Neutron-encoded mass signatures for multiplexed proteome quantification.

We describe a protein quantification method called neutron encoding that exploits the subtle mass differences caused by nuclear binding energy variation in stable isotopes. These mass differences are synthetically encoded into amino acids and incorporated into yeast and mouse proteins via metabolic labeling. Mass spectrometry analysis with high mass resolution (>200,000) reveals the isotopologue-embedded peptide signals, permitting quantification. Neutron encoding will enable highly multiplexed proteome analysis with excellent dynamic range and accuracy.

Quantification of mitochondrial acetylation dynamics highlights prominent sites of metabolic regulation.

Lysine acetylation is rapidly becoming established as a key post-translational modification for regulating mitochondrial metabolism. Nonetheless, distinguishing regulatory sites from among the thousands identified by mass spectrometry and elucidating how these modifications alter enzyme function remain primary challenges. Here, we performed multiplexed quantitative mass spectrometry to measure changes in the mouse liver mitochondrial acetylproteome in response to acute and chronic alterations in nutritional status, and integrated these data sets with our compendium of predicted Sirt3 targets. These analyses highlight a subset of mitochondrial proteins with dynamic acetylation sites, including acetyl-CoA acetyltransferase 1 (Acat1), an enzyme central to multiple metabolic pathways. We performed in vitro biochemistry and molecular modeling to demonstrate that acetylation of Acat1 decreases its activity by disrupting the binding of coenzyme A. Collectively, our data reveal an important new target of regulatory acetylation and provide a foundation for investigating the role of select mitochondrial protein acetylation sites in mediating acute and chronic metabolic transitions.

Second-site compensatory mutations of HIV-1 capsid mutations.

The human immunodeficiency virus (HIV) capsid (CA) protein assembles into a hexameric lattice that forms the mature virus core. Contacts between the CA N-terminal domain (NTD) of one monomer and the C-terminal domain (CTD) of the adjacent monomer are important for the assembly of this core. In this study, we have examined the effects of mutations in the NTD region associated with this interaction. We have found that such mutations yielded modest reductions of virus release but major effects on viral infectivity. Cell culture and in vitro assays indicate that the infectivity defects relate to abnormalities in the viral cores. We have selected second-site compensatory mutations that partially restored HIV infectivity. These mutations map to the CA CTD and to spacer peptide 1 (SP1), the portion of the precursor Gag protein immediately C terminal to the CTD. The compensatory mutations do not locate to the molecularly modeled intermolecular NTD-CTD interface. Rather, the compensatory mutations appear to act indirectly, possibly by realignment of the C-terminal helix of the CA CTD, which participates in the NTD-CTD interface and has been shown to serve an important role in the assembly of infectious virus.

Analysis of human immunodeficiency virus type 1 matrix binding to membranes and nucleic acids.

The human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein targets HIV-1 precursor Gag (PrGag) proteins to assembly sites at plasma membrane (PM) sites that are enriched in cholesterol and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)]. MA is myristoylated, which enhances membrane binding, and specifically binds PI(4,5)P(2) through headgroup and 2' acyl chain contacts. MA also binds nucleic acids, although the significance of this association with regard to the viral life cycle is unclear. We have devised a novel MA binding assay and used it to examine MA interactions with membranes and nucleic acids. Our results indicate that cholesterol increases the selectivity of MA for PI(4,5)P(2)-containing membranes, that PI(4,5)P(2) binding tolerates 2' acyl chain variation, and that the MA myristate enhances membrane binding efficiency but not selectivity. We also observed that soluble PI(4,5)P(2) analogues do not compete effectively with PI(4,5)P(2)-containing liposomes for MA binding but surprisingly do increase nonspecific binding to liposomes. Finally, we have demonstrated that PI(4,5)P(2)-containing liposomes successfully outcompete nucleic acids for MA binding, whereas other liposomes do not. These results support a model in which RNA binding protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to PM assembly sites.

Analysis of the N-terminal region of the murine leukemia virus nucleocapsid protein.

Lentiviruses such as the human immunodeficiency virus (HIV-1) and alpharetroviruses such as Rous Sarcoma virus encode an element that spans the precursor Gag (PrGag) protein capsid (CA) C-terminus, a spacer peptide (SP), and the N-terminus of nucleocapsid (NC). Perturbation of this element causes the assembly of aberrant, non-infectious virus particles. To determine whether this element is conserved in gammaretroviruses such as the Moloney murine leukemia virus (MLV), we examined the effects of insertion mutations in the N-terminal portion of the MLV NC coding region. Interestingly, we found that insertions of as many as twenty residues after the twelfth residue of MLV NC yielded proteins that directed the efficient assembly of virus particles. Virus morphologies and crosslink profiles appeared normal, and assembled viruses retained significant levels of infectivity in single cycle infection assays. Two variants were examined in the context of replicating virus constructs, and the mutations were found to be maintained during multiple rounds of infection in a cell culture system. These results suggest that the alpharetrovirus and lentivirus assembly elements either are not needed for gammaretroviruses, or are replaced by an alternative assembly element. Our results also indicate that the N-terminal region of MLV NC is amenable to genetic manipulation.

Determinants of the HIV-1 core assembly pathway.

Based on structural information, we have analyzed the mechanism of mature HIV-1 core assembly and the contributions of structural elements to the assembly process. Through the use of several in vitro assembly assay systems, we have examined details of how capsid (CA) protein helix 1, ß-hairpin and cyclophilin loop elements impact assembly-dependent protein interactions, and we present evidence for a contribution of CA helix 6 to the mature assembly-competent conformation of CA. Additional experiments with mixtures of proteins in assembly reactions provide novel analyses of the mature core assembly mechanism. Our results support a model in which initial assembly products serve as scaffolds for further assembly by converting incoming subunits to assembly proficient conformations, while mutant subunits increase the probability of assembly termination events.

Characterization of the in vitro HIV-1 capsid assembly pathway.

During the morphogenesis of mature human immunodeficiency virus-1 cores, viral capsid proteins assemble conical or tubular shells around viral ribonucleoprotein complexes. This assembly step is mimicked in vitro through reactions in which capsid proteins oligomerize to form long tubes, and this process can be modeled as consisting of a slow nucleation period, followed by a rapid phase of tube growth. We have developed a novel fluorescence microscopy approach to monitor in vitro assembly reactions and have employed it, along with electron microscopy analysis, to characterize the assembly process. Our results indicate that temperature, salt concentration, and pH changes have differential effects on tube nucleation and growth steps. We also demonstrate that assembly can be unidirectional or bidirectional, that growth can be capped, and that proteins can assemble onto the surfaces of tubes, yielding multiwalled or nested structures. Finally, experiments show that a peptide inhibitor of in vitro assembly also can dismantle preexisting tubes, suggesting that such reagents may possess antiviral effects against both viral assembly and uncoating. Our investigations help establish a basis for understanding the mechanism of mature human immunodeficiency virus-1 core assembly and avenues for antiviral inhibition.

Analysis of human immunodeficiency virus matrix domain replacements.

The matrix (MA) domain of the HIV-1 structural precursor Gag (PrGag) protein targets PrGag proteins to membrane assembly sites, and facilitates incorporation of envelope proteins into virions. To evaluate the specific requirements for the MA membrane-binding domain (MBD) in HIV-1 assembly and replication, we examined viruses in which MA was replaced by alternative MBDs. Results demonstrated that the pleckstrin homology domains of AKT protein kinase and phospholipase C delta1 efficiently directed the assembly and release of virus-like particles (VLPs) from cells expressing chimeric proteins. VLP assembly and release also were mediated in a phorbol ester-dependent fashion by the cysteine-rich binding domain of phosphokinase Cgamma. Although alternative MBDs promoted VLP assembly and release, the viruses were not infectious. Notably, PrGag processing was reduced, while cleavage of GagPol precursors resulted in the accumulation of Pol-derived intermediates within virions. Our results indicate that the HIV-1 assembly machinery is flexible with regard to its means of membrane association, but that alternative MBDs can interfere with the elaboration of infectious virus cores.

Sultam thiourea inhibition of West Nile virus.

We have identified sultam thioureas as novel inhibitors of West Nile virus (WNV) replication. One such compound inhibited WNV, with a 50% effective concentration of 0.7 microM, and reduced reporter expression from cells that harbored a WNV-based replicon. Our results demonstrate that sultam thioureas can block a postentry, preassembly step of WNV replication.

Analysis of human immunodeficiency virus type 1 Gag dimerization-induced assembly.

The nucleocapsid (NC) domains of retrovirus precursor Gag (PrGag) proteins play an essential role in virus assembly. Evidence suggests that NC binding to viral RNA promotes dimerization of PrGag capsid (CA) domains, which triggers assembly of CA N-terminal domains (NTDs) into hexamer rings that are interconnected by CA C-terminal domains. To examine the influence of dimerization on human immunodeficiency virus type 1 (HIV-1) Gag protein assembly in vitro, we analyzed the assembly properties of Gag proteins in which NC domains were replaced with cysteine residues that could be linked via chemical treatment. In accordance with the model that Gag protein pairing triggers assembly, we found that cysteine cross-linking or oxidation reagents induced the assembly of virus-like particles. However, efficient assembly also was observed to be temperature dependent or required the tethering of NTDs. Our results suggest a multistep pathway for HIV-1 Gag protein assembly. In the first step, Gag protein pairing through NC-RNA interactions or C-terminal cysteine linkage fosters dimerization. Next, a conformational change converts assembly-restricted dimers or small oligomers into assembly-competent ones. At the final stage, final particle assembly occurs, possibly through a set of larger intermediates.