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.     

A Quantitative and Kinetic Fusion Protein-Triggering Assay Can Discern Distinct Steps in the Nipah Virus Membrane Fusion Cascade

The deadly paramyxovirus Nipah virus (NiV) contains a fusion glycoprotein (F) with canonical structural and functional features common to its class. Receptor binding to the NiV attachment glycoprotein (G) triggers F to undergo a two-phase conformational cascade: the first phase progresses from a metastable prefusion state to a prehairpin intermediate (PHI), while the second phase is marked by transition from the PHI to the six-helix-bundle hairpin. The PHI can be captured with peptides that mimic F''s heptad repeat regions, and here we utilized a NiV heptad repeat peptide to quantify PHI formation and the half-lives (t_1/2) of the first and second fusion cascade phases. We found that ephrinB2 receptor binding to G triggered ∼2-fold more F than that triggered by ephrinB3, consistent with the increased rate and extent of fusion observed with ephrinB2- versus ephrinB3-expressing cells. In addition, for a series of hyper- and hypofusogenic F mutants, we quantified F-triggering capacities and measured the kinetics of their fusion cascade phases. Hyper- and hypofusogenicity can each be manifested through distinct stages of the fusion cascade, giving rise to vastly different half-lives for the first (t_1/2, 1.9 to 7.5 min) or second (t_1/2, 1.5 to 15.6 min) phase. While three mutants had a shorter first phase and a longer second phase than the wild-type protein, one mutant had the opposite phenotype. Thus, our results reveal multiple critical parameters that govern the paramyxovirus fusion cascade, and our assays should help efforts to elucidate other class I membrane fusion processes.Nipah (NiV) and Hendra (HeV) viruses are emerging members of the new Paramyxoviridae genus Henipavirus (12, 19). The Paramyxoviridae family comprises important viral pathogens, such as measles, mumps, human parainfluenza, respiratory syncytial, and Newcastle disease viruses and the henipaviruses (HNV), and NiV is its deadliest known member (4, 5). NiV has a broad host range and causes respiratory and neurological symptoms that often lead to encephalitis and a mortality rate of up to 75% in humans (21, 47). It can also spread efficiently and cause morbidity in economically important livestock (21). NiV is a biosafety level 4 (BSL4) pathogen and is considered a select agent with bio- and agro-terrorism potential. Both animal-to-human and human-to-human transmissions have been documented (4, 5), underscoring the need for research and treatment development. Since microvascular endothelial cell-cell fusion (syncytium formation) is a pathognomonic hallmark of NiV infection (50), understanding virus-cell and cell-cell membrane fusion should assist in the development of therapeutics to target this aspect of NiV pathobiology.Paramyxovirus membrane fusion requires the coordinated action of the attachment (G, HN, or H) and fusion (F) glycoproteins, and numerous canonical structural and functional features of G/HN/H and F proteins are conserved among paramyxoviruses (20, 23, 46, 48). G/HN/H proteins have a receptor-binding globular domain formed by a six-bladed beta-propeller connected to its transmembrane anchor via a flexible stalk domain (10, 51). For NiV and HeV, both ephrinB2 (B2) and ephrinB3 (B3) can be used as cell receptors (8, 33, 34), although B2 appears to be the higher-affinity receptor (34). B2 or B3 receptors bind to and activate G, which in turn triggers a conformation cascade in F that leads to membrane fusion (1). HNV F proteins are trimeric class I fusion proteins with structural/functional features common to their class (23, 52). HNV F proteins are synthesized as precursors that are cleaved and hence activated into a metastable conformation, poised for enabling membrane fusion. Cleavage generates a new N terminus that contains a hydrophobic fusion peptide (48). For NiV and HeV, the precursor (F₀) reaches the plasma membrane uncleaved, but endocytosis exposes F₀ to cathepsin L in the endosomes, cleaving F₀ to generate mature disulfide-linked F₁ and F₂ subunits that are trafficked back to the cell surface (14, 31). The structures of the retroviral Moloney murine leukemia virus p15E, lentiviral human immunodeficiency virus type 1 (HIV-1) gp41, Ebola virus GP2, influenza virus HA, and paramyxovirus SV5 and NiV-F fusion proteins all share similar trimeric coiled-coil core structures (6, 11, 17, 27, 53) and, in general, similar membrane fusion mechanisms (22, 23, 48).Receptor binding to paramyxoviral G/HN/H triggers a conformational cascade in F, leading to membrane fusion (Fig. ​(Fig.1).1). Although the determinants for F triggering on G/HN/H have not been defined clearly, evidence suggests that the stalk domain (7, 13, 24, 28, 29) and, at least for NiV, a region at the base of the globular domain of G (1) are involved in F triggering. Additionally, recent evidence indicates an interaction between the stalk region of the measles virus H protein and the globular domain of the cognate F protein (35). Once triggered, F progresses through a prehairpin intermediate (PHI) (Fig. 1A and B). In the PHI conformation, the fusion peptide is harpooned into the host cell membrane, and the N- and C-terminal heptad repeat domains (HR1 and HR2, respectively) are exposed. The HR domains then coalesce into the postfusion six-helix-bundle (6HB) hairpin conformation. In the 6HB, the transmembrane and fusion peptide domains are juxtaposed, bringing viral and target cell membranes together and driving membrane fusion (Fig. ​(Fig.1C)1C) (30, 48). Much evidence suggests that 6HB formation is coincident with membrane merger and that synthetic HR1 and HR2 peptides only bind to and inhibit fusion intermediates (e.g., PHI) prior to 6HB formation (9, 30, 37, 43, 48). Additionally, HR1 peptides can inhibit an earlier fusion intermediate than that inhibited by HR2 peptides (43), and HR2 peptides are invariably more potent inhibitors of fusion than HR1 peptides. HR2 peptides trap the PHI by binding to the radial interstices formed by the trimeric HR1 core, inhibiting 6HB formation and membrane fusion (22, 23, 48). Altogether, there is much evidence to support the fusion cascade shown in Fig. ​Fig.11 and the use of HR2 peptides to physically capture fusion intermediates (9, 30, 43, 48).Open in a separate windowFIG. 1.Nipah virus fusion cascade. The schematic shows the NiV fusion cascade broken down into three major stages. (A) EphrinB2 or ephrinB3 binding to NiV-G triggers the metastable NiV-F protein through allosteric mechanisms that are still being elucidated. (B) After F is triggered, it forms the PHI, in which a fusion peptide is harpooned into the host cell membrane. The PHI can be captured by peptides that mimic the NiV-F HR1 (orange-striped cylinder) or HR2 (green-striped cylinder) region and bind the F HR2 or HR1 region, respectively. (C) The HR1 and HR2 regions in the PHI coalesce to form the 6HB conformation, bringing the viral and cell membranes together and facilitating virus-host membrane fusion and viral entry. The viral membrane can be replaced by a cell membrane expressing the F and G glycoproteins in cell-cell fusion, resulting in syncytium formation. We term the transitions from A to B and from B to C phases I and II, respectively, of the fusion cascade. (D) Schematic representation of the F-triggering assay, showing its four main steps: (1) receptor binding at 4°C, (2) biotinylated HR2 peptide addition and induction of F triggering at 37°C, (3) fixation at 4°C with paraformaldehyde, and (4) signal amplification at 4°C. In the “time-of-addition” and “time-of-stopping” experiments, step 2 was modified as indicated in the text. The HR2 peptide (green hatched column) is shown with its N-terminal biotin modification (red star). Blue stars, streptavidin-APC; black, three-pronged symbols, activator; blue symbols with red octagons, enhancer.We previously developed a fluorescence-activated cell sorting (FACS)-based NiV-F-triggering assay by measuring the amount of HR2 peptide binding to F/G-expressing cells triggered by cell surface ephrinB2 (1). In this study, we further optimized our assay for robust quantification of HR2 peptide binding and used this assay to monitor the differential degree of F triggering induced by B2 or B3. In addition, through “time-of-addition” and “time-of-stopping” experiments (described below), we show that this HR2 binding assay can measure the half-lives of various fusion intermediates, i.e., the transition times from the prefusion (PF) state to PHI and from PHI to 6HB. Using a panel of hyper- and hypofusogenic mutants, we show that hyper- and hypofusogenicity can each be manifested through distinct effects on the half-lives of these fusion intermediates and/or the absolute amounts of F triggering. Thus, we elucidated the impacts of different mutations on individual steps of the fusion cascade. Since HR2 peptides can generally capture the PHI of class I fusion proteins, our assays should help efforts to understand fusion processes mediated by other class I fusion proteins.     

Reevaluating Herpes Simplex Virus Hemifusion

Membrane fusion induced by enveloped viruses proceeds through the actions of viral fusion proteins. Once activated, viral fusion proteins undergo large protein conformational changes to execute membrane fusion. Fusion is thought to proceed through a “hemifusion” intermediate in which the outer membrane leaflets of target and viral membranes mix (lipid mixing) prior to fusion pore formation, enlargement, and completion of fusion. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins—glycoprotein D (gD), glycoprotein B (gB), and a heterodimer of glycoprotein H and L (gH/gL)—to accomplish fusion. gD is primarily thought of as a receptor-binding protein and gB as a fusion protein. The role of gH/gL in fusion has remained enigmatic. Despite experimental evidence that gH/gL may be a fusion protein capable of inducing hemifusion in the absence of gB, the recently solved crystal structure of HSV-2 gH/gL has no structural homology to any known viral fusion protein. We found that in our hands, all HSV entry proteins—gD, gB, and gH/gL—were required to observe lipid mixing in both cell-cell- and virus-cell-based hemifusion assays. To verify that our hemifusion assay was capable of detecting hemifusion, we used glycosylphosphatidylinositol (GPI)-linked hemagglutinin (HA), a variant of the influenza virus fusion protein, HA, known to stall the fusion process before productive fusion pores are formed. Additionally, we found that a mutant carrying an insertion within the short gH cytoplasmic tail, 824L gH, is incapable of executing hemifusion despite normal cell surface expression. Collectively, our findings suggest that HSV gH/gL may not function as a fusion protein and that all HSV entry glycoproteins are required for both hemifusion and fusion. The previously described gH 824L mutation blocks gH/gL function prior to HSV-induced lipid mixing.Membrane fusion is an essential step during the entry process of enveloped viruses, such as herpes simplex virus (HSV), into target cells. The general pathway by which enveloped viruses fuse with target membranes through the action of fusion proteins is fairly well understood. Viral fusion proteins use the free energy liberated during their own protein conformational changes to draw the two membranes—viral and target—together. Fusion is thought to proceed through a “hemifusion” intermediate, in which the proximal leaflets of the two bilayers have merged but a viral pore has not yet formed and viral contents have not yet mixed with the cell cytoplasm (10, 38). Fusion proteins then drive the completion of fusion, which includes fusion pore formation, pore enlargement, and complete content mixing.HSV, an enveloped neurotropic virus, requires four glycoproteins—glycoprotein B (gB), glycoprotein D (gD), glycoprotein H (gH), and glycoprotein L (gL)—to execute fusion (9, 57, 60). gB, gD, and gH are membrane bound; gL is a soluble protein which complexes with gH to form a heterodimer (gH/gL). HSV-1 gH is not trafficked to the cell or virion surface in the absence of gL (32, 52). The requirement of four entry glycoproteins sets HSV apart from other enveloped viruses, most of which induce fusion through the activity of a single fusion protein. Although the specific mode of HSV entry is cell type dependent—fusion with neurons and Vero cells occurs at the plasma membrane at neutral pH; fusion with HeLa and CHO cells involves pH-dependent endocytosis, and fusion with C10 cells involves pH-independent endocytosis (42, 45)—all routes of entry require gD, gB, and gH/gL. Furthermore, although some discrepancies between virus-cell and cell-cell fusion have been observed (8, 44, 55, 58), both generally require the actions of gD, gB, and gH/gL.Much work has gone toward the understanding of how the required HSV entry glycoproteins work together to accomplish fusion, and many questions remain. After viral attachment, mediated by glycoprotein C and/or gB (54), the first step in HSV fusion is thought to be gD binding a host cell receptor (either herpesvirus entry mediator [HVEM], nectin-1, nectin-2, or heparan sulfate modified by specific 3-O-sulfotransferases) (56). The gD-receptor interaction induces a conformational change in gD (39) that is thought to trigger gD-gB and/or gD-gH/gL interactions that are required for the progression of fusion (1-4, 13, 18, 23, 49).gB and gH/gL are considered the core fusion machinery of most herpesviruses. The HSV-1 gB structure revealed surprising structural homology to the postfusion structures of two known viral fusion proteins (31, 35, 51). This structural homology indicates that despite not being sufficient for HSV fusion, gB is likely a fusion protein. Although the gB cytoplasmic tail (CT) is not included in the solved structure, it acts as a regulator of fusion, as CT truncations can cause either hyperfusion or fusion-null phenotypes (5, 17). The gB CT has been proposed to bind stably to lipid membranes and negatively regulate membrane fusion (12). Another proposed regulator of gB function is gH/gL. Despite conflicting accounts of whether gD and a gD receptor are required for the interaction of gH/gL and gB (1, 3, 4), a recent study indicates that gH/gL and gB interact prior to fusion and that gB may interact with target membranes prior to an interaction with gH/gL (2). The gB-gH/gL interaction seems to be required for the progression of fusion.Compared to the other required HSV entry glycoproteins, the role of gH/gL during fusion remains enigmatic. Mutational studies have revealed several regions of the gH ectodomain, transmembrane domain (TM), and CT that are required for its function (19, 25, 26, 30, 33). gH/gL of another herpesvirus, Epstein-Barr virus (EBV), have been shown to bind integrins during epithelial cell fusion, and soluble forms of HSV gH/gL have been shown to bind cells and inhibit viral entry in vitro (24, 46). However, the role of gH/gL binding to target cells in regard to the fusion process remains to be determined.There are some lines of evidence that suggest that gH/gL is a fusion protein. The gH/gL complexes of VZV and CMV have been reported to independently execute some level of cell-cell fusion (14, 37). HSV-1 gH/gL has been reported to independently mediate membrane fusion during nuclear egress (15). In silico analyses and studies of synthetic HSV gH peptides have proposed that gH has fusogenic properties (20, 21, 25-28). Finally, of most importance to the work we report here, gH/gL has been shown to be sufficient for induction of hemifusion in the presence of gD and a gD receptor, further promoting the premise that gH/gL is a fusion protein (59). However, the recently solved crystal structure of HSV-2 gH/gL revealed a tight complex of gH/gL in a “boot-like” structure, which bears no structural homology to any known fusion proteins (11). The HSV-2 gH/gL structure and research demonstrating that gH/gL and gB interactions are critical to fusion (2) have together prompted a new model of HSV fusion in which gH/gL is required to either negatively or positively regulate the activity of gB through direct binding.We wanted to investigate the ability of a previously reported gH CT mutant, 824L, to execute hemifusion. 824L gH contains a five-residue insertion at gH residue 824, just C-terminal of the TM domain. 824L is expressed on cell surfaces and incorporated into virions at levels indistinguishable from those of wild-type gH by either cell-based ELISA or immunoblotting, yet it is nonfunctional (33). We relied on a fusion assay capable of detecting hemifusion, developed by Subramanian et al. (59), which we modified to include an additional control for hemifusion or nonenlarging pore formation, glycosylphosphatidylinositol (GPI)-linked hemagglutinin (GPI-HA). GPI-HA is a variant of the influenza virus fusion protein, HA, that is known to stall the fusion process before enlarging fusion pores are formed.We were surprised to find that in our hands, gD, a gD receptor, and gH/gL were insufficient for the induction of hemifusion or lipid mixing in both cell-based and virus-based fusion assays. We found that gD, gB, and gH/gL are all required to observe lipid mixing. Further, we found that gB, gD, gL, and 824L gH are insufficient for lipid mixing. Our findings support the emerging view, based on gH/gL structure, that the gH/gL complex does not function as a fusion protein and does not insert into target membranes to initiate the process of fusion through a hemifusion intermediate. Our findings also further demonstrate that mutations in the CT of gH can have a dramatic effect on the ability of gH/gL to function in fusion.     

The Amino Terminus of Herpes Simplex Virus Type 1 Glycoprotein K (gK) Modulates gB-Mediated Virus-Induced Cell Fusion and Virion Egress

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKΔ31-47, gKΔ31-68, and gKΔ31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKΔ31-47 and gKΔ31-117 exhibited a gK-null (ΔgK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKΔ31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKΔ31-47 and gKΔ31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBΔ28syn) caused extensive virus-induced cell fusion. However, the gBΔ28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKΔ31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKΔ31-68, ΔgK, or UL20-null virus. The gKa peptide complemented the gKΔ31-47 and gKΔ31-68 mutant viruses for infectious-virus production and for gKΔ31-68/gBΔ28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.Herpes simplex virus type 1 (HSV-1) specifies at least 11 virally encoded glycoproteins, as well as several nonglycosylated and lipid-anchored membrane-associated proteins, which serve important functions in virion infectivity and virus spread. Although cell-free enveloped virions can efficiently spread viral infection, virions can also spread by causing cell fusion of adjacent cellular membranes. Virus-induced cell fusion, which is caused by viral glycoproteins expressed on infected cell surfaces, enables transmission of virions from one cell to another, avoiding extracellular spaces and exposure of free virions to neutralizing antibodies (reviewed in reference 56). Most mutations that cause extensive virus-induced cell-to-cell fusion (syncytial or syn mutations) have been mapped to at least four regions of the viral genome: the UL20 gene (5, 42, 44); the UL24 gene (37, 58); the UL27 gene, encoding glycoprotein B (gB) (9, 51); and the UL53 gene, coding for gK (7, 15, 35, 53, 54, 57).Increasing evidence suggests that virus-induced cell fusion is mediated by the concerted action of glycoproteins gD, gB, and gH/gL. Recent studies have shown that gD interacts with both gB and gH/gL (1, 2). Binding of gD to its cognate receptors, including Nectin-1, HVEM, and others (12, 29, 48, 59, 60, 62, 63), is thought to trigger conformation changes in gH/gL and gB that cause fusion of the viral envelope with cellular membranes during virus entry and virus-induced cell fusion (32, 34). Transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (49, 68). However, this phenomenon does not accurately model viral fusion, because other viral glycoproteins and membrane proteins known to be important for virus-induced cell fusion are not required (6, 14, 31). Specifically, gK and UL20 were shown to be absolutely required for virus-induced cell fusion (21, 46). Moreover, syncytial mutations within gK (7, 15, 35, 53, 54, 57) or UL20 (5, 42, 44) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than wild-type virus into susceptible cells (25). Furthermore, transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while coexpression of the wild-type gK with gB, gD, and gH/gL inhibited cell fusion (3).Glycoproteins gB and gH are highly conserved across all subfamilies of herpesviruses. gB forms a homotrimeric type I integral membrane protein, which is N glycosylated at multiple sites within the polypeptide. An unusual feature of gB is that syncytial mutations that enhance virus-induced cell fusion are located exclusively in the carboxyl terminus of gB, which is predicted to be located intracellularly (51). Single-amino-acid substitutions within two regions of the intracellular cytoplasmic domain of gB were shown to cause syncytium formation and were designated region I (amino acid [aa] positions 816 and 817) and region II (aa positions 853, 854, and 857) (9, 10, 28, 69). Furthermore, deletion of 28 aa from the carboxyl terminus of gB, disrupting the small predicted alpha-helical domain H17b, causes extensive virus-induced cell fusion as well as extensive glycoprotein-mediated cell fusion in the gB, gD, and gH/gL transient-coexpression system (22, 49, 68). The X-ray structure of the ectodomain of gB has been determined and is predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB. Therefore, perturbation of the carboxyl terminus of gB may alter the conformation of the amino terminus of gB, thus favoring one of the two predicted conformational structures that causes membrane fusion (34).The UL53 (gK) and UL20 genes encode multipass transmembrane proteins of 338 and 222 aa, respectively, which are conserved in all alphaherpesviruses (15, 42, 55). Both proteins have multiple sites where posttranslational modification can occur; however, only gK is posttranslationally modified by N-linked carbohydrate addition (15, 35, 55). The specific membrane topologies of both gK and UL20 protein (UL20p) have been predicted and experimentally confirmed using epitope tags inserted within predicted intracellular and extracellular domains (18, 21, 44). Syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (18), while syncytial mutations of UL20 are located within the amino terminus of UL20p, shown to be located intracellularly (44). A series of recent studies have shown that HSV-1 gK and UL20 functionally and physically interact and that these interactions are necessary for their coordinate intracellular transport and cell surface expression (16, 18, 21, 26, 45). Specifically, direct protein-protein interactions between the amino terminus of HSV-1 UL20 and gK domain III, both of which are localized intracellularly, were recently demonstrated by two-way coimmunoprecipitation experiments (19).According to the most prevalent model for herpesvirus intracellular morphogenesis, capsids initially assemble within the nuclei and acquire a primary envelope by budding into the perinuclear spaces. Subsequently, these virions lose their envelope through fusion with the outer nuclear lamellae. Within the cytoplasm, tegument proteins associate with the viral nucleocapsid and final envelopment occurs by budding of cytoplasmic capsids into specific trans-Golgi network (TGN)-associated membranes (8, 30, 47, 70). Mature virions traffic to cell surfaces, presumably following the cellular secretory pathway (33, 47, 61). In addition to their significant roles in virus-induced cell fusion, gK and UL20 are required for cytoplasmic virion envelopment. Viruses with deletions in either the gK or the UL20 gene are unable to translocate from the cytoplasm to extracellular spaces and accumulated as unenveloped virions in the cytoplasm (5, 15, 20, 21, 26, 35, 36, 38, 44, 55). Current evidence suggests that the functions of gK and UL20 in cytoplasmic virion envelopment and virus-induced cell fusion are carried out by different, genetically separable domains of UL20p. Specifically, UL20 mutations within the amino and carboxyl termini of UL20p allowed cotransport of gK and UL20p to cell surfaces, virus-induced cell fusion, and TGN localization, while effectively inhibiting cytoplasmic virion envelopment (44, 45).In this paper, we demonstrate that the amino terminus of gK expressed as a free peptide of 82 aa (gKa) is transported to infected cell surfaces by viral proteins other than gK or UL20p and facilitates virus-induced cell fusion caused by syncytial mutations in the carboxyl terminus of gB. Thus, functional domains of gK can be genetically separated, as we have shown previously (44, 45), as well as physically separated into different peptide portions that retain functional activities of gK. These results are consistent with the hypothesis that the amino terminus of gK directly or indirectly interacts with and modulates the fusogenic properties of gB.     

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.     

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.     

Characterization of a Highly Conserved Domain within the Severe Acute Respiratory Syndrome Coronavirus Spike Protein S2 Domain with Characteristics of a Viral Fusion Peptide

Many viral fusion proteins are primed by proteolytic cleavage near their fusion peptides. While the coronavirus (CoV) spike (S) protein is known to be cleaved at the S1/S2 boundary, this cleavage site is not closely linked to a fusion peptide. However, a second cleavage site has been identified in the severe acute respiratory syndrome CoV (SARS-CoV) S2 domain (R797). Here, we investigated whether this internal cleavage of S2 exposes a viral fusion peptide. We show that the residues immediately C-terminal to the SARS-CoV S2 cleavage site SFIEDLLFNKVTLADAGF are very highly conserved across all CoVs. Mutagenesis studies of these residues in SARS-CoV S, followed by cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for residues L803, L804, and F805 in membrane fusion. Mutation of the most N-terminal residue (S798) had little or no effect on membrane fusion. Biochemical analyses of synthetic peptides corresponding to the proposed S2 fusion peptide also showed an important role for this region in membrane fusion and indicated the presence of α-helical structure. We propose that proteolytic cleavage within S2 exposes a novel internal fusion peptide for SARS-CoV S, which may be conserved across the Coronaviridae.The severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2003 as a significant threat to human health, and CoVs still represent a leading source of novel viruses for emergence into the human population. The CoV spike (S) protein mediates both receptor binding (via the S1 domain) and membrane fusion (via the S2 domain) and shows many features of a class I fusion protein, including the presence of distinct heptad repeats within the fusion domain (37). A critical feature of any viral fusion protein is the so-called “fusion peptide,” which is a relatively apolar region of 15 to 25 amino acids that interacts with membranes and drives the fusion reaction (9, 34, 38). Fusion peptides can be classified as N-terminal or internal, depending on their location relative to the cleavage site of the virus fusion protein (23). One key feature of viral fusion peptides is that within a particular virus family, there is high conservation of amino acid residues; however, there is little similarity between fusion peptides of different virus families (26). Despite these differences, some common themes do emerge, including a high level of glycine and/or alanine residues, as well as critical bulky hydrophobic amino acids. In several cases, the fusion peptide is known to contain a central “kink.” In the case of influenza virus hemagglutinin (HA), which is a classic example of an N-terminal fusion peptide, the N- and C-terminal parts of the fusion peptide (which are α-helical) penetrate the outer leaflet of the target membrane, with the kink at the phospholipid surface. The inside of the kink contains hydrophobic amino acids, with charged residues on the outer face (18). Internal fusion peptides (such as Ebola virus [EBOV] GP) often contain a conserved proline near their centers but also require a mixture of hydrophobic and flexible residues similar to N-terminal fusion peptides (9, 11). It is believed that the kinked fusion peptide sits in the outer leaflet of the target membrane and possibly induces positive curvature to drive the fusion reaction (22). It is important to note that, despite the presence of key hydrophobic residues, viral fusion peptides often do not display extensive stretches of hydrophobicity and can contain one or more charged residues (8). Ultimately, fusion peptide identification must rely on an often complex set of criteria, including structures of the fusion protein in different conformations, biophysical measurements of peptide function in model membranes, and biological activity in the context of virus particles.To date, the exact location and sequence of the CoV fusion peptide are not known (4); however, by analogy with other class I viral fusion proteins, it is predicted to be in the S2 domain. Overall, three membranotropic regions in SARS-CoV S2 have been suggested as potential fusion peptides (14, 17). Based on sequence analysis and a hydrophobicity analysis of the S protein using the Wimley-White (WW) interfacial hydrophobic interface scale, initial indications were that the SARS-CoV fusion peptide resided in the N-terminal part of HR1 (heptad repeat 1) (5, 6), which is conserved across the Coronaviridae. Mutagenesis of this predicted fusion peptide inhibited fusion in syncytia assays of S-expressing cells (28). This region of SARS-CoV has also been analyzed by other groups in biochemical assays (16, 17, 29) and defined as the WW II region although Sainz et al. (29) actually identified another, less conserved and less hydrophobic, region (WW I) as being more important for fusion. Peptides corresponding to this region have also been studied in biochemical assays by other groups (13). In addition, a third, aromatic region adjacent to the transmembrane domain (the membrane-proximal domain) has been shown to be important in SARS-CoV fusion (15, 20, 25, 30). This membrane-proximal domain likely acts in concert with a fusion peptide in the S2 ectodomain to mediate final bilayer fusion once conformational changes have exposed the fusion peptide in the ectodomain. To date, there is little or no information on the fusion peptides of CoVs other than SARS-CoV, except for the identification of the N-terminal part of the mouse hepatitis virus (MHV) S HR1 domain as a putative fusion peptide based on sequence analysis (6). In none of these cases (for SARS-CoV or MHV) is the role of these sequences as bone fide fusion peptides established.The majority of class I fusion proteins prime fusion activation by proteolytic processing, with the cleavage event occurring immediately N-terminal to the fusion peptide (21). In the case of SARS-CoV, early reports analyzing heterologously expressed SARS-CoV spike protein indicated that most of the protein was not cleaved (31, 39) but that there was some possibility of limited cleavage at the S1-S2 boundary (39). However, it is generally considered that S1-S2 cleavage is not directly linked to fusion peptide exposure in the case of SARS-CoV or any other CoV (4). Recently, however, it has been shown that SARS-CoV S can be proteolytically cleaved at a downstream position in S2, at residue 797 (2, 36). Here, we investigated whether cleavage at this internal position in S2 might expose a domain with properties of a viral fusion peptide. We carried out a mutagenesis study of SARS-CoV S residues 798 to 815 using cell-cell fusion and pseudovirus assays, as well as lipid mixing and structural studies of an isolated peptide, and we show the importance of this region as a novel fusion peptide for SARS-CoV.     

Characterization of Lassa Virus Glycoprotein Oligomerization and Influence of Cholesterol on Virus Replication

Mature glycoprotein spikes are inserted in the Lassa virus envelope and consist of the distal subunit GP-1, the transmembrane-spanning subunit GP-2, and the signal peptide, which originate from the precursor glycoprotein pre-GP-C by proteolytic processing. In this study, we analyzed the oligomeric structure of the viral surface glycoprotein. Chemical cross-linking studies of mature glycoprotein spikes from purified virus revealed the formation of trimers. Interestingly, sucrose density gradient analysis of cellularly expressed glycoprotein showed that in contrast to trimeric mature glycoprotein complexes, the noncleaved glycoprotein forms monomers and oligomers spanning a wide size range, indicating that maturation cleavage of GP by the cellular subtilase SKI-1/S1P is critical for formation of the correct oligomeric state. To shed light on a potential relation between cholesterol and GP trimer stability, we performed cholesterol depletion experiments. Although depletion of cholesterol had no effect on trimerization of the glycoprotein spike complex, our studies revealed that the cholesterol content of the viral envelope is important for the infectivity of Lassa virus. Analyses of the distribution of viral proteins in cholesterol-rich detergent-resistant membrane areas showed that Lassa virus buds from membrane areas other than those responsible for impaired infectivity due to cholesterol depletion of lipid rafts. Thus, derivation of the viral envelope from cholesterol-rich membrane areas is not a prerequisite for the impact of cholesterol on virus infectivity.Lassa virus (LASV) is a member of the family Arenaviridae, of which Lymphocytic choriomeningitis virus (LCMV) is the prototype. Arenaviruses comprise more than 20 species, divided into the Old World and New World virus complexes (19). The Old World arenaviruses include the human pathogenic LASV strains, Lujo virus, which was first identified in late 2008 and is associated with an unprecedented high case fatality rate in humans, the nonhuman pathogenic Ippy, Mobala, and Mopeia viruses, and the recently described Kodoko virus (10, 30, 49). The New World virus complex contains, among others, the South American hemorrhagic fever-causing viruses Junín virus, Machupo virus, Guanarito virus, Sabiá virus, and the recently discovered Chapare virus (22).Arenaviruses contain a bisegmented single-stranded RNA genome encoding the polymerase L, matrix protein Z, nucleoprotein NP, and glycoprotein GP. The bipartite ribonucleoprotein of LASV is surrounded by a lipid envelope derived from the plasma membrane of the host cell. The matrix protein Z has been identified as a major budding factor, which lines the interior of the viral lipid membrane, in which GP spikes are inserted (61, 75). The glycoprotein is synthesized as precursor protein pre-GP-C and is cotranslationally cleaved by signal peptidase into GP-C and the signal peptide, which exhibits unusual length, stability, and topology (3, 27, 28, 33, 70, 87). Moreover, the arenaviral signal peptide functions as trans-acting maturation factor (2, 26, 33). After processing by signal peptidase, GP-C of both New World and Old World arenaviruses is cleaved by the cellular subtilase subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P) into the distal subunit GP-1 and the membrane-anchored subunit GP-2 within the secretory pathway (5, 52, 63). For LCMV, it has been shown that GP-1 subunits are linked to each other by disulfide bonds and are noncovalently connected to GP-2 subunits (14, 24, 31). GP-1 is responsible for binding to the host cell receptor, while GP-2 mediates fusion between the virus envelope and the endosomal membrane at low pH due to a bipartite fusion peptide near the amino terminus (24, 36, 44). Sequence analysis of the LCMV GP-2 ectodomain revealed two heptad repeats that most likely form amphipathic helices important for this process (34, 86).In general, viral class I fusion proteins have triplets of α-helical structures in common, which contain heptad repeats (47, 73). In contrast, class II fusion proteins are characterized by β-sheets that form dimers in the prefusion status and trimers in the postfusion status (43). The class III fusion proteins are trimers that, unlike class I fusion proteins, were not proteolytically processed N-terminally of the fusion peptide, resulting in a fusion-active membrane-anchored subunit (39, 62). Previous studies with LCMV described a tetrameric organization of the glycoprotein spikes (14), while more recent data using a bacterially expressed truncated ectodomain of the LCMV GP-2 subunit pointed toward a trimeric spike structure (31). Due to these conflicting data regarding the oligomerization status of LCMV GP, it remains unclear to which class of fusion proteins the arenaviral glycoproteins belong.The state of oligomerization and the correct conformation of viral glycoproteins are crucial for membrane fusion during virus entry. The early steps of infection have been shown for several viruses to be dependent on the cholesterol content of the participating membranes (i.e., either the virus envelope or the host cell membrane) (4, 9, 15, 20, 21, 23, 40, 42, 53, 56, 76, 78, 79). In fact, it has been shown previously that entry of both LASV and LCMV is susceptible to cholesterol depletion of the target host cell membrane using methyl-β-cyclodextrin (MβCD) treatment (64, 71). Moreover, cholesterol not only plays an important role in the early steps during entry in the viral life cycle but also is critical in the virus assembly and release process. Several viruses of various families, including influenza virus, human immunodeficiency virus type 1 (HIV-1), measles virus, and Ebola virus, use the ordered environment of lipid raft microdomains. Due to their high levels of glycosphingolipids and cholesterol, these domains are characterized by insolubility in nonionic detergents under cold conditions (60, 72). Recent observations have suggested that budding of the New World arenavirus Junin virus occurs from detergent-soluble membrane areas (1). Assembly and release from distinct membrane microdomains that are detergent soluble have also been described for vesicular stomatitis virus (VSV) (12, 38, 68). At present, however, it is not known whether LASV requires cholesterol in its viral envelope for successful virus entry or whether specific membrane microdomains are important for LASV assembly and release.In this study, we first investigated the oligomeric state of the premature and mature LASV glycoprotein complexes. Since it has been shown for several membrane proteins that the oligomerization and conformation are dependent on cholesterol (58, 59, 76, 78), we further analyzed the dependence of the cholesterol content of the virus envelope on glycoprotein oligomerization and virus infectivity. Finally, we characterized the lipid membrane areas from which LASV is released.     

The Human Papillomavirus Type 16 E5 Oncoprotein Inhibits Epidermal Growth Factor Trafficking Independently of Endosome Acidification

The human papillomavirus type 16 E5 oncoprotein (16E5) enhances acute, ligand-dependent activation of the epidermal growth factor receptor (EGFR) and concomitantly alkalinizes endosomes, presumably by binding to the 16-kDa “c” subunit of the V-ATPase proton pump (16K) and inhibiting V-ATPase function. However, the relationship between 16K binding, endosome alkalinization, and altered EGFR signaling remains unclear. Using an antibody that we generated against 16K, we found that 16E5 associated with only a small fraction of endogenous 16K in keratinocytes, suggesting that it was unlikely that E5 could significantly affect V-ATPase function by direct inhibition. Nevertheless, E5 inhibited the acidification of endosomes, as determined by a new assay using a biologically active, pH-sensitive fluorescent EGF conjugate. Since we also found that 16E5 did not alter cell surface EGF binding, the number of EGFRs on the cell surface, or the endocytosis of prebound EGF, we postulated that it might be blocking the fusion of early endosomes with acidified vesicles. Our studies with pH-sensitive and -insensitive fluorescent EGF conjugates and fluorescent dextran confirmed that E5 prevented endosome maturation (acidification and enlargement) by inhibiting endosome fusion. The E5-dependent defect in vesicle fusion was not due to detectable disruption of actin, tubulin, vimentin, or cytokeratin filaments, suggesting that membrane fusion was being directly affected rather than vesicle transport. Perhaps most importantly, while bafilomycin A₁ (like E5) binds to 16K and inhibits endosome acidification, it did not mimic the ability of E5 to inhibit endosome enlargement or the trafficking of EGF. Thus, 16E5 alters EGF endocytic trafficking via a pH-independent inhibition of vesicle fusion.High-risk human papillomaviruses (HPVs) are the causative agent of cervical cancer (63) and HPV type 16 (HPV-16) is associated with a majority of cervical malignancies worldwide (13). HPV-16 encodes three oncoproteins: E5, E6, and E7. While the contributions of E6 and E7 to cellular immortalization and transformation have been characterized in detail (20), the role of HPV-16 E5 (16E5) is poorly understood (53). Nevertheless, a number of studies suggest that 16E5 does contribute to the development of cervical cancer. Most high-risk HPV types encode an E5 protein (48), and targeted expression of the three HPV-16 oncogenes in basal epithelial cells of transgenic mice (4) leads to a higher incidence of cervical cancer than does the expression of E6 and E7 alone (44). In addition, targeted epithelial expression of 16E5 (without E6 and E7) in transgenic mice induces skin tumors (21). It may be noteworthy that unlike high-risk HPV-18, which integrates into the host DNA and potentially disrupts E5 gene expression (20, 64), the HPV-16 genome often persists in episomal form in malignant lesions (12, 16, 24, 36, 42).Biological activities of 16E5 that may facilitate carcinogenesis include evading host immune detection by interfering with the transport of antigen-presenting major histocompatibility complex (MHC) class I molecules to the cell surface (6), promoting anchorage-independent growth (33, 41, 52) and disrupting gap junctions responsible for cell-cell communication (37, 58). The 16E5 phenotype most frequently linked to the development of cancer is enhanced ligand-dependent activation of the epidermal growth factor receptor (EGFR) (15, 41, 46, 52). 16E5 stimulates EGF-dependent cell proliferation in vitro (7, 33, 40, 41, 52, 60) and in vivo (21), which might expand the population of basal or stemlike keratinocytes and thereby increase the probability that some of these cells would undergo malignant transformation. A number of studies indicate that 16E5 may enhance ligand-dependent EGFR activation by interfering with the acidification of early endosomes containing EGF bound to activated EGFRs (17, 51, 57). It has been hypothesized that 16E5 inhibits the H⁺ V-ATPase responsible for maintaining an acidic luminal pH in late endosomes and lysosomes (28) by associating with the V-ATPase 16-kDa “c” subunit (16K) (1, 5, 14, 22, 46) and disrupting assembly of the V-ATPase integral (V_o) and peripheral (V_i) subcomplexes (10). In contrast, Thomsen et al. (57) reported that 16E5 inhibits early endosome trafficking in fibroblasts by completely depolymerizing actin microfilaments.Due to the unavailability of antibodies that recognize native 16E5 and 16K, direct association of 16E5 with 16K has only been observed by overexpressing epitope-tagged forms of both proteins in vitro (5, 46) or in vivo (1, 14, 22). It is uncertain, therefore, whether these associations occur when the proteins are expressed at “physiological” levels. In yeast, both wild-type 16E5 (10) and several 16E5 mutants that associate with 16K in COS cells (1) inhibit vacuolar acidification, although another study in yeast concludes the opposite (5). 16K is a component of the V-ATPase V_o subcomplex, which is assembled in the endoplasmic reticulum (ER) (28), and 16E5 localizes to the ER and nuclear envelope in epithelial cells (32, 54). Thus, the export of V_o from the ER could potentially be inhibited by a significant level of 16K binding to 16E5, although the differential alkalinization of endosomes rather than the Golgi apparatus (17) would require specificity for those proton pumps directed to those sites.In the present study, we generated an antibody against native 16K and used it to determine whether 16K/16E5 complexes formed in primary keratinocytes. We also synthesized a new pH-sensitive fluorescent EGF conjugate to evaluate whether there was a correlation between E5-induced EGFR activation, trafficking and endosome alkalinization. Finally, we simultaneously monitored EGFR endocytic trafficking (using pH-insensitive fluorescent EGF), endosome fusion (using fluorescent EGF and dextran), and the status of cellular filaments and microtubules to evaluate whether E5 might disrupt some of these structures that mediate vesicle transport.     

A Novel Dehalobacter Species Is Involved in Extensive 4,5,6,7-Tetrachlorophthalide Dechlorination

The purpose of this study was the enrichment and phylogenetic identification of bacteria that dechlorinate 4,5,6,7-tetrachlorophthalide (commercially designated “fthalide”), an effective fungicide for rice blast disease. Sequential transfer culture of a paddy soil with lactate and fthalide produced a soil-free enrichment culture (designated the “KFL culture”) that dechlorinated fthalide by using hydrogen, which is produced from lactate. Phylogenetic analysis based on 16S rRNA genes revealed the dominance of two novel phylotypes of the genus Dehalobacter (FTH1 and FTH2) in the KFL culture. FTH1 and FTH2 disappeared during culture transfer in medium without fthalide and increased in abundance with the dechlorination of fthalide, indicating their growth dependence on the dechlorination of fthalide. Dehalobacter restrictus TEA is their closest relative, with 97.5% and 97.3% 16S rRNA gene similarities to FTH1 and FTH2, respectively.4,5,6,7-Tetrachlorophthalide (commercially designated “fthalide”) is an effective fungicide for rice blast disease, which inhibits melanin biosynthesis and the formation of the mature appressorial cells of the rice blast pathogen on the host plant (5, 16). Fthalide has been reported to be reductively dechlorinated in soil (16) and compost (28), although its fates in paddy soil and the fthalide-dechlorinating bacteria are unknown. Besides fthalide, polychlorinated aromatic compounds are known to be reductively dechlorinated by the bacteria of several phyla. Six strains of Desulfitobacterium spp. of the phylum Firmicutes (2, 3, 6, 10, 23, 29) and Desulfomonile tiedjei DCB-1 of the phylum Proteobacteria (21) can dechlorinate polychlorinated phenols. Three strains of the phylum Chloroflexi can dechlorinate a variety of compounds, including polychlorinated phenols, benzenes, biphenyls, or dibenzo-p-dioxins: Dehalococcoides ethenogenes 195 (9, 19), Dehalococcoides sp. strain CBDB1 (1, 4), and strain DF-1 of Chloroflexi, collectively called the “o-17/DF-1 group” (18). Dehalococcoides spp. utilize hydrogen as an electron donor and acetate as a carbon source for growth coupled to the reductive dechlorination of chlorinated compounds (1, 12, 13, 19, 26). In contrast, Desulfitobacterium spp. can dechlorinate chlorinated compounds not only with hydrogen, but also organic acids, such as formate, pyruvate, lactate, or butyrate (3, 10, 23). Strain DF-1 can utilize hydrogen and formate for the dechlorination of polychlorinated biphenyls (PCBs) (18).In this study, bacteria that dechlorinate fthalide were enriched from a paddy soil with sequentially transferred cultures using a soil-free medium supplemented with single organic acids. Acetate, formate, lactate, and butyrate were used in this study because they are frequently used in the enrichment of dechlorinators and release hydrogen at different concentrations (8, 11, 14). Fthalide-dechlorinating bacteria in the enriched culture were phylogenetically identified based on the 16S rRNA gene with PCR-denaturing gradient gel electrophoresis (DGGE), a 16S rRNA gene clone library, and quantitative real-time PCR (qPCR).     

The Longin Domain Regulates the Steady-State Dynamics of Sec22 in Plasmodium falciparum

The specificity of vesicle-mediated transport is largely regulated by the membrane-specific distribution of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. However, the signals and machineries involved in SNARE protein targeting to the respective intracellular locations are not fully understood. We have identified a Sec22 ortholog in Plasmodium falciparum (PfSec22) that contains an atypical insertion of the Plasmodium export element within the N-terminal longin domain. This Sec22 protein partially associates with membrane structures in the parasitized erythrocytes when expressed under the control of the endogenous promoter element. Our studies indicate that the atypical longin domain contains signals that are required for both endoplasmic reticulum (ER)/Golgi apparatus recycling of PfSec22 and partial export beyond the ER/Golgi apparatus interface. ER exit of PfSec22 is regulated by motifs within the α3 segment of the longin domain, whereas the recycling and export signals require residues within the N-terminal hydrophobic segment. Our data suggest that the longin domain of PfSec22 exhibits major differences from the yeast and mammalian orthologs, perhaps indicative of a novel mechanism for Sec22 trafficking in malaria parasites.Plasmodium falciparum exhibits a complex network of endomembrane organelles that are unique to this obligate intracellular parasite of human erythrocytes. They include parasite-induced tubules and vesicles in the infected host cell and specialized secretory structures collectively known as the apical complex. The asexual blood stages of the parasite develop within a parasitophorous vacuole (PV) and thus are separated from the external milieu by three lipid bilayers: the parasite plasma membrane (PPM), the PV membrane (PVM), and the erythrocyte plasma membrane. To survive inside these terminally differentiated human erythrocytes, P. falciparum remodels the host cell compartment by exporting numerous proteins into the erythrocyte cytoplasm (12, 15, 49, 50, 57). The mechanisms by which both soluble and membrane-bound proteins are transported, first into the PV lumen, followed by translocation across the PVM and transport within the erythrocyte cytosol, are not fully understood (9). A majority of the exported proteins contain bipartite signals that comprise a “recessed” N-terminal signal sequence and a Plasmodium export element/vacuolar translocation sequence (PEXEL/VTS) that is characterized by the consensus sequence RX(L/I)X(D/E/Q). These signals are predicted to facilitate the transport of proteins into the PV (using their recessed, or N-terminal, signal sequences) and translocation across the PVM (using their PEXEL/VTS motifs) (5, 23, 29, 34). However, a subset of the exported proteins lack either one or both signal elements and may require novel targeting motifs for transport beyond the PPM (20, 43). A majority of the proteins enter the parasite secretory system via the endoplasmic reticulum (ER), where they are incorporated into ER-derived vesicles and then transported through the “unstacked” Golgi bodies to their final destinations (45, 48, 55, 56). Membrane-bound vesicular elements have been detected in the infected host cell cytosol, suggesting the existence of an extraparasitic vesicle-mediated transport process in malaria parasites (22, 47, 52). How vesicle targeting is achieved in P. falciparum parasites remains elusive.Vesicle targeting and fusion in eukaryotic cells involves proteins of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family (25, 41, 42, 44). SNAREs are “tail-anchored” proteins that function by forming complexes that bridge vesicle and target membranes during fusion (6, 7, 24). Distinct sets of SNARE proteins localize to different intracellular transport pathways using processes that are not well understood. Increasing evidence suggests that the N-terminal regions of SNARE proteins contain signals required for their subcellular localization (4, 31, 53). These N-terminal regions include the three-helical Habc bundles of syntaxin SNAREs and the “profilin-like” folds of long VAMPs (vesicle-associated membrane proteins), also known as longin domains (7, 17, 33, 40, 46). The Sec22 gene products in mammals and yeast are longin domain-containing SNAREs that cycle between the ER and Golgi compartments (3, 19, 31, 32). We have identified a Sec22 ortholog in P. falciparum (PfSec22) that contains a PEXEL/VTS sequence insertion between the α2 and α3 segments of the longin domain preceded by a stretch of hydrophobic residues that spans a region between the β5 and α2 segments (2). In this study, we examined the distribution of PfSec22 in P. falciparum-infected erythrocytes and investigated the role of the atypical longin domain in its steady-state localization. Our data show that the P. falciparum ortholog of Sec22 partially associates with noncanonical destinations (tubovesicular network and intraerythrocytic vesicles) in the infected erythrocytes and that the N-terminal longin domain exhibits a dual function, mediating ER-to-Golgi apparatus trafficking, as well as retrieval from the Golgi apparatus.     

Balancing Reversion of Cytotoxic T-Lymphocyte and Neutralizing Antibody Escape Mutations within Human Immunodeficiency Virus Type 1 Env upon Transmission

Human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is subject to both neutralizing antibody (NAb) and CD8 T-cell (cytotoxic T-lymphocyte [CTL]) immune pressure. We studied the reversion of the Env CTL escape mutant virus to the wild type and the relationship between the reversion of CTL mutations with N-linked glycosylation site (NLGS)-driven NAb escape in pigtailed macaques. Env CTL mutations either did not revert to the wild type or only transiently reverted 5 to 7 weeks after infection. The CTL escape mutant reversion was coincident, for the same viral clones, with the loss of NLGS mutations. At one site studied, both CTL and NLGS mutations were needed to confer NAb escape. We conclude that CTL and NAb escape within Env can be tightly linked, suggesting opportunities to induce effective multicomponent anti-Env immunity.CD8 T-cell responses against human immunodeficiency virus (HIV) have long been observed to select for viral variants that avoid cytotoxic T-lymphocyte (CTL) recognition (2, 5, 15, 18, 27). These immune escape mutations may, however, result in reduced replication competence (“fitness cost”) (11, 20, 26). CTL escape variants have been shown to revert to the wild type (WT) upon passage to major histocompatibility complex-mismatched hosts, both in macaques with simian immunodeficiency virus (SIV) or chimeric SIV/HIV (SHIV) infection (11, 12) and in humans with HIV type 1 (HIV-1) infection (1, 19).Most analyses of CTL escape and reversion have studied Gag CTL epitopes known to facilitate control of viremia (7, 14, 21, 30). Fewer analyses have studied Env-specific CTL epitopes. Recent sequencing studies suggest the potential for mutations within predicted HIV-1 Env-specific CTL epitopes to undergo reversion to the WT (16, 23). Env-specific CTL responses may, however, have less impact on viral control of both HIV-1 and SIV/SHIV than do Gag CTL responses (17, 24, 25), presumably reflecting either less-potent inhibition of viral replication or minimal fitness cost of escape (9).Serial viral escape from antibody pressure also occurs in both macaques and humans (3, 13, 28). Env is extensively glycosylated, and this “evolving glycan shield” can sterically block antibody binding without mutation at the antibody-binding site (8, 16, 31). Mutations at glycosylation sites, as well as other mutations, are associated with escape from neutralizing antibody (NAb) responses (4, 13, 29). Mutations in the amino acid sequences of N-linked glycosylation sites (NLGS) can alter the packing of the glycan cloud that surrounds the virion, by a loss, gain, or shift of an NLGS (32), thus facilitating NAb escape.Env is the only viral protein targeted by both CTL and NAb responses. The serial viral escape from both Env-specific CTL and NAb responses could have implications for viral fitness and the reversion of multiple mutations upon transmission to naïve hosts.We previously identified three common HIV-1 Env-specific CD8 T cell epitopes, RY8_788-795, SP9_110-118, and NL9_671-679, and their immune escape patterns in pigtail macaques (Macaca nemestrina) infected with SHIV_mn229 (25). SHIV_mn229 is a chimeric virus constructed from an SIV_mac239 backbone and an HIV-1_HXB2 env fragment that was passaged through macaques to become pathogenic (11). This earlier work provided an opportunity for detailed studies of how viruses with Env-specific CTL escape mutations, as well as mutations in adjacent NLGS, evolve when transmitted to naïve pigtail macaques.     

Truncation of the Membrane-Spanning Domain of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Defines Elements Required for Fusion,Incorporation, and Infectivity

The membrane-spanning domain (MSD) of the envelope (Env) glycoprotein from human (HIV) and simian immunodeficiency viruses plays a key role in anchoring the Env complex into the viral membrane but also contributes to its biological function in fusion and virus entry. In HIV type 1 (HIV-1), it has been predicted to span 27 amino acids, from lysine residue 681 to arginine 707, and encompasses an internal arginine at residue 694. By examining a series of C-terminal-truncation mutants of the HIV-1 gp41 glycoprotein that substituted termination codons for amino acids 682 to 708, we show that this entire region is required for efficient viral infection of target cells. Truncation to the arginine at residue 694 resulted in an Env complex that was secreted from the cells. In contrast, a region from residues 681 to 698, which contains highly conserved hydrophobic residues and glycine motifs and extends 4 amino acids beyond 694R, can effectively anchor the protein in the membrane, allow efficient transport to the plasma membrane, and mediate wild-type levels of cell-cell fusion. However, these fusogenic truncated Env mutants are inefficiently incorporated into budding virions. Based on the analysis of these mutants, a “snorkeling” model, in which the flanking charged amino acid residues at 681 and 694 are buried in the lipid while their side chains interact with polar head groups, is proposed for the HIV-1 MSD.Human immunodeficiency virus type 1 (HIV-1) infection is initiated by fusion of the viral membrane with that of the target cell and is mediated by the viral envelope glycoprotein (Env). HIV-1 Env, a type 1 membrane-spanning glycoprotein, is a trimeric complex composed of three noncovalently linked heterodimers of gp120, the receptor-binding surface (SU) component, and gp41, the membrane-spanning, transmembrane (TM) component (12, 26, 44, 45). The gp120 and gp41 glycoproteins are synthesized as a precursor gp160 glycoprotein, which is encoded by the env gene. The gp160 precursor is cotranslationally glycosylated and, following transport to the trans-Golgi network, is cleaved into the mature products by a member of the furin family of endoproteases (45). Mature Env proteins are transported to the plasma membrane, where they are rapidly endocytosed or incorporated into virions (5, 33, 43). Recent evidence suggests that endocytosis and intracellular trafficking of Env is required for its interaction with Gag precursors and for efficient assembly into virions (20).HIV-1 Env molecules function as quasistable “spring-loaded” fusion machines. Recent studies have suggested that several regions of gp120 are reoriented following CD4 binding so that a planar “bridging sheet,” which forms the binding site for the coreceptor (CCR5 or CXCR4), can form (6, 7). Coreceptor binding is necessary for additional conformational changes in gp41 and for complete fusion (3). The gp41 monomer has three subdomains, an ectodomain, a membrane-spanning domain (MSD), and a cytoplasmic domain (39). The ectodomain of gp41, which mediates membrane fusion, is composed of a fusion peptide, two heptad repeats, and a tryptophan-rich membrane-proximal external region. Following the binding of gp120 to the CD4 receptor and the CCR5/CXCR4 coreceptor, conformational changes are induced in Env that result in the exposure of the gp41 fusion peptide (32). This peptide inserts into the target cell membrane, allowing gp41 to form a bridge between the viral and cellular membranes. Interaction of the heptad repeats to form a six-helix bundle then brings the target and viral membranes together, allowing membrane fusion to occur (24).While heptad repeat regions 1 and 2 in the N-terminal ectodomain play key roles in Env-mediated fusion by bringing the viral and cell membranes into close proximity, an important function of gp41 is to anchor the glycoprotein complex within the host-derived viral membrane (18). The precise boundaries of the HIV-1 MSD have not been clearly defined; however, the MSD is one of the most conserved regions in the gp41 sequence. Based on the initial functional studies of HIV-1, the MSD of Env was defined as a stretch of 25 predominantly hydrophobic amino acids that span residues K681 to R705 in the NL4-3 sequence (14, 16, 18). These residues were suggested to cross the viral membrane in the form of an alpha helix, the length of which is approximately equal to the theoretical depth of a membrane bilayer. A major caveat of this model is that it places a basic amino acid residue (R694) into the hydrophobic center of the lipid bilayer. While some transmembrane proteins do contain charged amino acid residues in their MSDs, it is normally considered to be energetically unfavorable without some mechanism to neutralize the charge (8, 13). Point mutation studies have yielded varying results, but in general, substitution of K681 is detrimental to fusion and infectivity while mutation of R694 or R705 has only a limited effect on these activities (16, 29). On the other hand, accumulating data argue for a different intramembrane structure of the HIV-1 MSD. Serial small deletions (3 amino acid residues) in the region between R694 and R705 showed normal cell-cell fusion, although larger deletions were detrimental (29), suggesting that, with respect to the biological functions of the Env glycoprotein, the length of this region is more important than its amino acid conservation.Previous C-terminal-truncation studies of simian immunodeficiency virus (SIV) Env (19, 41) suggested that the entire 27-amino-acid region is not required for the biological function of the protein. In the case of SIV, only the 15 apolar amino acids flanked by K689 and R705 (equivalent to K681 and R694 in HIV) and 6 additional amino acids (for a total of 23 amino acids) were required for near-wild-type (WT) fusion (19, 41). Two subsequent residues were required (total, 25 amino acids) for virus-cell entry and infectivity, while a length of 21 amino acid residues was sufficient for SIV Env to be incorporated into viral particles. These results led to a basic amino acid “snorkeling” model for the SIV MSD (41). In this model, the lysine and arginine (NL4-3 equivalents of K681 and R694) are buried in the lipid bilayer, while their long side chains are proposed to extend outward to the membrane surface and present the positively charged amino groups to the negatively charged head groups of the lipid bilayers. Applied to HIV-1 MSD, this model predicts a hydrophobic intramembrane core of only 12 amino acid residues (compared to 15 amino acid residues in the SIV MSD) between K681 and R694. The hydrophobic region C-terminal to K681 is not sufficient to effectively anchor the protein, since mutation of R694 to a stop codon yielded a nonfunctional protein that appeared to be retained in the endoplasmic reticulum (11). This contrasts with truncation experiments with the vesicular stomatitis virus (VSV) G glycoprotein, which have shown that a region of 12 hydrophobic amino acids flanked by basic residues is sufficient to anchor the protein in the membrane (1).In order to understand if the “snorkeling” model is applicable to the HIV-1 MSD, we constructed a series of nonsense mutants with HIV-1 gp41 truncated in single-amino-acid steps at the C terminus from residue R707 to residue R694. For each mutant Env, we determined the membrane stability, fusogenicity, and ability to mediate infectivity. The results of these studies suggest that the 12-residue “core” (36) plus three subsequent hydrophobic amino acids is the minimal anchor domain for HIV-1 Env, as well as the minimal sequence to mediate cell-cell fusion. In contrast to SIV Env, HIV-1 Env requires the entire 25-amino-acid region from K681 to R707 to mediate near-WT incorporation and infectivity.     

Environmental Factors Shape Sediment Anammox Bacterial Communities in Hypernutrified Jiaozhou Bay,China

Bacterial anaerobic ammonium oxidation (anammox) is an important process in the marine nitrogen cycle. Because ongoing eutrophication of coastal bays contributes significantly to the formation of low-oxygen zones, monitoring of the anammox bacterial community offers a unique opportunity for assessment of anthropogenic perturbations in these environments. The current study used targeting of 16S rRNA and hzo genes to characterize the composition and structure of the anammox bacterial community in the sediments of the eutrophic Jiaozhou Bay, thereby unraveling their diversity, abundance, and distribution. Abundance and distribution of hzo genes revealed a greater taxonomic diversity in Jiaozhou Bay, including several novel clades of anammox bacteria. In contrast, the targeting of 16S rRNA genes verified the presence of only “Candidatus Scalindua,” albeit with a high microdiversity. The genus “Ca. Scalindua” comprised the apparent majority of active sediment anammox bacteria. Multivariate statistical analyses indicated a heterogeneous distribution of the anammox bacterial assemblages in Jiaozhou Bay. Of all environmental parameters investigated, sediment organic C/organic N (OrgC/OrgN), nitrite concentration, and sediment median grain size were found to impact the composition, structure, and distribution of the sediment anammox bacterial community. Analysis of Pearson correlations between environmental factors and abundance of 16S rRNA and hzo genes as determined by fluorescent real-time PCR suggests that the local nitrite concentration is the key regulator of the abundance of anammox bacteria in Jiaozhou Bay sediments.Anaerobic ammonium oxidation (anammox, NH₄⁺ + NO₂⁻ → N₂ + 2H₂O) was proposed as a missing N transformation pathway decades ago. It was found 20 years later to be mediated by bacteria in artificial environments, such as anaerobic wastewater processing systems (see reference 32 and references therein). Anammox in natural environments was found even more recently, mainly in O₂-limited environments such as marine sediments (28, 51, 54, 67, 69) and hypoxic or anoxic waters (10, 25, 39-42). Because anammox may remove as much as 30 to 70% of fixed N from the oceans (3, 9, 64), this process is potentially as important as denitrification for N loss and bioremediation (41, 42, 73). These findings have significantly changed our understanding of the budget of the marine and global N cycles as well as involved pathways and their evolution (24, 32, 35, 72). Studies indicate variable anammox contributions to local or regional N loss (41, 42, 73), probably due to distinct environmental conditions that may influence the composition, abundance, and distribution of the anammox bacteria. However, the interactions of anammox bacteria with their environment are still poorly understood.The chemolithoautotrophic anammox bacteria (64, 66) comprise the new Brocadiaceae family in the Planctomycetales, for which five Candidatus genera have been described (see references 32 and 37 and references therein): “Candidatus Kuenenia,” “Candidatus Brocadia,” “Candidatus Scalindua,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia.” Due to the difficulty of cultivation and isolation, anammox bacteria are not yet in pure culture. Molecular detection by using DNA probes or PCR primers targeting the anammox bacterial 16S rRNA genes has thus been the main approach for the detection of anammox bacteria and community analyses (58). However, these studies revealed unexpected target sequence diversity and led to the realization that due to biased coverage and specificity of most of the PCR primers (2, 8), the in situ diversity of anammox bacteria was likely missed. Thus, the use of additional marker genes for phylogenetic analysis was suggested in hopes of better capturing the diversity of this environmentally important group of bacteria. By analogy to molecular ecological studies of aerobic ammonia oxidizers, most recent studies have attempted to include anammox bacterium-specific functional genes. All anammox bacteria employ hydrazine oxidoreductase (HZO) (= [Hzo]₃) to oxidize hydrazine to N₂ as the main source for a useable reductant, which enables them to generate proton-motive force for energy production (32, 36, 65). Phylogenetic analyses of Hzo protein sequences revealed three sequence clusters, of which the cladistic structure of cluster 1 is in agreement with the anammox bacterial 16S rRNA gene phylogeny (57). The hzo genes have emerged as an alternative phylogenetic and functional marker for characterization of anammox bacterial communities (43, 44, 57), allowing the 16S rRNA gene-based investigation methods to be corroborated and improved.The contribution of anammox to the removal of fixed N is highly variable in estuarine and coastal sediments (50). For instance, anammox may be an important pathway for the removal of excess N (23) or nearly negligible (48, 54, 67, 68). This difference may be attributable to a difference in the structure and composition of anammox bacterial communities, in particular how the abundance of individual cohorts depends on particular environmental conditions. Anthropogenic disturbance with variable source and intensity of eutrophication and pollution may further complicate the anammox bacterium-environment relationship.Jiaozhou Bay is a large semienclosed water body of the temperate Yellow Sea in China. Eutrophication has become its most serious environmental problem, along with red tides (harmful algal blooms), species loss, and contamination with toxic chemicals and harmful microbes (14, 15, 21, 61, 71). Due to different sources of pollution and various levels of eutrophication across Jiaozhou Bay (mariculture, municipal and industrial wastewater, crude oil shipyard, etc.), a wide spectrum of environmental conditions may contribute to a widely varying community structure of anammox bacteria. This study used both 16S rRNA and hzo genes as targets to measure their abundance, diversity, and spatial distribution and assess the response of the resident anammox bacterial community to different environmental conditions. Environmental factors with potential for regulating the sediment anammox microbiota are discussed.