Identification of a Novel WxSLVK Motif in the N Terminus of Human Immunodeficiency Virus and Simian Immunodeficiency Virus Vif That Is Critical for APOBEC3G and APOBEC3F Neutralization

The function of lentiviral Vif proteins is to neutralize the host antiviral cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F). Vif bridges a cullin 5-based E3 ubiquitin ligase with A3G and A3F and mediates their degradation by proteasomes. Recent studies have found that Vif uses different domains to bind to A3G and A3F. A ¹⁴DRMR¹⁷ domain binds to A3F, ⁴⁰YRHHY⁴⁴ binds to A3G, and ⁶⁹YxxL⁷² binds to both A3G and A3F. Here, we report another functional domain of Vif. Previously, we demonstrated that human immunodeficiency virus type 1 (HIV-1) Vif failed to mediate A3G proteasomal degradation when all 16 lysines were mutated to arginines. Here, we show that K26, and to a lesser extent K22, is critical for A3G neutralization. K22 and K26 are part of a conserved ²¹WxSLVK²⁶ (x represents N, K, or H) motif that is found in most primate lentiviruses and that shows species-specific variation. Both K22 and K26 in this motif regulated Vif specificity only for A3G, whereas the SLV residues regulated Vif specificity for both A3F and A3G. Interestingly, SLV and K26 in HIV-1 Vif did not directly mediate Vif interaction with either A3G or A3F. Previously, other groups have reported an important role for W21 in A3F and A3G neutralization. Thus, ²¹WxSLVK²⁶ is a novel functional domain that regulates Vif activity toward both A3F and A3G and is a potential drug target to inhibit Vif activity and block HIV-1 replication.The replication of human immunodeficiency virus type 1 (HIV-1) is seriously impaired in human primary lymphocytes when the viral protein Vif is not present (8, 38). The first cellular target of Vif was identified as APOBEC3G (A3G) (34), which belongs to the cytidine deaminase family known as APOBEC (apolipoprotein B mRNA-editing catalytic polypeptide) (14). This family consists of APOBEC1; activation-induced deaminase (AID); APOBEC2; a subgroup of APOBEC3 (A3) proteins, including A3A, A3B, A3C, A3DE, A3F, A3G, and A3H; and APOBEC4 in humans (12). They have one or two copies of a cytidine deaminase domain with a signature motif (HxEx_23-28PCx_2-4C), and normally only one of the cytidine deaminase domains has deaminase activity.All seven A3 genes have been shown to inhibit the replication of various types of retroviruses via cytidine deamination-dependent or -independent mechanisms (3). In particular, A3B, A3DE, A3F, and A3G inhibit HIV-1 replication, whereas A3A and A3C do not (1, 6, 7, 19, 34, 42, 50). Recently, it was shown that optimizing A3H expression in cell culture also inhibits HIV-1 replication (4, 10, 25, 39). Among these proteins, A3G and A3F have the most potent anti-HIV-1 activities. A3G and A3F share ∼50% sequence similarity but have different biochemical properties (41) and different target sequence preferences while catalyzing cytidine deamination of viral cDNAs (19).Nevertheless, HIV-1 is able to elude this defense mechanism and cause human disease for two reasons. First, A3B and A3H are expressed only at low levels in vivo (4, 7, 18, 26). Second, HIV-1 produces Vif, which is expressed in all lentiviruses except equine infectious anemia virus. Vif can destabilize A3DE, A3F, and A3G proteins by targeting them to the proteasomal degradation pathway (6, 22, 35, 37, 50). In addition, Vif may also inhibit A3 activity independently of proteasomal degradation (15, 16, 31).The action of Vif is highly species specific. Vif from HIV-1 inactivates only A3G from humans, and Vif from simian immunodeficiency virus (SIV) isolated from African green monkeys (AGM) does not inactivate A3G from humans. Nevertheless, Vif from SIV isolated from rhesus macaques (MAC) inactivates A3G from all humans, AGM, and MAC (21). A single residue in A3G at position 128, an aspartic acid in humans versus a lysine in AGM, determines A3G sensitivity to HIV-1 Vif (2, 32, 44). In addition, an N-terminal domain in HIV-1 Vif, ¹⁴DRMR¹⁷, determines Vif specificity for different A3G proteins (33).Vif targets A3G to the proteasome by acting as an adaptor protein that bridges A3G with a cullin 5 (Cul5)-based E3 ubiquitin ligase complex, which includes Cul5, elongin B (EloB), and EloC (46). Vif has a BC box motif (¹⁴⁴S¹⁴⁵L¹⁴⁶Q) that binds to EloC (23, 47) and an HCCH motif (¹¹⁴C/¹³³C) that binds to Cul5 (20, 24, 43). It has also been shown that Vif specifically binds to a region from amino acids 126 to 132 of A3G and to amino acids 283 to 300 of A3F (13, 30). It is believed that as a consequence of these interactions, A3G is polyubiquitylated and directed to 26S proteasomes for degradation.Several domains that determine Vif interactions with A3F and A3G have been identified. Analysis of HIV-1 patient-derived Vif sequences initially found that W11 is essential for A3F recognition and K22, Y40, and E45 are required for A3G recognition (36). The previously identified agmA3G-specific ¹⁴DRMR¹⁷ domain was also found to determine Vif specificity for A3F (33) by direct binding (29). An A3G-specific binding domain, ⁴⁰YRHHY⁴⁴, has also been identified (29), and a ⁶⁹YxxL⁷² domain interacts with both A3G and A3F (11, 28, 45).We have previously shown that Vif can mediate A3G proteasomal degradation in the absence of A3G polyubiquitylation and that, unexpectedly, this process is dependent on lysines in Vif (5). Here, we identify two N-terminal lysines that are important for Vif function. We show that these lysines are part of a ²¹WxSLVK²⁶ motif that is conserved in Vif from primate lentiviruses and that this motif regulates Vif activities against both A3G and A3F via different mechanisms.     

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.     

Identification of a Critical T(Q/D/E)x5ADx2(I/L) Motif from Primate Lentivirus Vif Proteins That Regulate APOBEC3G and APOBEC3F Neutralizing Activity

Primate lentiviruses are unique in that they produce several accessory proteins to help in the establishment of productive viral infection. The major function of these proteins is to clear host resistance factors that inhibit viral replication. Vif is one of these proteins. It functions as an adaptor that binds to the cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) and bridges them to a cullin 5 (Cul5) and elongin (Elo) B/C E3 ubiquitin ligase complex for proteasomal degradation. So far, 11 discontinuous domains in Vif have been identified that regulate this degradation process. Here we report another domain, T(Q/D/E)x₅ADx₂(I/L), which is located at residues 96 to 107 in the human immunodeficiency virus type 1 (HIV-1) Vif protein. This domain is conserved not only in all HIV-1 subtypes but also in other primate lentiviruses, including HIV-2 and simian immunodeficiency virus (SIV), which infects rhesus macaques (SIVmac) and African green monkeys (SIVagm). Mutations of the critical residues in this motif seriously disrupted Vif''s neutralizing activity toward both A3G and A3F. This motif regulates Vif interaction not only with A3G and A3F but also with Cul5. When this motif was inactivated in the HIV-1 genome, Vif failed to exclude A3G and A3F from virions, resulting in abortive HIV replication in nonpermissive human T cells. Thus, T(Q/D/E)x₅ADx₂(I/L) is a critical functional motif that directly supports the adaptor function of Vif and is an attractive target for inhibition of Vif function.Vif is a small viral protein that has 192 amino acids and is expressed by most lentiviruses, except for equine infectious anemia virus. It was first discovered in human immunodeficiency virus type 1 (HIV-1) (13, 14, 31), and its function in HIV-1 infection has been studied extensively (9, 34). Infection of human T-cell lines with vif-defective (ΔVif) HIV-1 identified two different cell types, namely, permissive cells that can be infected by ΔVif HIV-1 and nonpermissive cells, which are resistant to ΔVif HIV-1 (10, 36). Genomic complementation analysis indicated that these nonpermissive cells express a Vif-sensitive dominant viral inhibitor(s) (17, 27). The first inhibitor identified was APOBEC3G (A3G) (25), which belongs to the APOBEC (apolipoprotein B mRNA-editing catalytic polypeptide) family. In humans, this family consists of APOBEC1; activation-induced deaminase (AID); APOBEC2; a subgroup of APOBEC3 (A3) proteins, including A3A, A3B, A3C, A3DE, A3F, A3G, and A3H; and APOBEC4. All seven A3 genes have been shown to inhibit replication of various types of retrovirus by cytidine deamination-dependent and -independent mechanisms, as reviewed recently (21, 30, 35). In particular, human A3B, A3DE, A3F, A3G, and A3H inhibit HIV-1 replication, whereas A3A and A3C do not (2, 5, 6, 25, 39, 46). Among these, the protein expression of A3G and A3F in human primary tissues has been demonstrated, and in vitro studies indicate that these proteins have the most potent anti-HIV-1 activities. A3G and A3F share ∼50% sequence similarity but have different biochemical properties (38) and different target sequence preferences while catalyzing cytidine deamination of viral cDNAs (15).Vif hijacks the cellular proteasomal machinery to destroy A3G and A3F by the protein degradation pathway (18, 26, 33, 46). Vif acts as an adaptor protein that bridges A3 proteins to a cullin 5 (Cul5)-based E3 ubiquitin ligase complex, which includes Cul5, EloB, and EloC (44). These interactions trigger the polyubiquitylation of Vif, A3G, and A3F and direct them to 26S proteasomes for degradation. Thus, Vif binding to A3G or A3F as well as to Cul5/EloBC is a critical step for A3G and A3F degradation. Although A3G and A3F share a high level of homology, different surfaces are used for Vif interaction. Vif binds to the N-terminal region of A3G, from residues 126 to 132, and to the C-terminal region of A3F, from residues 283 to 300 (12, 24). In addition, 11 discontinuous motifs in the Vif protein have been identified as regulating Vif interactions with A3G, A3F, or the Cul5/EloBC E3 ligase complex. Three motifs determine Vif interaction with the E3 ligase. The ¹⁰⁸Hx₅Cx_17-18Cx_3-5H¹³⁹ motif, also called the HCCH zinc finger, binds to Cul5 (16, 20, 41); the ¹⁴⁴SLQYLA¹⁴⁹ motif, which is also called the BC box, binds to EloC (19, 45); and the ¹⁶¹PPLPx₄L¹⁶⁹ motif, which is also called the Cul box, binds to Cul5 (32, 45). The ¹⁶¹PPLP¹⁶⁴ subdomain has multiple activities, which not only determine Vif dimerization (43) but also regulate Vif binding to A3G (8, 37) and EloB (1). The other 8 motifs regulate the interaction between Vif and A3G/A3F. The ²¹WxSLVK²⁶ (3, 7) and ⁴⁰YRHHY⁴⁴ (23) motifs regulate Vif binding to A3G; the ¹¹Wx₂DRMR¹⁷ (23), ⁷⁴TGERxW⁷⁹ (11), and ¹⁷¹EDRW¹⁷⁴ (4) motifs regulate Vif binding to A3F; and the ⁵⁵VxIPLx₄L⁶⁴ (11), ⁶⁹YxxL⁷² (22), and ⁸¹LGxGx₂IxW⁸⁹ (4) motifs regulate Vif binding to both A3G and A3F. The ⁸¹LGxGx₂IxW⁸⁹ motif also regulates Vif binding to Cul5 (4). Thus, HIV has developed rather complicated mechanisms to assemble a protein degradation complex to neutralize these two critical host factors. A full understanding of these mechanisms is essential for pharmaceutical inhibition of Vif function to prevent HIV-1 infection. Here we report another functional motif from a previously uncharacterized region of HIV-1 Vif that regulates Vif interactions with A3G, A3F, and the Cul5/EloBC E3 ligase complex. Since this Vif region is the only one left uncharacterized, this is a significant step toward a complete understanding of this important host-pathogen interaction.     

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.     

Crystallographic Definition of the Epitope Promiscuity of the Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2F5: Vaccine Design Implications

The quest to create a human immunodeficiency virus type 1 (HIV-1) vaccine capable of eliciting broadly neutralizing antibodies against Env has been challenging. Among other problems, one difficulty in creating a potent immunogen resides in the substantial overall sequence variability of the HIV envelope protein. The membrane-proximal region (MPER) of gp41 is a particularly conserved tryptophan-rich region spanning residues 659 to 683, which is recognized by three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, Z13, and 4E10. In this study, we first describe the variability of residues in the gp41 MPER and report on the invariant nature of 15 out of 25 amino acids comprising this region. Subsequently, we evaluate the ability of the bnMAb 2F5 to recognize 31 varying sequences of the gp41 MPER at a molecular level. In 19 cases, resulting crystal structures show the various MPER peptides bound to the 2F5 Fab′. A variety of amino acid substitutions outside the ⁶⁶⁴DKW⁶⁶⁶ core epitope are tolerated. However, changes at the ⁶⁶⁴DKW⁶⁶⁶ motif itself are restricted to those residues that preserve the aspartate''s negative charge, the hydrophobic alkyl-π stacking arrangement between the β-turn lysine and tryptophan, and the positive charge of the former. We also characterize a possible molecular mechanism of 2F5 escape by sequence variability at position 667, which is often observed in HIV-1 clade C isolates. Based on our results, we propose a somewhat more flexible molecular model of epitope recognition by bnMAb 2F5, which could guide future attempts at designing small-molecule MPER-like vaccines capable of eliciting 2F5-like antibodies.Eliciting broadly neutralizing antibodies (bnAbs) against primary isolates of human immunodeficiency virus type I (HIV-1) has been identified as a major milestone to attain in the quest for a vaccine in the fight against AIDS (12, 28). These antibodies would need to interact with HIV-1 envelope glycoproteins gp41 and/or gp120 (Env), target conserved regions and functional conformations of gp41/gp120 trimeric complexes, and prevent new HIV-1 fusion events with target cells (21, 57, 70, 71). Although a humoral response generating neutralizing antibodies against HIV-1 can be detected in HIV-1-positive individuals, the titers are often very low, and virus control is seldom achieved by these neutralizing antibodies (22, 51, 52, 66, 67). The difficulty in eliciting a broad and potent neutralizing antibody response against HIV-1 is thought to reside in the high degree of genetic diversity of the virus, in the heterogeneity of Env on the surface of HIV-1, and in the masking of functional regions by conformational covering, by an extensive glycan shield, or by the ability of some conserved domains to partition to the viral membrane (24, 25, 29, 30, 38, 39, 56, 68, 69). So far, vaccine trials using as immunogens mimics of Env in different conformations have primarily elicited antibodies with only limited neutralization potency across different HIV-1 clades although recent work has demonstrated more encouraging results (4, 12, 61).The use of conserved regions on gp41 and gp120 Env as targets for vaccine design has been mostly characterized by the very few anti-HIV-1 broadly neutralizing monoclonal antibodies (bnMAbs) that recognize them: the CD4 binding-site on gp120 (bnMAb b12), a CD4-induced gp120 coreceptor binding site (bnMAbs 17b and X5), a mannose cluster on the outer face of gp120 (bnMAb 2G12), and the membrane proximal external region (MPER) of gp41 (bnMAbs 2F5, Z13 and 4E10) (13, 29, 44, 58, 73). The gp41 MPER region is a particularly conserved part of Env that spans residues 659 to 683 (HXB2 numbering) (37, 75). Substitution and deletion studies have linked this unusually tryptophan-rich region to the fusion process of HIV-1, possibly involving a series of conformational changes (5, 37, 41, 49, 54, 74). Additionally, the gp41 MPER has been implicated in gp41 oligomerization, membrane leakage ability facilitating pore formation, and binding to the galactosyl ceramide receptor on epithelial cells for initial mucosal infection mediated by transcytosis (2, 3, 40, 53, 63, 64, 72). This wide array of roles for the gp41 MPER will put considerable pressure on sequence conservation, and any change will certainly lead to a high cost in viral fitness.Monoclonal antibody 2F5 is a broadly neutralizing monoclonal anti-HIV-1 antibody isolated from a panel of sera from naturally infected asymptomatic individuals. It reacts with a core gp41 MPER epitope spanning residues 662 to 668 with the linear sequence ELDKWAS (6, 11, 42, 62, 75). 2F5 immunoglobulin G binding studies and screening of phage display libraries demonstrated that the DKW core is essential for 2F5 recognition and binding (15, 36, 50). Crystal structures of 2F5 with peptides representing its core gp41 epitope reveal a β-turn conformation involving the central DKW residues, flanked by an extended conformation and a canonical α-helical turn for residues located at the N terminus and C terminus of the core, respectively (9, 27, 45, 47). In addition to binding to its primary epitope, evidence is accumulating that 2F5 also undergoes secondary interactions: multiple reports have demonstrated affinity of 2F5 for membrane components, possibly through its partly hydrophobic flexible elongated complementarity-determining region (CDR) H3 loop, and it has also been suggested that 2F5 might interact in a secondary manner with other regions of gp41 (1, 10, 23, 32, 33, 55). Altogether, even though the characteristics of 2F5 interaction with its linear MPER consensus epitope have been described extensively, a number of questions persist about the exact mechanism of 2F5 neutralization at a molecular level.One such ambiguous area of the neutralization mechanism of 2F5 is investigated in this study. Indeed, compared to bnMAb 4E10, 2F5 is the more potent neutralizing antibody although its breadth across different HIV-1 isolates is more limited (6, 35). In an attempt to shed light on the exact molecular requirements for 2F5 recognition of its primary gp41 MPER epitope, we performed structural studies of 2F5 Fab′ with a variety of peptides. The remarkable breadth of possible 2F5 interactions reveals a somewhat surprising promiscuity of the 2F5 binding site. Furthermore, we link our structural observations with the natural variation observed within the gp41 MPER and discuss possible routes of 2F5 escape from a molecular standpoint. Finally, our discovery of 2F5''s ability to tolerate a rather broad spectrum of amino acids in its binding, a spectrum that even includes nonnatural amino acids, opens the door to new ways to design small-molecule immunogens potentially capable of eliciting 2F5-like neutralizing antibodies.     

HIV-1 gp120 Determinants Proximal to the CD4 Binding Site Shift Protective Glycans That Are Targeted by Monoclonal Antibody 2G12

HIV-1 R5 envelopes vary considerably in their capacities to exploit low CD4 levels on macrophages for infection and in their sensitivities to the CD4 binding site (CD4bs) monoclonal antibody (MAb) b12 and the glycan-specific MAb 2G12. Here, we show that nonglycan determinants flanking the CD4 binding loop, which affect exposure of the CD4bs, also modulate 2G12 neutralization. Our data indicate that such residues act via a mechanism that involves shifts in the orientation of proximal glycans, thus modulating the sensitivity of 2G12 neutralization and affecting the overall presentation and structure of the glycan shield.The trimeric envelope (Env) spikes on HIV-1 virions are comprised of gp120 and gp41 heterodimers. gp120 is coated extensively with glycans (9, 11, 15) that are believed to protect the envelope from neutralizing antibodies. The extents and locations of glycosylation are variable and evolving (15). Thus, while some glycans are conserved, others appear or disappear in a host over the course of infection. Such changes may result in exposure or protection of functional envelope sites and can result from selection by different environmental pressures in vivo, including neutralizing antibodies.We previously reported that HIV-1 R5 envelopes varied considerably in tropism and neutralization sensitivity (3, 4, 12-14). We showed that highly macrophage-tropic R5 envelopes were more frequently detected in brain than in semen, blood, and lymph node (LN) samples (12, 14). The capacity of R5 envelopes to infect macrophages correlated with their ability to exploit low levels of cell surface CD4 for infection (12, 14). Determinants within and proximal to the CD4 binding site (CD4bs) were shown to modulate macrophage infectivity (3, 4, 5, 12, 13) and presumably acted by altering the avidity of the trimer for cell surface CD4. These determinants include residues proximal to the CD4 binding loop, which is likely the first part of the CD4bs contacted by CD4 (1). We also observed that macrophage-tropic R5 envelopes were frequently more resistant to the glycan-specific monoclonal antibody (MAb) 2G12 than were non-macrophage-tropic R5 Envs (13).Here, we investigated the envelope determinants of 2G12 sensitivity by using two HIV-1 envelopes that we used previously to map macrophage tropism determinants (4), B33 from brain and LN40 from lymph node tissue of an AIDS patient with neurological complications. While B33 imparts high levels of macrophage infectivity and is resistant to 2G12, LN40 Env confers very inefficient macrophage infection and is 2G12 sensitive (12-14).     

Identification of 81LGxGxxIxW89 and 171EDRW174 Domains from Human Immunodeficiency Virus Type 1 Vif That Regulate APOBEC3G and APOBEC3F Neutralizing Activity

The human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) potently restrict human immunodeficiency virus type 1 (HIV-1) replication, but they are neutralized by the viral protein Vif. Vif bridges A3G and A3F with a Cullin 5 (Cul5)-based E3 ubiquitin ligase and mediates their proteasomal degradation. This mechanism has been extensively studied, and several Vif domains have been identified that are critical for A3G and A3F neutralization. Here, we identified two additional domains. Via sequence analysis of more than 2,000 different HIV-1 Vif proteins, we identified two highly conserved amino acid sequences, ⁸¹LGxGxSIEW⁸⁹ and ¹⁷¹EDRWN¹⁷⁵. Within the ⁸¹LGxGxSIEW⁸⁹ sequence, residues L81, G82, G84, and, to a lesser extent, I87 and W89 play very critical roles in A3G/A3F neutralization. In particular, residues L81 and G82 determine Vif binding to A3F, residue G84 determines Vif binding to both A3G and A3F, and residues ⁸⁶SIEW⁸⁹ affect Vif binding to A3F, A3G, and Cul5. Accordingly, this ⁸¹LGxGxSIEW⁸⁹ sequence was designated the ⁸¹LGxGxxIxW⁸⁹ domain. Within the ¹⁷¹EDRWN¹⁷⁵ sequence, all residues except N175 are almost equally important for regulation of A3F neutralization, and consistently, they determine Vif binding only to A3F. Accordingly, this domain was designated ¹⁷¹EDRW¹⁷⁴. The LGxGxxIxW domain is also partially conserved in simian immunodeficiency virus Vif from rhesus macaques (SIVmac239) and has a similar activity. Thus, ⁸¹LGxGxxIxW⁸⁹ and ¹⁷¹EDRW¹⁷⁴ are two novel functional domains that are very critical for Vif function. They could become new targets for inhibition of Vif activity during HIV replication.The function of the lentiviral protein Vif is to neutralize the major host antiretroviral cytidine deaminases that belong to the APOBEC (apolipoprotein B mRNA-editing catalytic polypeptide) family, as recently reviewed by several investigators (18, 29, 31). This family consists of APOBEC1; activation-induced deaminase (AID); APOBEC2; a subgroup of APOBEC3 (A3) proteins, including A3A, A3B, A3C, A3DE, A3F, A3G, and A3H; and APOBEC4 in humans. They have one or two copies of a cytidine deaminase (CDA) domain with a signature motif (HxEx_23-28PCx_2-4C), only one of which normally has deaminase activity.All seven A3 genes have been shown to inhibit replication of various types of retrovirus via cytidine deamination-dependent or -independent mechanisms. In particular, human A3B, A3DE, A3F, A3G, and A3H inhibit human immunodeficiency virus type 1 (HIV-1) replication, whereas A3A and A3C do not (1, 3, 5, 26, 33, 37). Among these proteins, the expression of human A3G and A3F in vivo has been demonstrated, and in vitro studies indicate that they have the most potent anti-HIV-1 activity. A3G and A3F share ∼50% sequence similarity but have different biochemical properties (32) and different target sequence preferences while catalyzing cytidine deamination of viral cDNAs (13). Expression of human A3B has not been detected (7), and a 29.5-kb deletion spanning from the 3′ end of the A3A gene to the 8th exon of the A3B gene, leading to the complete removal of the A3B gene, has been detected in certain human populations (12). Human A3H is also poorly expressed in vivo (20). It was reported that human A3H has four haplotypes (Hap I, II, III, and IV), and only Hap II, which is maintained primarily in African populations, could be stably expressed in vitro (19). However, expression of this protein has not been detected in any human populations. Thus, the primary function of HIV-1 Vif is to neutralize A3G, A3F, and, to a lesser extent, A3DE.Vif hijacks cellular proteasomal machinery to destroy these host cytidine deaminases by protein degradation (15, 27, 30). Vif acts as an adaptor protein that bridges A3 proteins with a Cullin 5-based E3 ubiquitin ligase complex, which includes Cul5, Elongin B (EloB), and Elongin C (EloC) (35). Vif has a BC-box motif (¹⁴⁴SLQYLALA¹⁴⁹) that binds to EloC (16, 36) and an HCCH motif (¹⁰⁸Hx₅Cx_17-18Cx_3-5H¹³⁹) that binds to Cul5 (14, 17, 34). On the other hand, Vif also interacts with A3G and A3F. As a consequence of these interactions, A3G and A3F are polyubiquitylated and directed to 26S proteasomes for degradation. In addition, Vif may also inhibit A3 activity independently of proteasomal degradation (10, 11, 24).Interactions between Vif and A3G/A3F are a key step for their proteasomal degradation, and this mechanism has been extensively studied. First, unique surfaces in A3G and A3F important for Vif interaction were identified, and interestingly, they are located in different regions of the two proteins (9, 23). Second, several discontinuous surfaces on Vif have been found to regulate A3G and/or A3F degradation. The ⁴⁰YRHHY⁴⁴ domain specifically binds to A3G and determines Vif specificity for A3G (22); the ¹¹WxxDRMR¹⁷ and ⁷⁴TGERxW⁷⁹ domains specifically bind to A3F and determine Vif specificity for A3F (8, 22); and the ²¹WxSLVK²⁶, ⁵⁵VxIPLx₄L⁶⁴, and ⁶⁹YxxL⁷²domains determine Vif specificity for both A3G and A3F (2, 6, 8, 21). These results indicate that the mechanism that regulates Vif recognition of A3G and A3F is quite complicated, and understanding this mechanism is critical for pharmaceutical protection of A3G and A3F from Vif-mediated proteasomal degradation.Based on our current knowledge of these functional domains, it has been thought that Vif interacts with A3G and A3F mainly via its N-terminal region and with Cul5 E3 ubiquitin ligase machinery via its C-terminal region. However, here we identify a new A3G and A3F regulatory domain from the central region and a new A3F regulatory domain from the C-terminal region of HIV-1 Vif. Our results indicate that A3G and A3F interaction surfaces on HIV-1 Vif are structurally complex, and more efforts are required for a complete understanding of this host-pathogen interactive mechanism.     

Viral Entry Inhibitors Targeted to the Membrane Site of Action

The fusion of enveloped viruses with the host cell is driven by specialized fusion proteins to initiate infection. The “class I” fusion proteins harbor two regions, typically two heptad repeat (HR) domains, which are central to the complex conformational changes leading to fusion: the first heptad repeat (HRN) is adjacent to the fusion peptide, while the second (HRC) immediately precedes the transmembrane domain. Peptides derived from the HR regions can inhibit fusion, and one HR peptide, T20 (enfuvirtide), is in clinical use for HIV-1. For paramyxoviruses, the activities of two membrane proteins, the receptor-binding protein (hemagglutinin-neuraminidase [HN] or G) and the fusion protein (F), initiate viral entry. The binding of HN or G to its receptor on a target cell triggers the activation of F, which then inserts into the target cell and mediates the membrane fusion that initiates infection. We have shown that for paramyxoviruses, the inhibitory efficacy of HR peptides is inversely proportional to the rate of F activation. For HIV-1, the antiviral potency of an HRC-derived peptide can be dramatically increased by targeting it to the membrane microdomains where fusion occurs, via the addition of a cholesterol group. We report here that for three paramyxoviruses—human parainfluenza virus type 3 (HPIV3), a major cause of lower respiratory tract diseases in infants, and the emerging zoonotic viruses Hendra virus (HeV) and Nipah virus (NiV), which cause lethal central nervous system diseases—the addition of cholesterol to a paramyxovirus HRC-derived peptide increased antiviral potency by 2 log units. Our data suggest that this enhanced activity is indeed the result of the targeting of the peptide to the plasma membrane, where fusion occurs. The cholesterol-tagged peptides on the cell surface create a protective antiviral shield, target the F protein directly at its site of action, and expand the potential utility of inhibitory peptides for paramyxoviruses.Fusion of enveloped viruses with the host cell is a key step in viral infectivity, and interference with this process can lead to highly effective antivirals. Viral fusion is driven by specialized proteins that undergo an ordered series of conformational changes. These changes facilitate the initial, close apposition of the viral and host membranes, and they ultimately result in the formation of a fusion pore (reviewed in reference 12). The “class I” fusion proteins harbor two regions, typically two heptad repeat (HR) domains: the first one (HRN) adjacent to the fusion peptide and the second one (HRC) immediately preceding the transmembrane domain. Peptides derived from the HR regions can inhibit fusion, and one of them, T20 (enfuvirtide), is in clinical use for HIV-1 (19). Peptides derived from the HRN and HRC regions of paramyxovirus fusion (F) proteins can interact with fusion intermediates of F (3, 20, 22, 37, 46, 49) and provide a promising antiviral strategy.The current model for class I-driven fusion postulates the existence of a so-called prehairpin intermediate, a high-energy structure that bridges the viral and cell membranes, where the HRN and the HRC are separated. The prehairpin intermediate spontaneously collapses into the postfusion structure—a six-helical bundle (6HB), with an inner trimeric coiled-coil formed by the HRN onto which the HRC folds (12, 14, 30, 40). The key to these events is the initial activation step, whereby HN triggers F to initiate the process. Structural and biophysical analyses of the paramyxovirus 6HB (30, 50, 51) suggest that inhibitors bind to the prehairpin intermediate and prevent its transition to the 6HB, thus inhibiting viral entry. The peptides bind to their complementary HR region and thereby prevent HRN and HRC from refolding into the stable 6HB structure required for fusion (3, 10, 40). The efficiency of F triggering by HN critically influences the degree of fusion mediated by F and thus the extent of viral entry (35). In addition, differences in the efficiency of triggering of the fusion process impact the efficacy of potential antiviral molecules that target intermediate states of the fusion protein (36).Paramyxoviruses cause important human illnesses, significantly contributing to global disease and mortality, ranging from lower-respiratory-tract diseases in infants caused by human parainfluenza virus types 1, 2, and 3 (HPIV1, -2, and -3) (9, 48), to highly lethal central nervous system diseases caused by the emerging paramyxoviruses HeV and NiV. No antiviral therapies or vaccines yet exist for these paramyxoviruses, and vaccines would be unlikely to protect the youngest infants. Antiviral agents, therefore, would be particularly beneficial. All paramyxoviruses possess two envelope glycoproteins directly involved in viral entry and pathogenesis: a fusion protein (F) and a receptor-binding protein (HN, H, or G). The paramyxovirus F proteins belong to the group of “class I” fusion proteins (44, 45), which also include the influenza virus hemagglutinin protein and the HIV-1 fusion protein gp120. The F protein is synthesized as a precursor protein (F0) that is proteolytically processed posttranslationally to form a trimer of disulfide-linked heterodimers (F1-F2). This cleavage event places the fusion peptide at the F1 terminus in the mature F protein and is essential for membrane fusion activity. The exact triggers that initiate a series of conformational changes in F leading to membrane fusion differ depending on the pathway the virus uses to enter the cell. In the case of HPIV, HeV, and NiV, the receptor-binding protein, hemagglutinin-neuraminidase (HN) (in HPIV3) or G (in HeV and NiV), binds to cellular surface receptors, brings the viral envelope into proximity with the plasma membrane, and activates the viral F protein. This receptor-ligand interaction is required for the F protein to mediate the fusion of the viral envelope with the host cell membrane (23, 33, 35).The HRC peptide regions of a number of paramyxoviruses, including Sendai virus, measles virus, Newcastle disease virus (NDV), respiratory syncytial virus (RSV), simian virus 5 (SV5), Hendra virus (HeV), and Nipah virus (NiV), can inhibit the infectivity of the homologous virus (17, 20, 31, 37, 47, 49, 52, 53). Recently, we showed that peptides derived from the HRC region of the F protein of HPIV3 are effective inhibitors of both HPIV and HeV/NiV fusion (31) and that, for HeV, the strength of HRC peptide binding to the corresponding HRN region correlates with the potency of fusion and infection inhibition (30). However, peptides derived from the HPIV3 F protein HRC region are more effective at inhibiting HeV/NiV fusion than HPIV3 fusion, despite a stronger homotypic HRN-HRC interaction for HPIV3 (30, 31). We showed (36) that the kinetics of fusion (kinetics of F activation) impacts sensitivity to inhibition by peptides, as is the case for HIV (39). Alterations in HPIV3 HN′s property of F activation affect the kinetics of F''s progression through its conformational changes, thus altering inhibitor efficacy. Once the extended intermediate stage of F has passed, and fusion proceeds, peptide inhibitors are ineffective. We have proposed that the design of effective inhibitors may require either targeting an earlier stage of F activation or increasing the concentration of inhibitor at the location of receptor binding, in order to enhance the access and association of the inhibitor with the intermediate-stage fusion protein (36).A substantial body of evidence supports the notion that viral fusion occurs in confined areas of the interacting viral and host membranes (26). For HIV-1, the lipid composition of the viral membrane is strikingly different from that of the host cell membrane; the former is particularly enriched in cholesterol and sphingomyelin (4, 5, 7, 8). Cholesterol and sphingolipids are often laterally segregated in membrane microdomains or “lipid rafts” (7, 11). In fact, the antiviral potency of the HIV-inhibitory HRC peptide C34 is dramatically increased by targeting it to the “lipid rafts” via the addition of a cholesterol group (16).We applied the targeting strategy based on cholesterol derivatization to paramyxoviruses, and we show here that by adding a cholesterol tag to HPIV3-derived HRC E459V (30) inhibitory peptides, we increased antiviral potency by 2 log units (50% inhibitory concentrations [IC₅₀], <2 nM). We chose to use the HPIV3-derived peptides for HeV/NiV, because we have previously shown that they are far more effective inhibitors of HeV and NiV than the homotypic peptides (30, 31). We propose that the enhanced activity resulting from the addition of a cholesterol tag is a result of the targeting of the peptide to the plasma membrane, where fusion occurs.     

An Antibody Directed against the Fusion Peptide of Junín Virus Envelope Glycoprotein GPC Inhibits pH-Induced Membrane Fusion

The arenavirus envelope glycoprotein (GPC) initiates infection in the host cell through pH-induced fusion of the viral and endosomal membranes. As in other class I viral fusion proteins, this process proceeds through a structural reorganization in GPC in which the ectodomain of the transmembrane fusion subunit (G2) engages the host cell membrane and subsequently refolds to form a highly stable six-helix bundle structure that brings the two membranes into apposition for fusion. Here, we describe a G2-directed monoclonal antibody, F100G5, that prevents membrane fusion by binding to an intermediate form of the protein on the fusion pathway. Inhibition of syncytium formation requires that F100G5 be present concomitant with exposure of GPC to acidic pH. We show that F100G5 recognizes neither the six-helix bundle nor the larger trimer-of-hairpins structure in the postfusion form of G2. Rather, Western blot analysis using recombinant proteins and a panel of alanine-scanning GPC mutants revealed that F100G5 binding is dependent on an invariant lysine residue (K283) near the N terminus of G2, in the so-called fusion peptide that inserts into the host cell membrane during the fusion process. The F100G5 epitope is located in the internal segment of the bipartite GPC fusion peptide, which also contains four conserved cysteine residues, raising the possibility that this fusion peptide may be highly structured. Collectively, our studies indicate that F100G5 identifies an on-path intermediate form of GPC. Binding to the transiently exposed fusion peptide may interfere with G2 insertion into the host cell membrane. Strategies to effectively target fusion peptide function in the endosome may lead to novel classes of antiviral agents.Enveloped viruses enter their target cells through fusion of the virus and cell membranes, in a process promoted by the viral envelope glycoprotein. For some viruses, such as human immunodeficiency virus (HIV), entry is initiated by interaction of the envelope glycoprotein with cell surface receptor proteins. Other viruses, such as influenza virus, are endocytosed and membrane fusion is triggered by exposure to acidic pH in the maturing endosome. The subsequent merger of the viral and cell membranes is accomplished through a major structural reorganization of the envelope glycoprotein. Antiviral strategies that target virus entry by using neutralizing antibodies or small-molecule fusion inhibitors can, in many cases, prevent virus infection and disease.The Arenaviridae comprise a diverse group of rodent-borne viruses, some of which are responsible for severe hemorrhagic fevers in humans. Lassa fever virus (LASV) is endemic in western Africa (59), and at least five New World species are recognized to cause fatal disease in the Americas, including the Argentine hemorrhagic fever virus Junín (JUNV) (63). New pathogenic arenavirus species continue to emerge from their distinct animal reservoirs (1, 11, 24). At present, there are no licensed vaccines or effective therapies to address the threat of arenavirus infection.Arenaviruses are enveloped, negative-strand RNA viruses whose bipartite genome encodes ambisense expression of four viral proteins (12, 22). The arenavirus envelope glycoprotein, GPC, is a member of the class I virus fusion proteins (33, 40, 75), a group that includes HIV Env, influenza virus hemagglutinin (HA), and paramyxovirus F protein. These envelope glycoproteins share several salient features. The precursor glycoproteins assemble as trimeric complexes and are subsequently rendered competent for membrane fusion by a proteolytic cleavage that results in the formation of the mature receptor-binding and transmembrane fusion subunits. The GPC precursor glycoprotein is cleaved by the cellular SKI-1/S1P protease (6, 51, 54) to generate the respective G1 and G2 subunits, which remain noncovalently associated. The ectodomain of the class I fusion subunit is distinguished by the presence of two 4-3 heptad repeat (HR1 and HR2) sequences that, in the course of membrane fusion, refold to form the now-classical six-helix bundle structure, which defines this class of envelope glycoproteins. Unlike other class I fusion proteins, GPC also contains a cleaved and stable signal peptide (SSP) as a third and essential subunit in the mature complex (2, 32, 69, 77, 81).Arenavirus infection is initiated by G1 binding to a cell surface receptor. The pathogenic clade B New World arenaviruses utilize transferrin receptor 1 (TfR1) for entry (1, 64, 65), whereas those in clades A and C, as well as the Old World viruses, bind α-dystroglycan and/or an unknown receptor (15, 34, 71). The virion particle is subsequently endocytosed (9), and membrane fusion is initiated by acidification in the maturing endosome (17, 28, 29). pH-dependent activation of GPC is modulated through a unique interaction between SSP and G2 (79, 80) and can be targeted by small-molecule inhibitors that block membrane fusion (76) and protect against arenavirus infection (8, 52).A generally accepted model for membrane fusion by the class I envelope glycoproteins (reviewed in references 45 and 73) posits that the native complex exists in a metastable state that is established on proteolytic maturation of the biosynthetic precursor. Upon activation, whether by acidic pH in the endosome or receptor binding at the plasma membrane, the fusion subunit that was sequestered in the prefusion state is exposed and undergoes a series of dramatic conformational changes leading to membrane fusion. In this process, a hydrophobic region at or near the N terminus of the fusion subunit (the fusion peptide) inserts into the host cell membrane, thus allowing the protein to bridge the two membranes. This so-called prehairpin intermediate subsequently collapses upon itself to form the highly stable six-helix bundle structure, in which the three HR2 helices pack into hydrophobic grooves on the trimeric HR1 coiled-coil in an antiparallel manner, bringing the virus and cell membranes into apposition. Free energy made available in the formation of this stable structure is thought to drive fusion of the lipid bilayers. Peptides that correspond in sequence to HR2 (C-peptides) bind to the putative prehairpin intermediate and interfere with its refolding, thereby preventing membrane fusion (18, 57, 74). While the structure of the six-helix bundle core has been elucidated in atomic detail (45, 73), information regarding the molecular pathway leading to this postfusion state is largely indirect. Indeed, the prehairpin intermediate is conceptualized through the activity of C-peptide fusion inhibitors (57, 74).In this report, we describe a G2-directed monoclonal antibody (MAb), F100G5, that recognizes a pH-induced intermediate of JUNV GPC and prevents GPC-mediated membrane fusion. This MAb binds at or near the internal fusion peptide of G2 and may act by interfering with its penetration into the host cell membrane. These studies highlight the feasibility of targeting short-lived GPC intermediates for inhibition of membrane fusion.     

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.     

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.