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Principal Investigator: ALAN REIN
Organization: DIVISION OF BASIC SCIENCES - NCI
Fiscal Year: 2022
Award: $616,301
Funding agency: National Cancer Institute
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. The interactions of retroviral Gag proteins with RNAs are remarkably complex. We believe that one role played by the viral RNA is to bring Gag proteins close together, as discussed in our project on Retrovirus Assembly and Maturation (ZIA BC 010511). At the same time, successful replication requires that viral genomic RNA (vRNA), which contains a packaging signal (termed "Psi"), be selected for encapsidation in preference to the thousands of cellular RNA species that are also potential substrates for packaging. Finally, both HIV-1 Gag and its cleavage product nucleocapsid (NC) possess nucleic acid chaperone activity, enabling them to catalyze rearrangements of nucleic acids to the conformation with the lowest free energy. It appears that this activity in Gag is responsible for annealing a tRNA molecule to vRNA, and the chaperone activity of NC plays several critical roles during reverse transcription. Our research is devoted to elucidating these diverse interactions at the molecular level. To better understand the relationship between HIV-1 RNA packaging and assembly, we are addressing the following questions: How does HIV-1 Gag discriminate between different RNAs, and how can we explain the selective packaging of Psi-containing RNA during particle assembly? _____ We have found that when Gag is expressed in vivo in the absence of vRNA, it can package almost any cellular mRNA. Similarly, addition of almost any nucleic acid to HIV-1 Gag will lead to virus-like particle (VLP) assembly in vitro. Thus, vRNA is in competition with mRNAs for packaging; the Psi packaging signal gives it an advantage in this competition. We are studying the binding of recombinant Gag to Psi-containing and control RNAs by using fluorescence correlation spectroscopy, microscale thermophoresis, SwitchSense instrumentation, and mass photometry. We find that Gag binds RNAs cooperatively. The affinity of Gag for Psi is only modestly higher than that for control RNA. Using several Gag mutants, we have found that binding to the control is largely attributable to the matrix domain. Notably, binding to Psi has a different character, as it is far more salt resistant than binding to control RNA, indicating a higher nonelectrostatic component. We are testing the idea that binding to Psi triggers the conformational shift that Gag undergoes to an assembly-ready state more efficiently than other RNAs. We have found that binding to Psi supports particle assembly more efficiently than binding to other RNAs. This would explain selective packaging of vRNA. The data also identify specific nucleotides within Psi that are required for this efficient particle assembly. _____Patents linked to this project: U.S. Patent #5,674,720: "Design and Construction of Noninfectious Human Retroviral Mutants Deficient in Genomic RNA"; issued October 7, 1997; Robert J. Gorelick, Larry O. Arthur, Alan Rein, Louis E. Henderson, and Stephen Oroszlan. This patent describes mutants of HIV-1 that are structurally normal but noninfectious; these mutants could potentially be considered as vaccine constituents. U.S. Patent #7,572,828: "Identification of Anti-HIV Compounds Inhibiting Virus Assembly and Binding of Nucleocapsid Protein to Nucleic Acid"; issued August 11, 2009; Robert Shoemaker, Michael Currens, Alan Rein, Ya-Xiong Feng, Robert Fisher, Andrew Stephen, Shizuko Sei, Bruce Crise, Louis Henderson, and Karen Worthy. This patent describes a class of compounds with anti-HIV-1 activity, which are under investigation for use in antiretroviral therapy.
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