Neutralization of Human Respiratory Syncytial Virus Infectivity by Antibodies and Low-Molecular-Weight Compounds Targeted against the Fusion Glycoprotein

Human respiratory syncytial virus (HRSV) fusion (F) protein is an essential component of the virus envelope that mediates fusion of the viral and cell membranes, and, therefore, it is an attractive target for drug and vaccine development. Our aim was to analyze the neutralizing mechanism of anti-F antibodies in comparison with other low-molecular-weight compounds targeted against the F molecule. It was found that neutralization by anti-F antibodies is related to epitope specificity. Thus, neutralizing and nonneutralizing antibodies could bind equally well to virions and remained bound after ultracentrifugation of the virus, but only the former inhibited virus infectivity. Neutralization by antibodies correlated with inhibition of cell-cell fusion in a syncytium formation assay, but not with inhibition of virus binding to cells. In contrast, a peptide (residues 478 to 516 of F protein [F478-516]) derived from the F protein heptad repeat B (HRB) or the organic compound BMS-433771 did not interfere with virus infectivity if incubated with virus before ultracentrifugation or during adsorption of virus to cells at 4°C. These inhibitors must be present during virus entry to effect HRSV neutralization. These results are best interpreted by asserting that neutralizing antibodies bind to the F protein in virions interfering with its activation for fusion. Binding of nonneutralizing antibodies is not enough to block this step. In contrast, the peptide F478-516 or BMS-433771 must bind to F protein intermediates generated during virus-cell membrane fusion, blocking further development of this process.Human respiratory syncytial virus (HRSV), a member of the Pneumovirus genus of the Paramyxoviridae family, is the main cause of severe lower respiratory tract infections in very young children (36), and it is a pathogen of considerable importance in the elderly (24, 26) and in immunocompromised adults (22). Currently, there is no effective vaccine against the virus although it is known that passive administration of neutralizing antibodies to individuals at high risk is an effective immunoprophylaxis (37, 38).The HRSV genome is a single-stranded negative-sense RNA molecule of approximately 15 kb that encodes 11 proteins (16, 53). Two of these proteins are the main surface glycoproteins of the virion. These are (i) the attachment (G) protein, which mediates virus binding to cells (44), and (ii) the fusion (F) protein, which promotes both fusion of the viral and cell membranes at the initial stages of the infectious cycle and fusion of the membrane of infected cells with those of adjacent cells to form characteristic syncytia (72). These two glycoproteins are the only targets of neutralizing antibodies either induced in animal models (19, 63, 65, 70) or present in human sera (62).The G protein is a highly variable type II glycoprotein that shares neither sequence identity nor structural features with the attachment protein of other paramyxoviruses (75). It is synthesized as a precursor of about 300 amino acids (depending on the strain) that is modified posttranslationally by the addition of a large number of N- and O-linked oligosaccharides and is also palmitoylated (17). The G protein is oligomeric (probably a homotetramer) (23) and promotes binding of HRSV to cell surface proteoglycans (35, 40, 49, 67). Whether this is the only interaction of G with cell surface components is presently unknown.The F protein is a type I glycoprotein that is synthesized as an inactive precursor of 574 amino acids (F0) which is cleaved by furin during transport to the cell surface to yield two disulfide-linked polypeptides, F2 from the N terminus and F1 from the C terminus (18). Like other viral type I fusion proteins, the mature F protein is a homotrimer which is in a prefusion, metastable, conformation in the virus particle. After fusion, the F protein adopts a highly stable postfusion conformation. Stability of the postfusion conformation is determined to great extent by two heptad repeat (HR) sequences, HRA and HRB, present in the F1 chain. Mixtures of HRA and HRB peptides form spontaneously heterotrimeric complexes (43, 51) that assemble in six-helix bundles (6HB), consisting of an internal core of three HRA helices surrounded by three antiparallel HRB helices, as determined by X-ray crystallography (79).The three-dimensional (3D) structure of the HRSV F protein has not been solved yet. Nevertheless, the structures of the pre- and postfusion forms of two paramyxovirus F proteins have revealed substantial conformational differences between the pre- and postfusion conformations (77, 78). The present hypothesis about the mechanism of membrane fusion mediated by paramyxovirus F proteins proposes that, following binding of the virus to the cell surface, the prefusion form of the F glycoprotein is activated, and membrane fusion is triggered. The F protein experiences then a series of conformational changes which include the exposure of a hydrophobic region, called the fusion peptide, and its insertion into the target membrane. Subsequent refolding of this intermediate leads to formation of the HRA and HRB six-helix bundle, concomitant with approximation of the viral and cell membranes that finally fuse, placing the fusion peptide and the transmembrane domain in the same membrane (4, 20). The formation of the 6HB and the associated free energy change are tightly linked to the merger of the viral and cellular membranes (60).Antibodies play a major role in protection against HRSV. Animal studies have demonstrated that immunization with either F or G glycoproteins induces neutralizing antibodies and protects against a viral challenge (19, 63, 70). Furthermore, transfer of these antibodies (31, 56) or of anti-F or anti-G monoclonal antibodies (MAbs) protects mice, cotton rats, or calves against either a human or bovine RSV challenge, respectively (65, 68, 73). Likewise, infants at high risk of severe HRSV disease are protected by the prophylactic administration of immunoglobulins with high anti-HRSV neutralizing titers (33). Finally, a positive correlation was found between high titers of serum neutralizing antibodies and protection in adult volunteers challenged with HRSV (34, 74), while an inverse correlation was found between high titers of neutralizing antibodies and risk of infection in children (29) and in the elderly (25).Whereas all the anti-G monoclonal antibodies reported to date are poorly neutralizing (1, 28, 48, 71), some anti-F monoclonal antibodies have strong neutralization activity (1, 3, 5, 28, 46). It is believed that HRSV neutralization by anti-G antibodies requires simultaneous binding of several antibodies to different epitopes, leading to steric hindrance for interaction of the G glycoprotein with the cell surface. Indeed, it has been shown that neutralization is enhanced by mixtures of anti-G monoclonal antibodies (1, 50), mimicking the effect of polyclonal anti-G antibodies. In contrast, highly neutralizing anti-F monoclonal antibodies do not require cooperation by other antibodies to block HRSV infectivity efficiently (1).In addition to neutralizing antibodies, other low-molecular-weight compounds directed against the F protein are potent inhibitors of HRSV infectivity. Synthetic peptides that reproduce sequences of heptad repeat B inhibit both membrane fusion promoted by the F protein and HRSV infectivity (42). Also, other small molecules obtained by chemical synthesis have been shown to interact with F protein and inhibit HRSV infectivity. These HRSV entry inhibitors have been the topic of intense research in recent years (55).This study explores the mechanisms of HRSV neutralization by different inhibitors of membrane fusion, including anti-F monoclonal antibodies, an HRB peptide, and the synthetic compound BMS-433771 (13-15). The results obtained indicate that antibodies and low-molecular-weight compounds block membrane fusion at different stages during virus entry.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Isolation of Human Monoclonal Antibodies That Potently Neutralize Human Cytomegalovirus Infection by Targeting Different Epitopes on the gH/gL/UL128-131A Complex

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC₉₀] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.Human cytomegalovirus (HCMV) is a member of the herpesvirus family which is widely distributed in the human population and can cause severe disease in immunocompromised patients and upon infection of the fetus. HCMV infection causes clinical disease in 75% of patients in the first year after transplantation (58), while primary maternal infection is a major cause of congenital birth defects including hearing loss and mental retardation (5, 33, 45). Because of the danger posed by this virus, development of an effective vaccine is considered of highest priority (51).HCMV infection requires initial interaction with the cell surface through binding to heparan sulfate proteoglycans (8) and possibly other surface receptors (12, 23, 64, 65). The virus displays a broad host cell range (24, 53), being able to infect several cell types such as endothelial cells, epithelial cells (including retinal cells), smooth muscle cells, fibroblasts, leukocytes, and dendritic cells (21, 37, 44, 54). Endothelial cell tropism has been regarded as a potential virulence factor that might influence the clinical course of infection (16, 55), whereas infection of leukocytes has been considered a mechanism of viral spread (17, 43, 44). Extensive propagation of HCMV laboratory strains in fibroblasts results in deletions or mutations of genes in the UL131A-128 locus (1, 18, 21, 36, 62, 63), which are associated with the loss of the ability to infect endothelial cells, epithelial cells, and leukocytes (15, 43, 55, 61). Consistent with this notion, mouse monoclonal antibodies (MAbs) to UL128 or UL130 block infection of epithelial and endothelial cells but not of fibroblasts (63). Recently, it has been shown that UL128, UL130, and UL131A assemble with gH and gL to form a five-protein complex (thereafter designated gH/gL/UL128-131A) that is an alternative to the previously described gCIII complex made of gH, gL, and gO (22, 28, 48, 63).In immunocompetent individuals T-cell and antibody responses efficiently control HCMV infection and reduce pathological consequences of maternal-fetal transmission (13, 67), although this is usually not sufficient to eradicate the virus. Albeit with controversial results, HCMV immunoglobulins (Igs) have been administered to transplant patients in association with immunosuppressive treatments for prophylaxis of HCMV disease (56, 57), and a recent report suggests that they may be effective in controlling congenital infection and preventing disease in newborns (32). These products are plasma derivatives with relatively low potency in vitro (46) and have to be administered by intravenous infusion at very high doses in order to deliver sufficient amounts of neutralizing antibodies (4, 9, 32, 56, 57, 66).The whole spectrum of antigens targeted by HCMV-neutralizing antibodies remains poorly characterized. Using specific immunoabsorption to recombinant antigens and neutralization assays using fibroblasts as model target cells, it was estimated that 40 to 70% of the serum neutralizing activity is directed against gB (6). Other studies described human neutralizing antibodies specific for gB, gH, or gM/gN viral glycoproteins (6, 14, 26, 29, 34, 41, 52, 60). Remarkably, we have recently shown that human sera exhibit a more-than-100-fold-higher potency in neutralizing infection of endothelial cells than infection of fibroblasts (20). Similarly, CMV hyperimmunoglobulins have on average 48-fold-higher neutralizing activities against epithelial cell entry than against fibroblast entry (10). However, epitopes that are targeted by the antibodies that comprise epithelial or endothelial cell-specific neutralizing activity of human immune sera remain unknown.In this study we report the isolation of a large panel of human monoclonal antibodies with extraordinarily high potency in neutralizing HCMV infection of endothelial and epithelial cells and myeloid cells. With the exception of a single antibody that recognized a conserved epitope of UL128, all other antibodies recognized conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex.     

Previously Unrecognized Amino Acid Substitutions in the Hemagglutinin and Fusion Proteins of Measles Virus Modulate Cell-Cell Fusion,Hemadsorption, Virus Growth,and Penetration Rate

Wild-type measles virus (MV) isolated in B95a cells could be adapted to Vero cells after several blind passages. In this study, we have determined the complete nucleotide sequences of the genomes of the wild type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strain and identified amino acid substitutions R516G, E271K, D439E and G464W (D439E/G464W), N481Y/H495R, and Y187H/L204F in the nucleocapsid, V, fusion (F), hemagglutinin (H), and large proteins, respectively. Expression of mutated H and F proteins from cDNA revealed that the H495R substitution, in addition to N481Y, in the H protein was necessary for the wild-type H protein to use CD46 efficiently as a receptor and that the G464W substitution in the F protein was important for enhanced cell-cell fusion. Recombinant wild-type MV strains harboring the F protein with the mutations D439E/G464W [F(D439E/G464W)] and/or H(N481Y/H495R) protein revealed that both mutated F and H proteins were required for efficient syncytium formation and virus growth in Vero cells. Interestingly, a recombinant wild-type MV strain harboring the H(N481Y/H495R) protein penetrated slowly into Vero cells, while a recombinant wild-type MV strain harboring both the F(D439E/G464W) and H(N481Y/H495R) proteins penetrated efficiently into Vero cells, indicating that the F(D439E/G464W) protein compensates for the inefficient penetration of a wild-type MV strain harboring the H(N481Y/H495R) protein. Thus, the F and H proteins synergistically function to ensure efficient wild-type MV growth in Vero cells.Measles virus (MV), which belongs to the genus Morbillivirus in the family Paramyxoviridae, is an enveloped virus with a nonsegmented negative-strand RNA genome. The MV genome encodes six structural proteins: the nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin (H), and large (L) proteins. The P gene also encodes two other accessory proteins, the C and V proteins. The C protein is translated from an alternative translational initiation site leading a different reading frame, and the V protein is synthesized from an edited mRNA. MV has two envelope glycoproteins, the F and H proteins. The former is responsible for envelope fusion, and the latter is responsible for receptor binding (12).Wild-type MV strains isolated in B95a cells and laboratory-adapted MV strains have distinct phenotypes (18). Wild-type MV strains can grow in B95a cells but not in Vero cells, while laboratory-adapted MV strains can grow in both B95a and Vero cells. Wild-type MV strains do not cause hemadsorption (HAd) in African green monkey red blood cells (AGM-RBC), while most of laboratory-adapted MV strains cause HAd. Importantly, wild-type MV strains are pathogenic and induce clinical signs that resemble human measles in experimentally infected monkeys while laboratory-adapted MV strains do not.One approach to identify amino acid substitutions responsible for these phenotypic differences is the comparison of a wild-type MV strain with a standard laboratory-adapted MV strain such as the Edmonston strain. With regard to the H protein, amino acid substitutions important for HAd activity and cell-cell fusion in tissue culture cells were identified by expressing the H proteins in mammalian cells (15, 21). Recently, Tahara et al. revealed that the M, H, and L proteins are responsible for efficient growth in Vero cells by constructing a series of recombinant viruses in which part of the genome of the wild-type MV was replaced with the corresponding sequences of the Edmonston strain (45, 46, 47).Another approach is the comparison of wild-type MV strains with their Vero cell-adapted MV strains. It was reported that Vero cell-adapted MV strains could be obtained by successive blind passages of wild-type MV strains in Vero cells (18, 24, 30, 43). Interestingly, in vivo and in vitro phenotypes of Vero cell-adapted MV strains were similar to those of laboratory-adapted standard MV strains (18, 19, 24, 30, 43). Comparison of the complete nucleotide sequences of the genomes of wild-type MV strains with those of Vero cell-adapted wild-type MV strains revealed amino acid substitutions in the P, C, V, M, H, and L proteins (27, 42, 48, 53).At present, these phenotypic differences are explained mainly by the receptor usage of MV. Wild-type MV strains can use signaling lymphocyte activation molecule (SLAM; also called CD150) but not CD46 as a cellular receptor, whereas laboratory-adapted MV strains can use both SLAM and CD46 as cellular receptors (7, 10, 16, 29, 56, 60).However, receptor usage per se cannot explain all of the phenotypic differences (20, 25, 48, 53). For example, recombinant Edmonston strains expressing wild-type H proteins can grow in Vero cells to some extent (17, 54). Several reports suggested the presence of the third MV receptor on Vero cells (14, 44, 54, 60). Other reports indicated the contribution of the M protein on cell-cell fusion and growth of MV in Vero cells (4, 27, 47). Recently, the unidentified epithelial cell receptor for MV was predicted in primary culture of human cells (1, 55) and several epithelial cell lines (23, 51). However, the identity of the third receptor on Vero cells and the unidentified epithelial cell receptor is not clear yet. Thus, the mechanism of Vero cell adaptation of wild-type MV is not completely understood.In order to understand the molecular mechanism of these phenotypic changes of wild-type MV strains during adaptation in Vero cells, we determined the complete nucleotide sequences of the genomes of the wild-type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strains (43) and examined the effect of individual amino acid substitutions using a mammalian cell expression system and reverse genetics. We show here that previously unrecognized new amino acid substitutions in the H and F proteins are important for MV adaptation and HAd activity.     

Probing the Spatial Organization of Measles Virus Fusion Complexes

The spatial organization of metastable paramyxovirus fusion (F) and attachment glycoprotein hetero-oligomers is largely unknown. To further elucidate the organization of functional fusion complexes of measles virus (MeV), an archetype of the paramyxovirus family, we subjected central predictions of alternative docking models to experimental testing using three distinct approaches. Carbohydrate shielding through engineered N-glycans indicates close proximity of a membrane-distal, but not membrane-proximal, section of the MeV attachment (H) protein stalk domain to F. Directed mutagenesis of this section identified residues 111, 114, and 118 as modulators of avidity of glycoprotein interactions and determinants of F triggering. Stalk-length variation through deletion or insertion of HR elements at positions flanking this section demonstrates that the location of the stalk segment containing these residues cannot be altered in functional fusion complexes. In contrast, increasing the distance between the H head domains harboring the receptor binding sites and this section through insertion of structurally rigid α-helical domains with a pitch of up to approximately 75 Å downstream of stalk position 118 partially maintains functionality in transient expression assays and supports efficient growth of recombinant virions. In aggregate, these findings argue against specific protein-protein contacts between the H head and F head domains but instead support a docking model that is characterized by short-range contacts between the prefusion F head and the attachment protein stalk, possibly involving H residues 111, 114, and 118, and extension of the head domain of the attachment protein above prefusion F.Paramyxoviruses infect cells through fusion of the viral envelope with target cell membranes. For all members of the Paramyxovirinae subfamily, this involves the concerted action of two envelope glycoproteins, the fusion (F) and attachment (H, HN, or G, depending on the Paramyxovirinae genus) proteins. Both proteins feature short lumenal tails, a single transmembrane domain, and large ectodomains. The F protein, in type I orientation, forms homotrimers, while homodimers or homotetramers have been suggested as functional units for attachment proteins of different Paramyxovirinae subfamily members (7, 14, 28, 41, 49, 50, 66). For entry, upon receptor binding, the attachment protein is considered to initiate a series of conformational rearrangements in the metastable prefusion F protein (15, 77), which ultimately brings together transmembrane domains and fusion peptides and, thus, donor and target membranes (3, 32, 45, 53, 80).Multiple studies have demonstrated that specific interactions between compatible F and attachment proteins of paramyxovirinae are imperative for the formation of functional fusion complexes (6, 29, 36, 42, 43, 56, 75). However, the molecular nature of these interactions and the spatial organization of functional glycoprotein hetero-oligomers remain largely unknown. Individual ectodomain and partial ectodomain crystal structures have been obtained for different paramyxovirus F (13, 76, 77) and attachment (8, 14, 17, 28, 35, 79) proteins, respectively. For F, a stabilized human parainfluenza virus type 5 (HPIV5) ectodomain that is believed to represent a prefusion conformation folds into a globular head structure that is attached to the transmembrane domains through a helical stalk consisting of the membrane-proximal heptad repeat B (HR-B) domains (77). For the attachment protein, a globular head that harbors the receptor binding sites is considered to be connected to the transmembrane region through extended stalk domains (34, 78). Crystal structures of isolated head domains have been solved for several paramyxovirus attachment proteins, including measles virus (MeV) H, and reveal the six-blade propeller fold typical of sialidase structures (8, 14, 17, 28, 79). However, morbilliviruses recognize proteinaceous receptors (for MeV, the regulator of complement activation [CD46] and/or signaling lymphocytic activation molecule [SLAM], depending on the virus strain) (21, 40, 46, 51, 64, 65). X-ray data do not extend to the stalk domains, but circular dichroism analysis (78) and structure predictions (36, 78) support an α-helical coiled-coil configuration of the stalk.The nature of individual residues that engage in specific intermolecular interactions between glycoproteins of paramyxovirinae prior to refolding has been studied most extensively for the attachment protein. The stalk domains of several paramyxovirus HN proteins have been implicated in mediating specificity for their homotypic F proteins (18, 20, 43, 63, 70, 72). We have found that this extends to MeV and canine distemper virus H and, thus, to paramyxovirinae recognizing proteinaceous receptors (36), supporting the general hypothesis that F-interacting residues may reside in the stalk region of the attachment protein (30, 78).Considerably less information concerning the nature of F microdomains that mediate attachment protein specificity is available. Among the few exceptions are peptides derived from Newcastle disease virus (NDV) and Sendai virus F HR-B domains, which interact with soluble variants of the respective HN proteins in vitro (25, 67). Multiple domains have been suggested to mediate specificity of HPIV2 F for its HN (69). However, a conclusive N-glycan shielding study (43) and structural information (77, 78) argue against direct contacts between NDV F HR-B domains and HN in native glycoprotein complexes. Thus, the role of individual HPIV2 F residues in HN binding is unclear (25, 43).Building on the observation that MeV H is able to engage in productive heterotypic interactions with F proteins derived from some but not all isolates of closely related canine distemper virus, we have recently identified residues in morbillivirus F (MeV F residue 121) and H (H stalk residues 110 to 114) that interdependently contribute to physical MeV glycoprotein interaction and F triggering for fusion (36). While these residues could mediate reciprocal glycoprotein specificity through long-range effects, molecular modeling of the MeV H stalk in an α-helical conformation has posited F residue 121 at the same level above the viral envelope as H residues 110 to 114, making direct contacts structurally conceivable (36). This spatial organization of functional fusion complexes furthermore provides a comprehensive explanation for previous demonstrations of a specific role for attachment protein stalk domains of paramyxovirinae in functional and physical interactions with F (18, 43, 63, 70, 72). However, this “staggered-head” model mandates positioning the globular head of the attachment protein above the prefusion F trimer (36), as opposed to a suggested “parallel-head” alignment of the glycoproteins (31, 47). The latter is mostly based on transmission electron microscopy micrographs of viral particles apparently showing glycoprotein spikes of equal length (33). Unfortunately, these images lack the resolution for an identification of the molecular nature of the spikes (attachment or F protein) or the distinguishing between densely packaged H and F head domains of different heights and laterally aligned head domains. Indeed, a recent single-particle reconstruction based on cryo-electron microscopy images of HPIV5 particles revealed that defined spikes correspond to F in a postfusion conformation, which was interpreted as a product of possible premature F refolding (38). These two-dimensional images of heavy-metal-stained particles did not reveal F spikes in a prefusion conformation. Rather, a dense surface layer was considered to correspond to prefusion glycoprotein hetero-oligomers (38). In addition to further-advanced image reconstructions, biochemical assessment of alternative docking modes is imperative for the elucidation of the organization of functional fusion complexes of paramyxovirinae.In this study, we subjected central predictions of the hypothetical alignment models to experimental analysis. By employing carbohydrate shielding, directed mutagenesis, and variation of the length of the H stalk domain, we examined the proximity of different regions of the H stalk to F, probed a role of individual residues around the previously identified H stalk section from positions 110 to 114 in the formation of functional fusion complexes, tested the effect of varying the length of the H stalk membrane proximal and membrane distal to this section, and explored the general possibility of whether specific contacts between the prefusion F and H head domains are required for F triggering. Experimental data were interpreted in the light of a working model of MeV glycoprotein hetero-oligomers prior to receptor binding.     

Prophylactic Treatment with a G Glycoprotein Monoclonal Antibody Reduces Pulmonary Inflammation in Respiratory Syncytial Virus (RSV)-Challenged Na?ve and Formalin-Inactivated RSV-Immunized BALB/c Mice

We examined whether prophylactically administered anti-respiratory syncytial virus (anti-RSV) G monoclonal antibody (MAb) would decrease the pulmonary inflammation associated with primary RSV infection and formalin-inactivated RSV (FI-RSV)-enhanced disease in mice. MAb 131-2G administration 1 day prior to primary infection reduced the pulmonary inflammatory response and the level of RSV replication. Further, intact or F(ab′)₂ forms of MAb 131-2G administered 1 day prior to infection in FI-RSV-vaccinated mice reduced enhanced inflammation and disease. This study shows that an anti-RSV G protein MAb might provide prophylaxis against both primary infection and FI-RSV-associated enhanced disease. It is possible that antibodies with similar reactivities might prevent enhanced disease and improve the safety of nonlive virus vaccines.Respiratory syncytial virus (RSV) infection in infants and young children causes substantial bronchiolitis and pneumonia (11, 27, 28, 40) resulting in 40,000 to 125,000 hospitalizations in the United States each year (27). RSV is also a prominent cause of respiratory illness in older children; those of any age with compromised cardiac, pulmonary, or immune systems; and the elderly (6, 7, 11, 17, 18, 39). Despite extensive efforts toward vaccine development (3, 5, 8, 20, 30, 38), none is yet available. Currently, only preventive measures are available that focus on infection control to decrease transmission and prophylactic administration of a humanized IgG monoclonal antibody (MAb) directed against the F protein of RSV (palivizumab) that is recommended for high-risk infants and young children (4, 7, 17). To date, no treatment has been highly effective for active RSV infection (17, 21).The first candidate vaccine, a formalin-inactivated RSV (FI-RSV) vaccine developed in the 1960s, not only failed to protect against disease but led to severe RSV-associated lower respiratory tract infection in young vaccine recipients upon subsequent natural infection (8, 16). The experience with FI-RSV has limited nonlive RSV vaccine development for the RSV-naïve infant and young child. Understanding the factors contributing to disease pathogenesis and FI-RSV vaccine-enhanced disease may identify ways to prevent such a response and to help achieve a safe and effective vaccine.The RSV G, or attachment, protein has been implicated in the pathogenesis of disease after primary infection and FI-RSV-enhanced disease (2, 26, 31). The central conserved region of the G protein contains four evolutionarily conserved cysteines in a cysteine noose structure, within which lies a CX3C chemokine motif (9, 29, 34). The G protein CX3C motif is also immunoactive, as suggested by studies with the mouse model that show that G protein CX3C motif interaction with CX3CR1 alters pulmonary inflammation (41), RSV-specific T-cell responses (12), FI-RSV vaccine-enhanced disease, and expression of the neurokinin substance P (14) and also depresses respiratory rates (32). Recent studies demonstrated that therapeutic treatment with a murine anti-RSV G protein monoclonal antibody (MAb 131-2G) which blocks binding to CX3CR1 can reduce pulmonary inflammation associated with primary infection (13, 23). These findings led us to hypothesize that prophylactic administration of this anti-RSV G monoclonal antibody may also diminish pulmonary inflammation associated with RSV infection in naïve and in FI-RSV-vaccinated mice. In this study, we evaluate the impact of prophylactic administration of MAb 131-2G on the pulmonary inflammatory response to primary infection and to RSV challenge following FI-RSV immunization in mice.     

Lipid Phosphate Phosphatase 3 Stabilization of β-Catenin Induces Endothelial Cell Migration and Formation of Branching Point Structures

Endothelial cell (EC) migration, cell-cell adhesion, and the formation of branching point structures are considered hallmarks of angiogenesis; however, the underlying mechanisms of these processes are not well understood. Lipid phosphate phosphatase 3 (LPP3) is a recently described p120-catenin-associated integrin ligand localized in adherens junctions (AJs) of ECs. Here, we tested the hypothesis that LPP3 stimulates β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) to induce EC migration and formation of branching point structures. In subconfluent ECs, LPP3 induced expression of fibronectin via β-catenin/LEF-1 signaling in a phosphatase and tensin homologue (PTEN)-dependent manner. In confluent ECs, depletion of p120-catenin restored LPP3-mediated β-catenin/LEF-1 signaling. Depletion of LPP3 resulted in destabilization of β-catenin, which in turn reduced fibronectin synthesis and deposition, which resulted in inhibition of EC migration. Accordingly, reexpression of β-catenin but not p120-catenin in LPP3-depleted ECs restored de novo synthesis of fibronectin, which mediated EC migration and formation of branching point structures. In confluent ECs, however, a fraction of p120-catenin associated and colocalized with LPP3 at the plasma membrane, via the C-terminal cytoplasmic domain, thereby limiting the ability of LPP3 to stimulate β-catenin/LEF-1 signaling. Thus, our study identified a key role for LPP3 in orchestrating PTEN-mediated β-catenin/LEF-1 signaling in EC migration, cell-cell adhesion, and formation of branching point structures.Angiogenesis, the formation of new blood vessels, involves several well-coordinated cellular processes, including endothelial cell (EC) migration, synthesis and deposition of extracellular matrix proteins, such as fibronectin, cell-cell adhesion, and formation of branching point structures (1-3, 19, 33); however, less is known about the underlying mechanisms of these processes (6, 8, 12, 14, 16, 17). For example, adherens junctions (AJs), which mediate cell-cell adhesion between ECs, may be involved in limiting the extent of cell migration (2, 14, 38, 40). VE-cadherin, a protein found in AJs, is a single-pass transmembrane polypeptide responsible for calcium-dependent homophilic interactions through its extracellular domains (2, 38, 40). The VE-cadherin cytoplasmic domain interacts with the Armadillo domain-containing proteins, β-catenin, γ-catenin (plakoglobin), and p120-catenin (p120ctn) (2, 15, 38, 40, 43). Genetic and biochemical evidence documents a crucial role of β-catenin in regulating cell adhesion as well as proliferation secondary to the central position of β-catenin in the Wnt signaling pathway (13, 16, 25, 31, 44). In addition, the juxtamembrane protein p120ctn regulates AJ stability via binding to VE-cadherin (2, 7, 9, 15, 21, 28, 32, 43). The absence of regulation or inappropriate regulation of β-catenin and VE-cadherin functions is linked to cardiovascular disease and tumor progression (2, 6).We previously identified lipid phosphate phosphatase 3 (LPP3), also known as phosphatidic acid phosphatase 2b (PAP2b), in a functional assay of angiogenesis (18, 19, 41, 42). LPP3 not only exhibits lipid phosphatase activity but also functions as a cell-associated integrin ligand (18, 19, 35, 41, 42). The known LPPs (LPP1, LPP2, and LPP3) (20-23) are six transmembrane domain-containing plasma membrane-bound enzymes that dephosphorylate sphingosine-1-phosphate (S1P) and its structural homologues, and thus, these phosphatases generate lipid mediators (4, 5, 23, 35, 39). All LPPs, which contain a single N-glycosylation site and a putative lipid phosphatase motif, are situated such that their N and C termini are within the cell (4, 5, 22, 23, 35, 39). Only the LPP3 isoform contains an Arg-Gly-Asp (RGD) sequence in the second extracellular loop, and this RGD sequence enables LPP3 to bind integrins (18, 19, 22). Transfection experiments with green fluorescent protein (GFP)-tagged LPP1 and LPP3 showed that LPP1 is apically sorted, whereas LPP3 colocalized with E-cadherin at cell-cell contact sites with other Madin-Darby canine kidney (MDCK) cells (22). Mutagenesis and domain swapping experiments established that LPP1 contains an apical targeting signal sequence (FDKTRL) in its N-terminal segment. In contrast, LPP3 contains a dityrosine (109Y/110Y) basolateral sorting motif (22). Interestingly, conventional deletion of Lpp3 is embryonic lethal, since the Lpp3 gene plays a critical role in extraembryonic vasculogenesis independent of its lipid phosphatase activity (11). In addition, an LPP3-neutralizing antibody was shown to prevent cell-cell interactions (19, 42) and angiogenesis (42). Here, we addressed the hypothesis that LPP3 plays a key role in EC migration, cell-cell adhesion, and formation of branching point structures by stimulating β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) signaling.     

Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

HIV-1 possesses an exquisite ability to infect cells independently from their cycling status by undergoing an active phase of nuclear import through the nuclear pore. This property has been ascribed to the presence of karyophilic elements present in viral nucleoprotein complexes, such as the matrix protein (MA); Vpr; the integrase (IN); and a cis-acting structure present in the newly synthesized DNA, the DNA flap. However, their role in nuclear import remains controversial at best. In the present study, we carried out a comprehensive analysis of the role of these elements in nuclear import in a comparison between several primary cell types, including stimulated lymphocytes, macrophages, and dendritic cells. We show that despite the fact that none of these elements is absolutely required for nuclear import, disruption of the central polypurine tract-central termination sequence (cPPT-CTS) clearly affects the kinetics of viral DNA entry into the nucleus. This effect is independent of the cell cycle status of the target cells and is observed in cycling as well as in nondividing primary cells, suggesting that nuclear import of viral DNA may occur similarly under both conditions. Nonetheless, this study indicates that other components are utilized along with the cPPT-CTS for an efficient entry of viral DNA into the nucleus.Lentiviruses display an exquisite ability to infect dividing and nondividing cells alike that is unequalled among Retroviridae. This property is thought to be due to the particular behavior or composition of the viral nucleoprotein complexes (NPCs) that are liberated into the cytoplasm of target cells upon virus-to-cell membrane fusion and that allow lentiviruses to traverse an intact nuclear membrane (17, 28, 29, 39, 52, 55, 67, 79). In the case of the human immunodeficiency type I virus (HIV-1), several studies over the years identified viral components of such structures with intrinsic karyophilic properties and thus perfect candidates for mediation of the passage of viral DNA (vDNA) through the nuclear pore: the matrix protein (MA); Vpr; the integrase (IN); and a three-stranded DNA flap, a structure present in neo-synthesized viral DNA, specified by the central polypurine tract-central termination sequence (cPPT-CTS). It is clear that these elements may mediate nuclear import directly or via the recruitment of the host''s proteins, and indeed, several cellular proteins have been found to influence HIV-1 infection during nuclear import, like the karyopherin α2 Rch1 (38); importin 7 (3, 30, 93); the transportin SR-2 (13, 20); or the nucleoporins Nup98 (27), Nup358/RANBP2, and Nup153 (13, 56).More recently, the capsid protein (CA), the main structural component of viral nucleoprotein complexes at least upon their cytoplasmic entry, has also been suggested to be involved in nuclear import or in postnuclear entry steps (14, 25, 74, 90, 92). Whether this is due to a role for CA in the shaping of viral nucleoprotein complexes or to a direct interaction between CA and proteins involved in nuclear import remains at present unknown.Despite a large number of reports, no single viral or cellular element has been described as absolutely necessary or sufficient to mediate lentiviral nuclear import, and important controversies as to the experimental evidences linking these elements to this step exist. For example, MA was among the first viral protein of HIV-1 described to be involved in nuclear import, and 2 transferable nuclear localization signals (NLSs) have been described to occur at its N and C termini (40). However, despite the fact that early studies indicated that the mutation of these NLSs perturbed HIV-1 nuclear import and infection specifically in nondividing cells, such as macrophages (86), these findings failed to be confirmed in more-recent studies (23, 33, 34, 57, 65, 75).Similarly, Vpr has been implicated by several studies of the nuclear import of HIV-1 DNA (1, 10, 21, 43, 45, 47, 64, 69, 72, 73, 85). Vpr does not possess classical NLSs, yet it displays a transferable nucleophilic activity when fused to heterologous proteins (49-51, 53, 77, 81) and has been shown to line onto the nuclear envelope (32, 36, 47, 51, 58), where it can truly facilitate the passage of the viral genome into the nucleus. However, the role of Vpr in this step remains controversial, as in some instances Vpr is not even required for viral replication in nondividing cells (1, 59).Conflicting results concerning the role of IN during HIV-1 nuclear import also exist. Indeed, several transferable NLSs have been described to occur in the catalytic core and the C-terminal DNA binding domains of IN, but for some of these, initial reports of nuclear entry defects (2, 9, 22, 46, 71) were later shown to result from defects at steps other than nuclear import (60, 62, 70, 83). These reports do not exclude a role for the remaining NLSs in IN during nuclear import, and they do not exclude the possibility that IN may mediate this step by associating with components of the cellular nuclear import machinery, such as importin alpha and beta (41), importin 7 (3, 30, 93, 98), and, more recently, transportin-SR2 (20).The central DNA flap, a structure present in lentiviruses and in at least 1 yeast retroelement (44), but not in other orthoretroviruses, has also been involved in the nuclear import of viral DNA (4, 6, 7, 31, 78, 84, 95, 96), and more recently, it has been proposed to provide a signal for viral nucleoprotein complexes uncoating in the proximity of the nuclear pore, with the consequence of providing a signal for import (8). However, various studies showed an absence or weakness of nuclear entry defects in viruses devoid of the DNA flap (24, 26, 44, 61).Overall, the importance of viral factors in HIV-1 nuclear import is still unclear. The discrepancies concerning the role of MA, IN, Vpr, and cPPT-CTS in HIV-1 nuclear import could in part be explained by their possible redundancy. To date, only one comprehensive study analyzed the role of these four viral potentially karyophilic elements together (91). This study showed that an HIV-1 chimera where these elements were either deleted or replaced by their murine leukemia virus (MLV) counterparts was, in spite of an important infectivity defect, still able to infect cycling and cell cycle-arrested cell lines to similar efficiencies. If this result indicated that the examined viral elements of HIV-1 were dispensable for the cell cycle independence of HIV, as infections proceeded equally in cycling and arrested cells, they did not prove that they were not required in nuclear import, because chimeras displayed a severe infectivity defect that precluded their comparison with the wild type (WT).Nuclear import and cell cycle independence may not be as simply linked as previously thought. On the one hand, there has been no formal demonstration that the passage through the nuclear pore, and thus nuclear import, is restricted to nondividing cells, and for what we know, this passage may be an obligatory step in HIV infection in all cells, irrespective of their cycling status. In support of this possibility, certain mutations in viral elements of HIV affect nuclear import in dividing as well as in nondividing cells (4, 6, 7, 31, 84, 95). On the other hand, cell cycle-independent infection may be a complex phenomenon that is made possible not only by the ability of viral DNA to traverse the nuclear membrane but also by its ability to cope with pre- and postnuclear entry events, as suggested by the phenotypes of certain CA mutants (74, 92).Given that the cellular environment plays an important role during the early steps of viral infection, we chose to analyze the role of the four karyophilic viral elements of HIV-1 during infection either alone or combined in a wide comparison between cells highly susceptible to infection and more-restrictive primary cell targets of HIV-1 in vivo, such as primary blood lymphocytes (PBLs), monocyte-derived macrophages (MDM), and dendritic cells (DCs).In this study, we show that an HIV-1-derived virus in which the 2 NLSs of MA are mutated and the IN, Vpr, and cPPT-CTS elements are removed displays no detectable nuclear import defect in HeLa cells independently of their cycling status. However, this mutant virus is partially impaired for nuclear entry in primary cells and more specifically in DCs and PBLs. We found that this partial defect is specified by the cPPT-CTS, while the 3 remaining elements seem to play no role in nuclear import. Thus, our study indicates that the central DNA flap specifies the most important role among the viral elements involved thus far in nuclear import. However, it also clearly indicates that the role played by the central DNA flap is not absolute and that its importance varies depending on the cell type, independently from the dividing status of the cell.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding

The human immunodeficiency virus type 1 structural polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.The human immunodeficiency virus type 1 (HIV-1) structural precursor polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles (VLPs). Gag is composed of four major structural domains, matrix (MA), capsid (CA), nucleocapsid (NC), and p6, as well as two spacer peptides, SP1 and SP2 (3, 30, 94). Following particle assembly and release, cleavage by HIV-1 protease separates these domains. However, these domains must work together in the context of the full-length Gag polyprotein to drive particle assembly.Previous studies have mapped two major functional domains involved in the early steps of assembly: first, Gag associates with cellular membranes via basic residues and N-terminal myristoylation of the MA domain (10, 17, 20, 35, 39, 87, 91, 106); second, the Gag-Gag interaction domains that span the CA C-terminal domain (CA-CTD) and NC domain promote Gag multimerization (3, 11, 14, 16, 18, 23, 27, 29, 30, 33, 36, 46, 64, 88, 94, 102, 103). Structural and genetic studies have identified two residues (W184 and M185) within a dimerization interface in the CA-CTD that are critical to CA-CA interactions (33, 51, 74, 96). Analytical ultracentrifugation of heterodimers formed between wild-type (WT) Gag and Gag mutants with changes at these residues suggests that the dimerization interface consists of two reciprocal interactions, one of which can be disrupted to form a “half-interface” (22).In addition to the CA-CTD, NC contributes to assembly via 15 basic residues (8, 9, 11, 14, 18, 23, 25, 28, 34, 40, 43, 54, 57, 58, 74, 79, 88, 97, 104, 105), although some researchers have suggested that NC instead contributes to the stability of mature virions after assembly (75, 98, 99). It is thought that the contribution of NC to assembly is due to its ability to bind RNA, since the addition of RNA promotes the formation of particles in vitro (14-16, 37, 46), and RNase treatment disrupts Gag-Gag interactions (11) and immature viral cores (67). However, RNA is not necessary per se, since dimerization motifs can substitute for NC (1, 4, 19, 49, 105). This suggests a model in which RNA serves a structural role, such as a scaffold, to promote Gag-Gag interactions through NC. Based on in vitro studies, it has been suggested that this RNA scaffolding interaction facilitates the low-order Gag multimerization mediated by CA-CTD dimerization (4, 37, 49, 62, 63, 85). Despite a wealth of biochemical data, the relative contributions of the CA-CTD and NC to Gag multimerization leading to assembly are yet to be determined in cells.Mutations in Gag interaction domains alter membrane binding in addition to affecting Gag multimerization. In particular, mutations or truncations of CA reduce membrane binding (21, 74, 82), and others previously reported that mutations or truncations of NC affect membrane binding (13, 78, 89, 107). These findings are consistent with a myristoyl switch model of membrane binding in which Gag can switch between high- and low-membrane-affinity states (38, 71, 76, 83, 86, 87, 92, 95, 107). Many have proposed, and some have provided direct evidence (95), that Gag multimerization mediated by CA or NC interactions promotes the exposure of the myristoyl moiety to facilitate membrane associations.Gag membrane binding and multimerization appear to be interrelated steps of virus assembly, since membrane binding also facilitates Gag multimerization. Unlike betaretroviruses that fully assemble prior to membrane targeting and envelopment (type B/D), lentiviruses, such as HIV, assemble only on cellular membranes at normal Gag expression levels (type C), although non-membrane-bound Gag complexes exist (45, 58, 60, 61, 65). Consistent with this finding, mutations that reduce Gag membrane associations cause a defect in Gag multimerization (59, 74). Therefore, in addition to their primary effects on Gag-Gag interactions, mutations in Gag interaction domains cause a defect in membrane binding, which, in turn, causes a secondary multimerization defect. To determine the relative contributions of the CA-CTD and the NC domain to Gag-Gag interactions at the plasma membrane, it is essential to eliminate secondary effects due to a modulation of membrane binding.Except for studies using a His-tag-mediated membrane binding system (5, 46), biochemical studies of C-type Gag multimerization typically lack membranes. Therefore, these studies do not fully represent particle assembly, which occurs on biological membranes in cells. Furthermore, many biochemical and structural approaches are limited to isolated domains or truncated Gag constructs. Thus, some of these studies are perhaps more relevant to the behavior of protease-cleaved Gag in mature virions. With few exceptions (47, 74), cell-based studies of Gag multimerization have typically been limited to measuring how well mutant Gag is incorporated into VLPs when coexpressed or not with WT Gag. Since VLP production is a complex multistep process, effects of mutations on other steps in the process can confound this indirect measure. For example, NC contributes to VLP production by both promoting multimerization and interacting with the host factor ALIX to promote VLP release (26, 80). To directly assay Gag multimerization in cells, several groups (24, 45, 52, 56) developed microscopy assays based on fluorescence resonance energy transfer (FRET). These assays measure the transfer of energy between donor and acceptor fluorescent molecules that are brought within ∼5 nm by the association of the proteins to which they are attached (41, 48, 90). However, these microscopy-based Gag FRET assays have not been used to fully elucidate several fundamental aspects of HIV-1 Gag multimerization at the plasma membrane of cells, such as the relative contributions of the CA-CTD and NC and the effect of membrane binding on Gag-Gag interactions. In this study, we used a FRET stoichiometry method based on calibrated spectral analysis of fluorescence microscopy images (41). This algorithm determines the fractions of both donor and acceptor fluorescent protein-tagged Gag molecules participating in FRET. For cells expressing Gag molecules tagged with donor (cyan fluorescent protein [CFP]) and acceptor (yellow fluorescent protein [YFP]) molecules, this method measures the apparent FRET efficiency, which is proportional to the mole fraction of Gag constructs in complex. By measuring apparent FRET efficiencies, quantitative estimates of the mole fractions of interacting proteins can be obtained.Using this FRET-based assay, we aim to answer two questions: (i) what are the relative contributions of CA-CTD and NC domains to Gag multimerization when secondary effects via membrane binding are held constant, and (ii) what is the effect of modulating membrane binding on the ability of Gag mutants to interact with WT Gag?Our data demonstrate that the CA-CTD dimerization interface is essential for Gag multimerization at the plasma membrane, as fully disrupting the CA-CTD interaction abolishes FRET, whereas a modest level of FRET is still detected in the absence of NC. We also present evidence that the CA-CTD dimerization interface consists of two reciprocal interactions, allowing the formation of a half-interface that can still contribute to Gag multimerization. Notably, when Gag derivatives with an intact CA-CTD were coexpressed with WT Gag, either membrane binding ability or NC was required for the Gag mutants to interact with WT Gag, suggesting functional compensation between these factors.