Discrimination between Functional and Non-functional Cellular Gag Complexes involved in HIV-1 Assembly.

HIV-1 Gag and Gag-Pol are responsible for viral assembly and maturation and represent a major paradigm for enveloped virus assembly. Numerous intracellular Gag-containing complexes (GCCs) have been identified in cellular lysates using sucrose gradient ultracentrifugation. While these complexes are universally present in Gag-expressing cells, their roles in virus assembly are not well understood. Here we demonstrate that most GCC species are predominantly comprised of monomeric or dimeric Gag molecules bound to ribosomal complexes, and as such, are not on-pathway intermediates in HIV assembly. Rather, these GCCs represent a population of Gag that is not yet functionally committed for incorporation into a viable virion precursor. We hypothesize that these complexes act as a reservoir of monomeric Gag that can incorporate into assembling viruses, and serve to mitigate non-specific intracellular Gag oligomerization. We have identified a subset of large GCC complexes, comprising more than 20 Gag molecules, that may be equivalent to membrane-associated puncta previously shown to be bona fide assembling-virus intermediates. This work provides a clear rationale for the existence of diverse GCCs, and serves as the foundation for characterizing on-pathway intermediates early in virus assembly.

Novel Intersubunit Interaction Critical for HIV-1 Core Assembly Defines a Potentially Targetable Inhibitor Binding Pocket.

HIV-1 capsid protein (CA) plays critical roles in both early and late stages of the viral replication cycle. Mutagenesis and structural experiments have revealed that capsid core stability significantly affects uncoating and initiation of reverse transcription in host cells. This has led to efforts in developing antivirals targeting CA and its assembly, although none of the currently identified compounds are used in the clinic for treatment of HIV infection. A specific interaction that is primarily present in pentameric interfaces in the HIV-1 capsid core was identified and is reported to be important for CA assembly. This is shown by multidisciplinary characterization of CA site-directed mutants using biochemical analysis of virus-like particle formation, transmission electron microscopy of in vitro assembly, crystallographic studies, and molecular dynamic simulations. The data are consistent with a model where a hydrogen bond between CA residues E28 and K30' from neighboring N-terminal domains (CANTDs) is important for CA pentamer interactions during core assembly. This pentamer-preferred interaction forms part of an N-terminal domain interface (NDI) pocket that is amenable to antiviral targeting.IMPORTANCE Precise assembly and disassembly of the HIV-1 capsid core are key to the success of viral replication. The forces that govern capsid core formation and dissociation involve intricate interactions between pentamers and hexamers formed by HIV-1 CA. We identified one particular interaction between E28 of one CA and K30' of the adjacent CA that appears more frequently in pentamers than in hexamers and that is important for capsid assembly. Targeting the corresponding site could lead to the development of antivirals which disrupt this interaction and affect capsid assembly.

Ca2+-Induced Rigidity Change of the Myosin VIIa IQ Motif-Single α Helix Lever Arm Extension.

Several unconventional myosins contain a highly charged single α helix (SAH) immediately following the calmodulin (CaM) binding IQ motifs, functioning to extend lever arms of these myosins. How such SAH is connected to the IQ motifs and whether the conformation of the IQ motifs-SAH segments are regulated by Ca2+ fluctuations are not known. Here, we demonstrate by solving its crystal structure that the predicted SAH of myosin VIIa (Myo7a) forms a stable SAH. The structure of Myo7a IQ5-SAH segment in complex with apo-CaM reveals that the SAH sequence can extend the length of the Myo7a lever arm. Although Ca2+-CaM remains bound to IQ5-SAH, the Ca2+-induced CaM binding mode change softens the conformation of the IQ5-SAH junction, revealing a Ca2+-induced lever arm flexibility change for Myo7a. We further demonstrate that the last IQ motif of several other myosins also binds to both apo- and Ca2+-CaM, suggesting a common Ca2+-induced conformational regulation mechanism.

Non-canonical antagonism of PI3K by the kinase Itpkb delays thymocyte ß-selection and renders it Notch-dependent.

ß-selection is the most pivotal event determining αß T cell fate. Here, surface-expression of a pre-T cell receptor (pre-TCR) induces thymocyte metabolic activation, proliferation, survival and differentiation. Besides the pre-TCR, ß-selection also requires co-stimulatory signals from Notch receptors - key cell fate determinants in eukaryotes. Here, we show that this Notch-dependence is established through antagonistic signaling by the pre-TCR/Notch effector, phosphoinositide 3-kinase (PI3K), and by inositol-trisphosphate 3-kinase B (Itpkb). Canonically, PI3K is counteracted by the lipid-phosphatases Pten and Inpp5d/SHIP-1. In contrast, Itpkb dampens pre-TCR induced PI3K/Akt signaling by producing IP4, a soluble antagonist of the Akt-activating PI3K-product PIP3. Itpkb(-/-) thymocytes are pre-TCR hyperresponsive, hyperactivate Akt, downstream mTOR and metabolism, undergo an accelerated ß-selection and can develop to CD4(+)CD8(+) cells without Notch. This is reversed by inhibition of Akt, mTOR or glucose metabolism. Thus, non-canonical PI3K-antagonism by Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement.