vaccine approaches against AIDS and in identifying and validating new targets for antiviral therapies. Highlights of recent accomplishments are described below.
HIV RNA Expression Regulation
Expression of HIV-1 RNA is totally dependent on the presence of the viral Rev protein. In the absence of Rev, instability elements (INS) scattered throughout the HIV-1 mRNA trap these messages within the nucleus, thus interfering with their expression. We have demonstrated that these INS elements are targets for binding the poly(A)-binding protein (PABP1). Binding of PABP1 and other factors in the nucleus interfere with mRNA transport and lead to more splicing or degradation. We eliminated the INS sequences from the HIV-1 messages by using multiple point mutations that maintain the amino acid composition of the corresponding protein. This technique resulted in high HIV-1 mRNA expression levels even in the absence of Rev, and led to the development of simple expression vectors that produce large amounts of the Gag, Pol, and Env proteins in many cell types and in animals. Increased expression of these proteins in mice and primates produced a strong immune response. Both cellular and humoral immunity components were activated in these animals after DNA injection, indicating the potential of this approach in AIDS vaccine applications. Currently, several vaccination protocols are under way that use these vectors.
We are studying the complex interactions of HIV-1 with cytokines such as interleukin 4 (IL-4) and tumor necrosis factors (TNFs). We have found dichotomous effects on the propagation of HIV strains with different receptor specificity due to the regulation of both virus and receptor gene expression by these cytokines. These studies have led to the proposal that both IL-4 and TNF are important regulators of HIV in vivo and that they participate in the long-term selection in the body leading to altered receptor utilization. Because receptor-blocking drugs are now under development, receptor switching by HIV needs to be better studied and understood.
Protein Localization and Trafficking
We have developed and applied new technologies in our study of the localization and trafficking of proteins in live cells. In particular, we generated several mutants of the Aequorea victoria green fluorescent protein (GFP) with enhanced fluorescence. Crystallization and analysis of these mutants led to the identification of the molecular basis for spectral variation in GFP and facilitated the design of additional beneficial mutants. We have used these mutants extensively in tagging proteins, cells, virions or viruses, and in gene expression studies. Among our findings was the identification of a new trafficking pattern for primarily cytoplasmic proteins such as PABP1 and the product of the von Hippel-Lindau tumor suppressor gene (pVHL).
We are also studying the function of several HIV-1 proteins. We have elucidated the parameters of rapid and continuous trafficking between the nucleus and cytoplasm for the Rev protein. We have shown that the mechanism of transdominance of a Rev mutant over the normal protein involves the binding of Rev to transdominant Rev in the nucleolus and the subsequent inhibition of export. We have demonstrated that the HIV-1 Vpr protein is a transcriptional coactivator of nuclear receptors. This finding, in turn, explains several actions of Vpr on the expression of cellular genes. We also showed that the HIV-1 Nef protein colocalizes with the AP2 adaptor protein complex, which is involved in protein sorting from the plasma membrane.
In addition to the above techniques, we have developed improved cationic liposome formulations for in vivo gene delivery. This method opens up new approaches for gene delivery and has applications in gene therapy and gene vaccine methods.
Our collaborators include George Chrousos, Tomoshige Kino, Richard Klausner, Stephen Lee, and Alexander Wlodawer, NIH; Markus Neumann and Ralf Schneider, GSF-National Research Center for Environment and Health, Germany; and Xiao-Fang Yu, Johns Hopkins University.
This page was last updated on 2/21/2013.