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Alan Rein, Ph.D.
Mechanisms in Retroviral Replication and Pathogenesis
The goal of the research efforts in the Retroviral Assembly Section is to extend our understanding of basic mechanisms in retroviral replication and pathogenesis. Our hope is that this understanding will lead to new methods of combatting retrovirus-induced disease, including AIDS.
There appear to be several different modes of interaction between retroviral proteins and nucleic acids, each with important functional consequences for viral replication. First, an exquisitely specific recognition by the Gag polyprotein (the structural protein of the virus particle) selects the viral RNA for packaging during virus assembly. This recognition involves zinc fingers in the protein. We are studying the mechanism by which the Gag protein recognizes and packages the genomic RNA of the virus during assembly in vivo. Our research strongly suggests that the recognition signal involves the three-dimensional structure formed by a dimer of genomic RNA molecules. We are studying the structure of the dimer linkage and its possible role in packaging of viral RNA. Our mutational studies also show that the zinc fingers have other crucial functions, in addition to their role in the recognition process. These additional functions are now under investigation. We have found that virus particles lacking genomic RNA contain cellular mRNA molecules in place of the viral RNA. While the majority of mRNA species are packaged into the virions nonselectively, there are a few species that are significantly enriched. We are currently analyzing the mechanism of this enrichment; it is possible that this will help us understand how Gag normally recognizes the viral RNA during wild-type particle assembly.
We have analyzed the properties of recombinant HIV-1 Gag protein in some detail. When nucleic acid is added to Gag, the protein assembles into virus-like particles. This is a very nonspecific interaction: almost any nucleic acid will support assembly. These particles are significantly smaller than authentic viral particles, but particles of the correct size are formed if inositol phosphates, as well as nucleic acid, are provided to the protein. In the absence of nucleic acid, the protein is in monomer-dimer equilibrium, using the previously described interface within the C-terminal domain of its capsid moiety. We generated a mutant of Gag that remains monomeric at relatively high concentrations. We have subjected this mutant protein to a series of hydrodynamic and biophysical analyses. These results, together with modeling studies, indicate that the protein is folded over in solution, with its N and C termini relatively close together in three-dimensional space. Since Gag molecules are extended rods in immature virus particles, they must undergo a drastic conformational change during particle assembly. Collaborative studies using neutron reflectivity and other techniques have now shown that Gag extends when preferred ligands are available for both its N-terminal matrix domain and its C-terminal nucleocapsid domain. Interestingly, these properties of HIV-1 Gag are not shared with the Gag protein from another retrovirus, Moloney murine leukemia virus.
Gag is also a nucleic acid chaperone, and participates in the annealing of a cellular tRNA molecule to the viral RNA, where the tRNA will serve as primer for viral DNA synthesis.
The mechanism by which nucleic acid promotes particle assembly by the Gag protein is not known. We recently reported that a very short region of Gag, termed 'SP1,' undergoes a conformational change when it is at high concentration. We proposed that this region 'senses' the local Gag concentration, and that the increased Gag concentration resulting from cooperative binding to nucleic acid would thus lead to a change in Gag conformation, exposing new interfaces required for particle assembly. This hypothesis would explain the contribution of nucleic acid to particle assembly. Current studies are devoted to testing this hypothesis and exploring its implications.
We are also studying the mechanism by which the restriction factor mouse APOBEC3 blocks infection by murine retroviruses. This restriction, unlike retroviral restriction by human APOBEC3G, does not involve hypermutation of G to A in the viral genome.
Unspliced HIV-1 RNA is exported from the nucleus of the infected cell by binding of a structural element within the RNA, the 'Rev Response Element' or RRE, to the viral protein Rev. Our colleague Yun-Xing Wang recently determined the overall three-dimensional structure of the RRE, and we tested a series of RRE mutants for their functional properties. The results showed that the new RRE structure explains the specificity of Rev for viral RNA.
It has been suggested that retroviruses such as mouse mammary tumor virus play a role in human breast cancer. We are testing clinical samples for the presence of this virus.
Our collaborators include Judith Levin, Ph.D., National Institute of Child Health and Human Development; Mark Yeager, M.D., Ph.D., University of Virginia; Karin Musier-Forsyth, Ph.D., Ohio State University; Kevin Weeks, Ph.D., University of North Carolina; Hirsh Nanda, Ph.D., and Matthias Losche, Ph.D., Carnegie Mellon University; Bogdan Dragnea, Ph.D., University of Indiana; Adam Zlotnick, Ph.D., University of Indiana; Hung Fan, Ph.D., University of California at Irvine; Volker M. Vogt, Cornell University; Edward Gabrielson, M.D., Johns Hopkins Oncology Center; and Yun-Xing Wang, Ph.D., Center for Cancer Research, NCI.
This page was last updated on 11/19/2013.