The 5´-leader of the HIV-1 genome regulates multiple functions during viral replication by mechanisms that have yet to be established. We developed an NMR approach that enabled direct detection of structural elements within the intact leader (712 nucleotide dimer) that are critical for genome packaging. Residues spanning the gag start codon (AUG) form a hairpin in the monomeric leader and base pair with residues of the Unique-5´ region (U5) in the dimer. U5:AUG formation promotes dimerization by displacing and exposing a dimer-promoting hairpin, and enhances binding by the nucleocapsid protein (NC), the cognate domain of the viral Gag polyprotein that directs packaging. Our findings support a packaging mechanism in which translation, dimerization, NC binding, and packaging are regulated by a common RNA structural switch.

Assembly of Human Immunodeficiency Virus Type-1 (HIV-1) particles is initiated in the cytoplasm by the formation of a ribonucleoprotein complex comprising the dimeric RNA genome and a small number of viral Gag polyproteins. Genomes are recognized by the nucleocapsid (NC) domains of Gag, which interact with packaging elements believed to be located primarily within the 5´-leader of the viral RNA. Recent studies revealed that the native 5´-leader exists as an equilibrium of two conformers, one in which dimer-promoting residues and NC binding sites are sequestered and packaging is attenuated, and one in which these sites are exposed and packaging is promoted. To identify the elements within the dimeric 5´-leader that are important for packaging, we generated HIV-1 5´-leader RNAs containing mutations and deletions designed to eliminate substructures without perturbing the overall structure of the leader and examined effects of the mutations on RNA dimerization, NC binding and packaging. Our findings identify a 159 residue RNA packaging signal that possesses dimerization and NC binding properties similar to those of the intact 5´-leader and contains elements required for efficient RNA packaging.

SUMMARY Synaptic transmission is bioenergetically demanding, and the diverse processes underlying synaptic plasticity elevate these demands. Therefore, mitochondrial functions including ATP synthesis and Ca2+ handling, are likely essential for plasticity. Although axonal mitochondria have been extensively analyzed, LTP is predominantly induced postsynaptically, where mitochondria are understudied. Additionally, though mitochondrial fission is essential for their function, signaling pathways that regulate fission in neurons remain poorly understood. We found that NMDAR-dependent LTP induction prompted a rapid burst of dendritic mitochondrial fission, and elevations of mitochondrial matrix Ca2+. The fission burst was triggered by cytosolic Ca2+ elevation, and required CaMKII, actin, and Drp1, as well as dynamin 2. Preventing fission impaired mitochondrial matrix Ca2+ elevations, structural LTP in cultured neurons, and electrophysiological LTP in hippocampal slices. These data illustrate a novel pathway whereby synaptic activity controls mitochondrial fission, and show that dynamic control of fission regulates plasticity induction perhaps by modulating mitochondrial Ca2+ handling.