The roles of lipids and nucleic acids in HIV-1 assembly

During HIV-1 assembly, precursor Gag (PrGag) proteins are delivered to plasma membrane (PM) assembly sites, where they are triggered to oligomerize and bud from cells as immature virus particles. The delivery and triggering processes are coordinated by the PrGag matrix (MA) and nucleocapsid (NC) domains. Targeting of PrGag proteins to membranes enriched in cholesterol and phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2) is mediated by the MA domain, which also has been shown to bind both RNA and DNA. Evidence suggests that the nucleic acid-binding function of MA serves to inhibit PrGag binding to inappropriate intracellular membranes, prior to delivery to the PM. At the PM, MA domains putatively trade RNA ligands for PI(4,5)P2 ligands, fostering high affinity membrane binding. Triggering of oligomerization, budding and virus particle release results when NC domains on adjacent PrGag proteins bind to viral RNA, leading to capsid (CA) domain oligomerization. This process leads to the assembly of immature virus shells in which hexamers of membrane-bound MA trimers appear to organize above interlinked CA hexamers. Here we review the functions of retroviral MA proteins, with an emphasis on the nucleic acid binding capability of the HIV-1 MA protein, and its effects on membrane binding. <br/

Hexagonal Organization of Moloney Murine Leukemia Virus Capsid Proteins

AbstractTo help elucidate the mechanisms by which retrovirus structural proteins associate to form virus particles, we have examined membrane-bound assemblies of Moloney murine leukemia virus (M-MuLV) capsid (CA) proteins. Electron microscopy and image reconstruction techniques showed that CA dimers appear to function as organizational subunits of the cage-like, membrane-bound protein arrays. However, new three-dimensional (3D) data also were consistent with hexagonal (p6) assembly models. The p6 3D reconstructions of membrane-bound M-MuLV CA proteins gave unit cells of a = b = 80.3 Å, c = 110 Å, γ = 120°, in which six dimer units formed a cage lattice. Neighbor cage hole-to-hole distances were 45 Å, while distances between hexagonal cage holes corresponded to unit cell lengths (80.3 Å). The hexagonal model predicts two types of cage holes (trimer and hexamer holes), uses symmetric head-to-head dimer building blocks, and permits the introduction of lattice curvature by conversion of hexamer to pentamer units. The M-MuLV CA lattice is similar to those formed in helical tubes by HIV CA in that hexamer units surround cage holes of 25–30 Å, but differs in that M-MuLV hexamer units appear to be CA dimers, whereas HIV CA units appear to be monomers. These results suggest that while general assembly principles apply to different retroviruses, clear assembly distinctions exist between these virus types

Mutations affecting cleavage at the p10-capsid protease cleavage site block Rous sarcoma virus replication

A series of amino acid substitutions (M239F, M239G, P240F, V241G) were placed in the p10-CA protease cleavage site (VVAM*PVVI) to change the rate of cleavage of the junction. The effects of these substitutions on p10-CA cleavage by RSV PR were confirmed by measuring the kinetics of cleavage of model peptide substrates containing the wild type and mutant p10-CA sites. The effects of these substitutions on processing of the Gag polyprotein were determined by labeling Gag transfected COS-1 cells with (35)S-Met and -Cys, and immunoprecipitation of Gag and its cleavage products from the media and lysate fractions. All substitutions except M239F caused decreases in detectable Gag processing and subsequent release from cells. Several of the mutants also caused defects in production of the three CA proteins. The p10-CA mutations were subcloned into an RSV proviral vector (RCAN) and introduced into a chick embryo fibroblast cell line (DF-1). All of the mutations except M239F blocked RSV replication. In addition, the effects of the M239F and M239G substitutions on the morphology of released virus particles were examined by electron microscopy. While the M239F particles appeared similar to wild type particles, M239G particles contained cores that were large and misshapen. These results suggest that mutations affecting cleavage at the p10-CA protease cleavage site block RSV replication and can have a negative impact on virus particle morphology

Small Molecule Inhibition of HIV-1–Induced MHC-I Down-Regulation Identifies a Temporally Regulated Switch in Nef Action

Nef assembles a multi-kinase complex triggering MHC-I down-regulation. We identify an inhibitor that blocks MHC-I down-regulation, identifying a temporally regulated switch in Nef action from directing MHC-I endocytosis to blocking cell surface delivery. These findings challenge current dogma and reveal a regulated immune evasion program

Regional distribution of maternal messenger RNA in the amphibian oocyte

We show that there is a non-random distribution of information in the form of mRNAs along the animal/vegetal axis of Xenopus oocytes. Polyadenylated RNA isolated from different regions along this axis was translated in a cell-free system, and the products were fractionated by two-dimensional gel electrophoresis. A comparison of the resulting protein products using a computer program has established that specific translatable mRNAs are regionally localized within the oocyte prior to fertilization. Seventeen mRNAs show significant qualitative or quantitative differences in their subcellular location. These sequences comprise as much as 4% of the total detected translatable polyadenylated RNA. Asymmetric segregation of these messages during Xenopus development may be involved in the determination of cell fate

Stem cell factor binding to retrovirus primer binding site silencers.

Using modified nuclear lysis and binding conditions, we have examined the binding of an embryonal carcinoma (EC) cell factor, binding factor A, to a stem cell-specific silencer which acts at the DNA level and overlaps the Moloney murine leukemia virus (M-MuLV) proline primer binding site (PBS). Following our protocol, we found that in vitro binding of factor A correlated with the in vivo activity of the M-MuLV silencer. Factor A bound specifically to the wild-type silencer element at room temperature and 30 degrees C, but not at 4 degrees C, and bound 10-fold better to the full-length silencer than to a minimal silencer core element. The factor was enriched in nuclear compared with cytosolic extracts and in undifferentiated EC cells compared with differentiated cells in which the silencer is nonfunctional. Salt and ion requirements for factor A binding were investigated, and partial purification steps indicated the factor to be a heparin-Sepharose-binding moiety of greater than 100 kDa. To examine possible relationships between silencer and PBS activities, sequences representing phenylalanine, isoleucine, lysine-1,2, lysine-3, methionine, and tryptophan PBS DNA fragments were tested in vivo for stem cell-specific repression of M-MuLV expression and in vitro in DNA binding assays. Of these PBS elements, only the lysine-1,2 PBS DNA fragment showed consistently high levels of repression. Interestingly, the lysine-1,2 PBS DNA fragment also formed a complex with an EC cell factor with characteristics similar to those of factor A. However, the two factors did not cross-compete in binding studies, suggesting that they may be different but related factors. Our results suggest that expression of Mason-Pfizer monkey virus, visna virus, and spumavirus, which use the lysine-1,2 PBS, may be inhibited in undifferentiated stem cells

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)P2]. MA is myristoylated, which enhances membrane binding, and specifically binds PI(4,5)P2 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)P2-containing membranes, that PI(4,5)P2 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)P2 analogues do not compete effectively with PI(4,5)P2-containing liposomes for MA binding but surprisingly do increase nonspecific binding to liposomes. Finally, we have demonstrated that PI(4,5)P2-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

Virus Particle Core Defects Caused by Mutations in the Human Immunodeficiency Virus Capsid N-Terminal Domain

The N-terminal domains (NTDs) of the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein have been modeled to form hexamer rings in the mature cores of virions. In vitro, hexamer ring units organize into either tubes or spheres, in a pH-dependent fashion. To probe factors which might govern hexamer assembly preferences in vivo, we examined the effects of mutations at CA histidine residue 84 (H84), modeled at the outer edges of NTD hexamers, as well as a nearby histidine (H87) in the cyclophilin A (CypA) binding loop. Although mutations at H87 yielded infectious virions, mutations at H84 produced assembly-competent but poorly infectious virions. The H84 mutant viruses incorporated wild-type levels of CypA and viral RNAs and showed nearly normal signals in virus entry assays. However, mutant CA proteins assembled aberrant virus cores, and mutant core fractions retained abnormally high levels of CA but reduced reverse transcriptase activities. Our results suggest that HIV-1 CA residue 84 contributes to a structure which helps control either NTD hexamer assembly or the organization of hexamers into higher-order structures