C/EBPα, C/EBPα oncoproteins, or C/EBPß preferentially bind NF-κB p50 compared with p65, focusing therapeutic targeting on the C/EBP:p50 interaction.

Canonical nuclear factor kappaB (NF-κB) activation signals stimulate nuclear translocation of p50:p65, replacing inhibitory p50:p50 with activating complexes on chromatin. C/EBP interaction with p50 homodimers provides an alternative pathway for NF-κB target gene activation, and interaction with p50:p65 may enhance gene activation. We previously found that C/EBPα cooperates with p50, but not p65, to induce Bcl-2 transcription and that C/EBPα induces Nfkb1/p50, but not RelA/p65, transcription. Using p50 and p65 variants containing the FLAG epitope at their N- or C-termini, we now show that C/EBPα, C/EBPα myeloid oncoproteins, or the LAP1, LAP2, or LIP isoforms of C/EBPß have markedly higher affinity for p50 than for p65. Deletion of the p65 transactivation domain did not increase p65 affinity for C/EBPs, suggesting that unique residues in p50 account for specificity, and clustered mutation of HSDL in the "p50 insert" lacking in p65 weakens interaction. Also, in contrast to Nfkb1 gene deletion, absence of the RelA gene does not reduce Bcl-2 or Cebpa RNA in unstimulated cells or prevent interaction of C/EBPα with the Bcl-2 promoter. Saturating mutagenesis of the C/EBPα basic region identifies R300 and nearby residues, identical in C/EBPß, as critical for interaction with p50. These findings support the conclusion that C/EBPs activate NF-κB target genes via contact with p50 even in the absence of canonical NF-κB activation and indicate that targeting C/EBP:p50 rather than C/EBP:p65 interaction in the nucleus will prove effective for inflammatory or malignant conditions, alone or synergistically with agents acting in the cytoplasm to reduce canonical NF-κB activation.

Host ABCE1 is at plasma membrane HIV assembly sites and its dissociation from Gag is linked to subsequent events of virus production.

In primate cells, assembly of a single HIV-1 capsid involves multimerization of thousands of Gag polypeptides, typically at the plasma membrane. Although studies support a model in which HIV-1 assembly proceeds through complexes containing Gag and the cellular adenosine triphosphatase ABCE1 (also termed HP68 or ribonuclease L inhibitor), whether these complexes constitute true assembly intermediates remains controversial. Here we demonstrate by pulse labeling in primate cells that a population of Gag associates with endogenous ABCE1 within minutes of translation. In the next approximately 2 h, Gag-ABCE1 complexes increase in size to approximately that of immature capsids. Dissociation of ABCE1 from Gag correlates closely with Gag processing during virion maturation and occurs much less efficiently when the HIV-1 protease is inactivated. Finally, quantitative double-label immunogold electron microscopy reveals that ABCE1 is recruited to sites of assembling wild-type Gag at the plasma membrane but not to sites of an assembly-defective Gag mutant at the plasma membrane. Together these findings demonstrate that a population of Gag present at plasma membrane sites of assembly associates with ABCE1 throughout capsid formation until the onset of virus maturation, which is then followed by virus release. Moreover, the data suggest a linkage between Gag-ABCE1 dissociation and subsequent events of virion production.

Basic residues in the nucleocapsid domain of Gag are required for interaction of HIV-1 gag with ABCE1 (HP68), a cellular protein important for HIV-1 capsid assembly.

During human immunodeficiency virus, type 1 (HIV-1) assembly, Gag polypeptides multimerize into immature HIV-1 capsids. The cellular ATP-binding protein ABCE1 (also called HP68 or RNase L inhibitor) appears to be critical for proper assembly of the HIV-1 capsid. In primate cells, ABCE1 associates with Gag polypeptides present in immature capsid assembly intermediates. Here we demonstrate that the NC domain of Gag is critical for interaction with endogenous primate ABCE1, whereas other domains in Gag can be deleted without eliminating the association of Gag with ABCE1. NC contains two Cys-His boxes that form zinc finger motifs and are responsible for encapsidation of HIV-1 genomic RNA. In addition, NC contains basic residues known to play a critical role in nonspecific RNA binding, Gag-Gag interactions, and particle formation. We demonstrate that basic residues in NC are needed for the Gag-ABCE1 interaction, whereas the cysteine and histidine residues in the zinc fingers are dispensable. Constructs that fail to interact with primate ABCE1 or interact poorly also fail to form capsids and are arrested at an early point in the immature capsid assembly pathway. Whereas others have shown that basic residues in NC bind nonspecifically to RNA, which in turn scaffolds or nucleates assembly, our data demonstrate that the same basic residues in NC act either directly or indirectly to recruit a cellular protein that also promotes capsid formation. Thus, in cells, basic residues in NC appear to act by two mechanisms, recruiting both RNA and a cellular ATPase in order to facilitate efficient assembly of HIV-1 capsids.

Comparing capsid assembly of primate lentiviruses and hepatitis B virus using cell-free systems.

Many viruses that assemble their capsids in the eukaryotic cytoplasm require a threshold concentration of capsid protein to achieve capsid assembly. Strategies for achieving this include maintaining high levels of capsid protein synthesis and targeting to specific sites to raise the effective concentration of capsid polypeptides. To understand how different viruses achieve the threshold capsid protein concentration required for assembly, we used cell-free systems to compare capsid assembly of hepatitis B virus (HBV) and three primate lentiviruses. Capsid formation of these diverse viruses in a common eukaryotic extract was dependent on capsid protein concentration. HBV capsid assembly was also dependent on the presence of intact membrane surfaces. Surprisingly, not all of the primate lentiviral capsid proteins examined required myristoylation and intact membranes for assembly, even though all contain a myristoylation signal. These findings reveal significant diversity in how different capsid proteins assemble in the same cellular extract.

Cell-free systems for capsid assembly of primate lentiviruses from three different lineages.

We recently demonstrated that capsids from three main primate lentiviral lineages appear to form via a pathway of assembly intermediates in primate cells. Retroviral capsid assembly intermediates were initially identified and characterized using a cell-free system for assembly of immature HIV-1 capsids. Because cell-free capsid assembly systems are useful tools, we are interested in developing such systems for other primate lentiviruses besides HIV-1. Here we extend previous cell-free studies by showing that Gag proteins of HIV-2, from a second primate lentiviral lineage, progress from early intermediates to late intermediates and completed capsids over time. Additionally, we demonstrate that Gag proteins of SIVagm, from a third primate lentiviral lineage, associate with the cellular factor HP68 and complete assembly in this system. Therefore, cell-free systems reproduce assembly of Gag from three main primate lentiviral lineages, and can be used to compare mechanistic features of capsid assembly of genetically divergent primate lentiviruses.

Conservation of a stepwise, energy-sensitive pathway involving HP68 for assembly of primate lentivirus capsids in cells.

Previously we have described a stepwise, energy-dependent pathway for human immunodeficiency virus type 1 (HIV-1) capsid assembly in a cell-free system. In this pathway, Gag polypeptides utilize the cellular factor HP68 and assemble into immature capsids by way of assembly intermediates that have defined biochemical characteristics. Here we address whether this pathway is universally conserved among primate lentiviruses and can be observed in mammalian cells. We demonstrate that HIV-2 Gag associates with human HP68 in a cell-free system and that Gag proteins of HIV-2, simian immunodeficiency virus SIVmac239, and SIVagm associate with endogenous HP68 in primate cells, as is seen for HIV-1. Analysis of primate cells expressing lentivirus Gag proteins revealed Gag-containing complexes with the same sedimentation values as seen for previously described HIV-1 assembly intermediates in the cell-free system (10S, 80-150S, and 500S). These complexes fit criteria for assembly intermediates as judged by energy sensitivity, pattern of HP68 association, and the failure of specific complexes to be formed by assembly-incompetent Gag mutants. We also demonstrate that virus-like particles released from cells do not appear to contain HP68, suggesting that HP68 is released from Gag upon completion of capsid assembly in cells, as was observed previously in the cell-free system. Together these findings support a model in which all primate lentivirus capsids assemble by a conserved pathway of HP68-containing, energy-dependent assembly intermediates that have specific biochemical features.

Characterization of virus infectivity and cell-free capsid assembly of SIVMneCL8.

We have previously described a cell-free system that reconstitutes immature capsid assembly of Gag polypeptides from viruses belonging to three major primate lentiviral lineages, including HIV-1, HIV-2 and SIVagm. Studies described here examine a member of the SIVmac/Mne lineage, SIVMneCL8, using assays for virus production and infectivity as well as cellular events in capsid formation. We report that SIVMneCL8, a molecular clone with properties typical of transmitted viral variants, is less infectious per unit p27 Gag than another member of the SIVmac/Mne lineage, SIVmac239. SIVMneCL8 Gag polypeptides are arrested at an early stage of capsid assembly in the cell-free system. Additionally, SIVMneCL8 Gag polypeptides associate minimally with the host factor human HP68. This is the first report of a primate lentivirus that does not complete capsid assembly in the cell-free system.