All normal retroviral particles contain a dimer of genomic RNA. The physical structure of the dimer, and of the linkage between the two monomers, is not understood. We reported some years ago that the viral nucleocapsid protein alters the linkage between the monomers when it is released from Gag during viral maturation. This stabilization of the dimeric linkage results from the nucleic acid chaperone activity of nucleocapsid. We have continued to probe the structure of viral RNA within authentic virus particles. We have recently analyzed the RNA by cleaving it at specific sites using RNase H and short oligodeoxynucleotides complementary to specific sequences in the RNA. The results of these experiments, for the first time, localized the most stable linkage between the monomers to the region near their 5'ends, in both immature and mature murine leukemia virus (MLV) particles. The experiments also revealed the existence of additional, less stable linkages between the monomers. Retroviral nucleocapsid proteins are highly active nucleic acid chaperones. The mechanism of this crucial activity is not well understood. We have analyzed the binding of HIV-1 nucleocapsid to a very short (8-base) oligodeoxynucleotide in great detail, using several biophysical techniques. We found that a single nucleocapsid molecule can bind simultaneously to two nucleic acid molecules;conversely, a single nucleic acid molecule can bind two nucleocapsid molecules. It seems likely that the ability of the protein to interact with more than one nucleic acid molecule is a critical element in its nucleic acid chaperone activity. We are currently analyzing packaging signals in both HIV-1 and MLV viral RNAs, using both genetic and structural methods. Recent Accomplishments and Current Research: a. Genetic analysis of gammaretroviral 5'untranslated regions In collaboration with Drs. Kevin Weeks and Robert Gorelick, we have defined the dimerization/packaging signal of MLV RNA in unprecedented detail. We will perform further mutagenic analysis and do comparative studies on other gammaretroviruses. We will also perform mutagenic studies on the packaging signal of HIV-1 RNA. b. Characterization of nucleic acid chaperone activity of MLV Gag protein In collaboration with Dr. Karin Musier-Forsyth, we have found that the MA domain can interfere with the annealing activity of HIV-1 Gag. We will test whether this is also true of MLV Gag, despite the extended conformation of the latter. c. Analysis of selective packaging of cellular mRNAs A few cellular mRNAs are greatly enriched in retrovirus particles. We will try to identify the "packaging signal" in one of them, viz. ASB-1 mRNA, and to determine whether it is packaged in dimeric form. d. Structural studies on dimers in MLV and HIV-1 In collaboration with Dr. Yun-Xing Wang, we will study the three-dimensional structure of dimeric RNAs of MLV and HIV-1, relying heavily on small-angle X-ray scattering. e. Solution studies of binding of HIV-1 Gag to nucleic acids The binding of Gag proteins to nucleic acids is very poorly characterized to date. We will make basic measurements of affinity at high ionic strength, in hopes that the binding reactions will be simplified under these conditions. Patent Linked to 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. [Corresponds to Rein Project 2 in the November 2011 site visit report of the HIV Drug Resistance Program]

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010512-11
Application #
8763124
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
2013
Total Cost
$408,847
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Grohman, Jacob K; Gorelick, Robert J; Kottegoda, Sumith et al. (2014) An immature retroviral RNA genome resembles a kinetically trapped intermediate state. J Virol 88:6061-8
Ricci, Emiliano P; Herbreteau, Cecile H; Decimo, Didier et al. (2008) In vitro expression of the HIV-2 genomic RNA is controlled by three distinct internal ribosome entry segments that are regulated by the HIV protease and the Gag polyprotein. RNA 14:1443-55