Effects of Ammonium and Nitrite on Growth and Competitive Fitness of Cultivated Methanotrophic Bacteria

The effects of nitrite and ammonium on cultivated methanotrophic bacteria were investigated. Methylomicrobium album ATCC 33003 outcompeted Methylocystis sp. strain ATCC 49242 in cultures with high nitrite levels, whereas cultures with high ammonium levels allowed Methylocystis sp. to compete more easily. M. album pure cultures and cocultures consumed nitrite and produced nitrous oxide, suggesting a connection between denitrification and nitrite tolerance.The application of ammonium-based fertilizers has been shown to immediately reduce the uptake of methane in a number of diverse ecological systems (3, 5, 7, 8, 11-13, 16, 27, 28), due likely to competitive inhibition of methane monooxygenase enzymes by ammonia and production of nitrite (1). Longer-term inhibition of methane uptake by ammonium has been attributed to changes in methanotrophic community composition, often favoring activity and/or growth of type I Gammaproteobacteria methanotrophs (i.e., Gammaproteobacteria methane-oxidizing bacteria [gamma-MOB]) over type II Alphaproteobacteria methanotrophs (alpha-MOB) (19-23, 25, 26, 30). It has been argued previously that gamma-MOB likely thrive in the presence of high N loads because they rapidly assimilate N and synthesize ribosomes whereas alpha-MOB thrive best under conditions of N limitation and low oxygen levels (10, 21, 23).Findings from studies with rice paddies indicate that N fertilization stimulates methane oxidation through ammonium acting as a nutrient, not as an inhibitor (2). Therefore, the actual effect of ammonium on growth and activity of methanotrophs depends largely on how much ammonia-N is used for assimilation versus cometabolism. Many methanotrophs can also oxidize ammonia into nitrite via hydroxylamine (24, 29). Nitrite was shown previously to inhibit methane consumption by cultivated methanotrophs and by organisms in soils through an uncharacterized mechanism (9, 17, 24), although nitrite inhibits purified formate dehydrogenase from Methylosinus trichosporium OB3b (15). Together, the data from these studies show that ammonium and nitrite have significant effects on methanotroph activity and community composition and reveal the complexity of ammonia as both a nutrient and a competitive inhibitor. The present study demonstrates the differential influences of high ammonium or nitrite loads on the competitive fitness of a gamma-MOB versus an alpha-MOB strain.     

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

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

Human Immunodeficiency Virus Type 1 Nucleocapsid p1 Confers ESCRT Pathway Dependence

To facilitate the release of infectious progeny virions, human immunodeficiency virus type 1 (HIV-1) exploits the Endosomal Sorting Complex Required for Transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in the C-terminal p6 domain of Gag. However, the L domains in p6 are known to be dispensable for efficient particle production by certain HIV-1 Gag constructs that have the nucleocapsid (NC) domain replaced by a foreign dimerization domain to substitute for the assembly function of NC. We now show that one such L domain-independent HIV-1 Gag construct (termed Z_WT) that has NC-p1-p6 replaced by a leucine zipper domain is resistant to dominant-negative inhibitors of the ESCRT pathway that block HIV-1 particle production. However, Z_WT became dependent on the presence of an L domain when NC-p1-p6 was restored to its C terminus. Furthermore, when the NC domain was replaced by a leucine zipper, the p1-p6 region, but not p6 alone, conferred sensitivity to inhibition of the ESCRT pathway. In an authentic HIV-1 Gag context, the effect of an inhibitor of the ESCRT pathway on particle production could be alleviated by deleting a portion of the NC domain together with p1. Together, these results indicate that the ESCRT pathway dependence of HIV-1 budding is determined, at least in part, by the NC-p1 region of Gag.Human immunodeficiency virus type 1 (HIV-1) and other retroviruses hijack the cellular Endosomal Sorting Complex Required for Transport (ESCRT) pathway to promote the detachment of virions from the cell surface and from each other (3, 21, 42, 44, 47). The ESCRT pathway was initially identified based on its requirement for the sorting of ubiquitinated cargo into multivesicular bodies (MVB) (50, 51). During MVB biogenesis, the ESCRT pathway drives the membrane deformation and fission events required for the inward vesiculation of the limiting membrane of this organelle (26, 29, 50, 51). More recently, it emerged that the ESCRT pathway is also essential for the normal abscission of daughter cells during the final stage of cell division (10, 43). Most of the components of the ESCRT pathway are involved in the formation of four heteromeric protein complexes termed ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. Additional components include ALIX, which interacts both with ESCRT-I and ESCRT-III, and the AAA ATPase Vps4, which mediates the disassembly of ESCRT-III (29, 42).The deformation and scission of endocytic membranes is thought to be mediated by ESCRT-III, which, together with Vps4, constitutes the most conserved element of the pathway (23, 26, 42). Indeed, it was recently shown that purified yeast ESCRT-III induces membrane deformation (52), and in another study three subunits of yeast ESCRT-III were sufficient to promote the formation of intralumenal vesicles in an in vitro assay (61). In mammals, ESCRT-III is formed by the charged MVB proteins (CHMPs), which are structurally related and tightly regulated through autoinhibition (2, 33, 46, 53, 62). The removal of an inhibitory C-terminal domain induces polymerization and association with endosomal membranes and converts CHMPs into potent inhibitors of retroviral budding (34, 46, 53, 60, 62). Alternatively, CHMPs can be converted into strong inhibitors of the ESCRT pathway and of HIV-1 budding through the addition of a bulky tag such as green fluorescent protein (GFP) or red fluorescent protein (RFP) (27, 36, 39, 54). Retroviral budding in general is also strongly inhibited by catalytically inactive Vps4 (22, 41, 55), or upon Vsp4B depletion (31), confirming the crucial role of ESCRT-III.Retroviruses engage the ESCRT pathway through the activity of so-called late assembly (L) domains in Gag. In the case of HIV-1, the primary L domain maps to a conserved PTAP motif in the C-terminal p6 domain of Gag (24, 28) and interacts with the ESCRT-I component Tsg101 (15, 22, 40, 58). HIV-1 p6 also harbors an auxiliary L domain of the LYPx_nL type, which interacts with the V domain of ALIX (20, 35, 39, 54, 59, 63). Interestingly, Tsg101 binding site mutants of HIV-1 can be fully rescued through the overexpression of ALIX, and this rescue depends on the ALIX binding site in p6 (20, 56). In contrast, the overexpression of a specific splice variant of the ubiquitin ligase Nedd4-2 has been shown to rescue the release and infectivity of HIV-1 mutants lacking all known L domains in p6 (12, 57). Nedd4 family ubiquitin ligases had previously been implicated in the function of PPxY-type L domains, which also depend on an intact ESCRT pathway for function (4, 32, 38). However, HIV-1 Gag lacks PPxY motifs, and the WW domains of Nedd4-2, which mediate its interaction with PPxY motifs, are dispensable for the rescue of HIV-1 L domain mutants (57).ALIX also interacts with the nucleocapsid (NC) region of HIV-1 Gag (18, 49), which is located upstream of p6 and the p1 spacer peptide. ALIX binds HIV-1 NC via its Bro1 domain, and the capacity to interact with NC and to stimulate the release of a minimal HIV-1 Gag construct is shared among widely divergent Bro1 domain proteins (48). Based on these findings and the observation that certain mutations in NC cause a phenotype that resembles that of L domain mutants, it has been proposed that NC cooperates with p6 to recruit the machinery required for normal HIV-1 budding (18, 49).NC also plays a role in Gag polyprotein multimerization, and this function of NC depends on its RNA-binding activity (5-8). It has been proposed that the role of the NC-nucleic acid interaction during assembly is to promote the formation of Gag dimers (37), and HIV-1 assembly in the absence of NC can indeed be efficiently rescued by leucine zipper dimerization domains (65). Surprisingly, in this setting the L domains in p6 also became dispensable, since particle production remained efficient even when the entire NC-p1-p6 region of HIV-1 Gag was replaced by a leucine zipper (1, 65). These findings raised the possibility that the reliance of wild-type (WT) HIV-1 Gag on a functional ESCRT pathway is, at least in part, specified by NC-p1-p6. However, it also remained possible that the chimeric Gag constructs engaged the ESCRT pathway in an alternative manner.In the present report, we provide evidence supporting the first of those two possibilities. Particle production became independent of ESCRT when the entire NC-p1-p6 region was replaced by a leucine zipper, and reversion to ESCRT dependence was shown to occur as a result of restoration of p1-p6 but not of p6 alone. Furthermore, although the deletion of p1 alone had little effect in an authentic HIV-1 Gag context, the additional removal of a portion of NC improved particle production in the presence of an inhibitor of the ESCRT pathway. Together, these data imply that the NC-p1 region plays an important role in the ESCRT-dependence of HIV-1 particle production.     

Identification of Novel Methane-, Ethane-, and Propane-Oxidizing Bacteria at Marine Hydrocarbon Seeps by Stable Isotope Probing

Marine hydrocarbon seeps supply oil and gas to microorganisms in sediments and overlying water. We used stable isotope probing (SIP) to identify aerobic bacteria oxidizing gaseous hydrocarbons in surface sediment from the Coal Oil Point seep field located offshore of Santa Barbara, California. After incubating sediment with ¹³C-labeled methane, ethane, or propane, we confirmed the incorporation of ¹³C into fatty acids and DNA. Terminal restriction fragment length polymorphism (T-RFLP) analysis and sequencing of the 16S rRNA and particulate methane monooxygenase (pmoA) genes in ¹³C-DNA revealed groups of microbes not previously thought to contribute to methane, ethane, or propane oxidation. First, ¹³C methane was primarily assimilated by Gammaproteobacteria species from the family Methylococcaceae, Gammaproteobacteria related to Methylophaga, and Betaproteobacteria from the family Methylophilaceae. Species of the latter two genera have not been previously shown to oxidize methane and may have been cross-feeding on methanol, but species of both genera were heavily labeled after just 3 days. pmoA sequences were affiliated with species of Methylococcaceae, but most were not closely related to cultured methanotrophs. Second, ¹³C ethane was consumed by members of a novel group of Methylococcaceae. Growth with ethane as the major carbon source has not previously been observed in members of the Methylococcaceae; a highly divergent pmoA-like gene detected in the ¹³C-labeled DNA may encode an ethane monooxygenase. Third, ¹³C propane was consumed by members of a group of unclassified Gammaproteobacteria species not previously linked to propane oxidation. This study identifies several bacterial lineages as participants in the oxidation of gaseous hydrocarbons in marine seeps and supports the idea of an alternate function for some pmoA-like genes.Hydrocarbon seeps are widespread along continental margins and emit large amounts of oil and gas into the surrounding environment. This gas is primarily composed of methane, a powerful greenhouse gas, and marine hydrocarbon seeps are estimated to contribute 20 Tg year⁻¹ methane to the atmosphere, representing about 5% of the total atmospheric flux (21, 39). Seeps of thermogenic gas also release an estimated 0.45 Tg year⁻¹ ethane and 0.09 Tg year⁻¹ propane to the atmosphere (20). Each of these three fluxes would be substantially larger if not for microbial oxidation in the sediments and water column (68). Methane, ethane, and propane are subject to anaerobic oxidation in anoxic sediments and water columns (44, 53, 68) or to aerobic oxidation in oxic and suboxic water columns and oxygenated surface sediment (10, 47, 53, 80). We focus here on aerobic oxidation.The majority of known aerobic methane-oxidizing bacteria are members of either Gammaproteobacteria (type I) or Alphaproteobacteria (type II) (29), though several strains of highly acidophilic methanotrophic Verrucomicrobia have also been recently isolated (63). Most methanotrophs are capable of growth only on methane or other one-carbon compounds (17, 29), using a methane monooxygenase (MMO) enzyme to oxidize methane to methanol. There are two known forms of this enzyme: soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO). sMMO is a soluble, di-iron-containing monooxygenase found only in certain methanotrophs and typically expressed only under low-copper conditions (57). In contrast, pMMO is a membrane-bound enzyme believed to contain copper and iron (26). It is found in all known methanotrophs, with the exception of species of the genus Methylocella (16). pmoA, the gene encoding the α subunit of pMMO, is often used to identify methanotrophic bacteria (54). Very few methanotrophs from marine environments have been cultured (22, 49, 72, 74), but several previous studies of marine methanotrophs (35, 62, 77, 82, 85) have been performed with culture-independent methods and have almost exclusively detected type I methanotrophs. Many of the pmoA sequences from methane seep sites are quite different from those of cultured organisms, suggesting that these environments may contain many novel methanotrophs (77, 82, 85).Even less is known about the organisms that oxidize ethane or propane in marine environments. The number of such isolates, which primarily represent high G+C Gram-positive bacteria (Nocardia, Pseudonocardia, Gordonia, Mycobacterium, and Rhodococcus) or Pseudomonas species, is limited (70). Nearly all of these strains were isolated from soil and selected for their ability to grow on propane or n-butane as the sole carbon source. Most propane-oxidizing strains can oxidize butane, as well as a range of longer chain n-alkanes, but differ in the ability to oxidize ethane. These strains show little, if any, ability to oxidize methane, and none have been shown to grow with methane as the sole carbon source (13, 27, 38, 45, 65). As with methane metabolism, the first step in aerobic ethane and propane metabolism is the oxidation of the alkane to an alcohol (70). Several different enzymes are known to catalyze this step. Thauera butanivorans uses a soluble di-iron butane monooxygenase related to sMMO to oxidize C₂ through C₉ n-alkanes (18, 73). Gordonia sp. strain TY-5, Mycobacterium sp. strain TY-6, and Pseudonocardia sp. strain TY-7 contain soluble di-iron propane monooxygenases that are capable of both terminal and subterminal propane oxidation and differ in their substrate ranges (45, 46). Nocardioides sp. strain CF8 is believed to possess a copper-containing monooxygenase similar to pMMO and ammonia monooxygenase (27, 28). An alkane hydroxylase typically used to oxidize longer-chain n-alkanes has also shown some ability to oxidize propane and butane but not ethane (38). The variety of enzymes and their substrate ranges make it difficult to identify ethane or propane oxidizers with a single functional gene.In order to identify the organisms responsible for methane, ethane, and propane oxidation at hydrocarbon seeps, we used stable isotope probing (SIP). SIP allows the identification of organisms actively consuming a ¹³C-labeled substrate of interest, based on the incorporation of ¹³C into biomass, including DNA and lipids (67). We collected sediment from the Coal Oil Point seep field and incubated sediment-seawater slurries with ¹³C methane, ethane, or propane. Samples were removed at three time points, chosen to ensure sufficient ¹³C incorporation into DNA while minimizing the spread of ¹³C through the community as a result of cross-feeding on metabolic byproducts. ¹³C-DNA was separated from ¹²C-DNA by CsCl density gradient ultracentrifugation, and we used the fractionated DNA for terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis. We also measured ¹³C incorporation into fatty acids in order to confirm significant ¹³C enrichment in membrane lipids, to determine the carbon labeling pattern for each substrate and lipid, and to further characterize the composition of the microbial community.     

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

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

Vertical Distribution of Ammonia-Oxidizing Crenarchaeota and Methanogens in the Epipelagic Waters of Lake Kivu (Rwanda-Democratic Republic of the Congo)

Four stratified basins in Lake Kivu (Rwanda-Democratic Republic of the Congo) were sampled in March 2007 to investigate the abundance, distribution, and potential biogeochemical role of planktonic archaea. We used fluorescence in situ hybridization with catalyzed-reported deposition microscopic counts (CARD-FISH), denaturing gradient gel electrophoresis (DGGE) fingerprinting, and quantitative PCR (qPCR) of signature genes for ammonia-oxidizing archaea (16S rRNA for marine Crenarchaeota group 1.1a [MCG1] and ammonia monooxygenase subunit A [amoA]). Abundance of archaea ranged from 1 to 4.5% of total DAPI (4′,6-diamidino-2-phenylindole) counts with maximal concentrations at the oxic-anoxic transition zone (∼50-m depth). Phylogenetic analysis of the archaeal planktonic community revealed a higher level of richness of crenarchaeal 16S rRNA gene sequences (21 of the 28 operational taxonomic units [OTUs] identified [75%]) over euryarchaeotal ones (7 OTUs). Sequences affiliated with the kingdom Euryarchaeota were mainly recovered from the anoxic water compartment and mostly grouped into methanogenic lineages (Methanosarcinales and Methanocellales). In turn, crenarchaeal phylotypes were recovered throughout the sampled epipelagic waters (0- to 100-m depth), with clear phylogenetic segregation along the transition from oxic to anoxic water masses. Thus, whereas in the anoxic hypolimnion crenarchaeotal OTUs were mainly assigned to the miscellaneous crenarchaeotic group, the OTUs from the oxic-anoxic transition and above belonged to Crenarchaeota groups 1.1a and 1.1b, two lineages containing most of the ammonia-oxidizing representatives known so far. The concomitant vertical distribution of both nitrite and nitrate maxima and the copy numbers of both MCG1 16S rRNA and amoA genes suggest the potential implication of Crenarchaeota in nitrification processes occurring in the epilimnetic waters of the lake.Lake Kivu is a meromictic lake located in the volcanic region between Rwanda and the Democratic Republic of the Congo and is the smallest of the African Great Rift Lakes. The monimolimnion of the lake contains a large amount of dissolved CO₂ and methane (300 km³ and 60 km³, respectively) as a result of geological and biological activity (24, 73, 85). This massive accumulation converts Lake Kivu into one of the largest methane reservoirs in the world and into a unique ecosystem for geomicrobiologists interested in the methane cycle and in risk assessment and management (34, 71, 72, 85). Comprehensive studies on the diversity and activity of planktonic populations of both large and small eukaryotes and their trophic interplay operating in the epilimnetic waters of the lake are available (33, 39, 49). Recent surveys have also provided a deeper insight into the seasonal variations of photosynthetic and heterotrophic picoplankton (67, 68), although very few data exist on the composition, diversity, and spatial distribution of bacterial and archaeal communities. In this regard, the studies conducted so far of the bacterial/archaeal ecology in Lake Kivu have been mostly focused on the implications on the methane cycle (34, 73), but none have addressed the presence and distribution of additional archaeal populations in the lake.During the last few years, microbial ecology studies carried out in a wide variety of habitats have provided compelling evidence of the ubiquity and abundance of mesophilic archaea (4, 10, 13, 19). Moreover, the discovery of genes encoding enzymes related to nitrification and denitrification in archaeal metagenomes from soil and marine waters (29, 86, 88) and the isolation of the first autotrophic archaeal nitrifier (40) demonstrated that some archaeal groups actively participate in the carbon and nitrogen cycles (56, 64, 69). In relation to aquatic environments, genetic markers of ammonia-oxidizing archaea (AOA) of the marine Crenarchaeota group 1.1a (MCG1) have consistently been found in water masses of several oceanic regions (6, 14, 17, 26, 28, 30, 37, 42, 51, 52, 89), estuaries (5, 9, 26, 53), coastal aquifers (26, 66), and stratified marine basins (15, 41, 44). Although less information is available for freshwater habitats, recent studies carried out in oligotrophic high-mountain and arctic lakes showed an important contribution of AOA in both the planktonic and the neustonic microbial assemblages (4, 61, 89).The oligotrophic nature of Lake Kivu and the presence of a well-defined redoxcline may provide an optimal niche for the development of autotrophic AOA populations. Unfortunately, no studies of the involvement of microbial planktonic populations in cycling nitrogen in the lake exist, and only data on the distribution of dissolved inorganic nitrogen species in relation to phytoplankton ecology (67, 68) and nutrient loading are available (54, 58). Our goals here were to ascertain whether or not archaeal populations other than methane-related lineages were relevant components of the planktonic microbial community and to determine whether the redox gradient imposed by the oxic-anoxic interphase acts as a threshold for their vertical distribution in epipelagic waters (0- to 100-m depth). To further explore the presence and potential activity of nitrifying archaeal populations in Lake Kivu, samples were analyzed for the abundance and vertical distribution of signature genes for these microorganisms, i.e., the 16S rRNA of MCG1 and the ammonia monooxygenase subunit A (amoA) gene by quantitative PCR.     

Investigation of Clade B New World Arenavirus Tropism by Using Chimeric GP1 Proteins

Clade B of the New World arenaviruses contains both pathogenic and nonpathogenic members, whose surface glycoproteins (GPs) are characterized by different abilities to use the human transferrin receptor type 1 (hTfR1) protein as a receptor. Using closely related pairs of pathogenic and nonpathogenic viruses, we investigated the determinants of the GP1 subunit that confer these different characteristics. We identified a central region (residues 85 to 221) in the Guanarito virus GP1 that was sufficient to interact with hTfR1, with residues 159 to 221 being essential. The recently solved structure of part of the Machupo virus GP1 suggests an explanation for these requirements.Arenaviruses are bisegmented, single-stranded RNA viruses that use an ambisense coding strategy to express four proteins: NP (nucleoprotein), Z (matrix protein), L (polymerase), and GP (glycoprotein). The viral GP is sufficient to direct entry into host cells, and retroviral vectors pseudotyped with GP recapitulate the entry pathway of these viruses (5, 13, 24, 31). GP is a class I fusion protein comprising two subunits, GP1 and GP2, cleaved from the precursor protein GPC (4, 14, 16, 18, 21). GP1 contains the receptor binding domain (19, 28), while GP2 contains structural elements characteristic of viral membrane fusion proteins (8, 18, 20, 38). The N-terminal stable signal peptide (SSP) remains associated with the mature glycoprotein after cleavage (2, 39) and plays a role in transport, maturation, and pH-dependent fusion (17, 35, 36, 37).The New World arenaviruses are divided into clades A, B, and C based on phylogenetic relatedness (7, 9, 11). Clade B contains the human pathogenic viruses Junin (JUNV), Machupo (MACV), Guanarito (GTOV), Sabia, and Chapare, which cause severe hemorrhagic fevers in South America (1, 10, 15, 26, 34). Clade B also contains the nonpathogenic viruses Amapari (AMAV), Cupixi, and Tacaribe (TCRV), although mild disease has been reported for a laboratory worker infected with TCRV (29).Studies with both viruses and GP-pseudotyped retroviral vectors have shown that the pathogenic clade B arenaviruses use the human transferrin receptor type 1 (hTfR1) to gain entry into human cells (19, 30). In contrast, GPs from nonpathogenic viruses, although capable of using TfR1 orthologs from other species (1), cannot use hTfR1 (1, 19) and instead enter human cells through as-yet-uncharacterized hTfR1-independent pathways (19). In addition, human T-cell lines serve as useful tools to distinguish these GPs, since JUNV, GTOV, and MACV pseudotyped vectors readily transduce CEM cells, while TCRV and AMAV GP vectors do not (27; also unpublished data). These properties of the GPs do not necessarily reflect a tropism of the pathogenic viruses for human T cells, since viral tropism is influenced by many factors and T cells are not a target for JUNV replication in vivo (3, 22, 25).     

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

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

DivIC Stabilizes FtsL against RasP Cleavage

The essential cell division protein FtsL is a substrate of the intramembrane protease RasP. Using heterologous coexpression experiments, we show here that the division protein DivIC stabilizes FtsL against RasP cleavage. Degradation seems to be initiated upon accessibility of a cytosolic substrate recognition motif.Cell division in bacteria is a highly regulated process (1). The division site selection as well as assembly and disassembly of the divisome have to be strictly controlled (1, 4). Although the spatial control of the divisome is relatively well understood (2, 4, 14, 17), mechanisms governing the temporal control of division are still mainly elusive. Regulatory proteolysis was thought to be a potential modulatory mechanism (8, 9). The highly unstable division protein FtsL was shown to be rate limiting for division and would make an ideal candidate for a regulatory factor in the timing of bacterial cell division (7, 9). In Bacillus subtilis, FtsL is an essential protein of the membrane part of the divisome (5, 7, 8). It is necessary for the assembly of the membrane-spanning division proteins, and a knockout is lethal (8, 9, 12). We have previously reported that FtsL is a substrate of the intramembrane protease RasP (5).These findings raised the question of whether RasP can regulate cell division by cleaving FtsL from the division complex. In order to mimic the situation in which FtsL is bound to at least one of its interaction partners, we used a heterologous coexpression system in which we synthesized FtsL and DivIC. It has been reported before that DivIC and FtsL are intimate binding partners in various organisms (6, 9, 15, 21, 22, 26) and that FtsL and DivIC (together with DivIB) can form complexes even in the absence of the other divisome components (6, 21). We therefore asked whether RasP is able to cleave FtsL in the presence of its major interaction partner DivIC, which would argue for the possibility that RasP could cleave FtsL within a mature divisome. In contrast, if interaction with DivIC could stabilize FtsL against RasP cleavage, this result would bring such a model into question. An alternative option for the role of RasP might be the removal of FtsL from the membrane. It has been shown that divisome disassembly and prevention of reassembly are crucial to prevent minicell formation close to the new cell poles (3, 16).     

Heavily Isotype-Dependent Protective Activities of Human Antibodies against Vaccinia Virus Extracellular Virion Antigen B5

Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro—and protection in vivo in a mouse model—by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.The smallpox vaccine, live vaccinia virus (VACV), is frequently considered the gold standard of human vaccines and has been enormously effective in preventing smallpox disease. The smallpox vaccine led to the worldwide eradication of the disease via massive vaccination campaigns in the 1960s and 1970s, one of the greatest successes of modern medicine (30). However, despite the efficacy of the smallpox vaccine, the mechanisms of protection remain unclear. Understanding those mechanisms is key for developing immunologically sound vaccinology principles that can be applied to the design of future vaccines for other infectious diseases (3, 101).Clinical studies of fatal human cases of smallpox disease (variola virus infection) have shown that neutralizing antibody titers were either low or absent in patient serum (24, 68). In contrast, neutralizing antibody titers for the VACV intracellular mature virion (MV or IMV) were correlated with protection of vaccinees against smallpox (68). VACV immune globulin (VIG) (human polyclonal antibodies) is a promising treatment against smallpox (47), since it was able to reduce the number of smallpox cases ∼80% among variola-exposed individuals in four case-controlled clinical studies (43, 47, 52, 53, 69). In animal studies, neutralizing antibodies are crucial for protecting primates and mice against pathogenic poxviruses (3, 7, 17, 21, 27, 35, 61, 66, 85).The specificities and the functions of protective antipoxvirus antibodies have been areas of intensive research, and the mechanics of poxvirus neutralization have been debated for years. There are several interesting features and problems associated with the antibody response to variola virus and related poxviruses, including the large size of the viral particles and the various abundances of many distinct surface proteins (18, 75, 91, 93). Furthermore, poxviruses have two distinct virion forms, intracellular MV and extracellular enveloped virions (EV or EEV), each with a unique biology. Most importantly, MV and EV virions share no surface proteins (18, 93), and therefore, there is no single neutralizing antibody that can neutralize both virion forms. As such, an understanding of virion structure is required to develop knowledge regarding the targets of protective antibodies.Neutralizing antibodies confer protection mainly through the recognition of antigens on the surface of a virus. A number of groups have discovered neutralizing antibody targets of poxviruses in animals and humans (3). The relative roles of antibodies against MV and EV in protective immunity still remain somewhat unclear. There are compelling data that antibodies against MV (21, 35, 39, 66, 85, 90, 91) or EV (7, 16, 17, 36, 66, 91) are sufficient for protection, and a combination of antibodies against both targets is most protective (66). It remains controversial whether antibodies to one virion form are more important than those to the other (3, 61, 66). The most abundant viral particles are MV, which accumulate in infected cells and are released as cells die (75). Neutralization of MV is relatively well characterized (3, 8, 21, 35). EV, while less abundant, are critical for viral spread and virulence in vivo (93, 108). Neutralization of EV has remained more enigmatic (3).B5R (also known as B5 or WR187), one of five known EV-specific proteins, is highly conserved among different strains of VACV and in other orthopoxviruses (28, 49). B5 was identified as a protective antigen by Galmiche et al., and the available evidence indicated that the protection was mediated by anti-B5 antibodies (36). Since then, a series of studies have examined B5 as a potential recombinant vaccine antigen or as a target of therapeutic monoclonal antibodies (MAbs) (1, 2, 7, 17, 40, 46, 66, 91, 110). It is known that humans immunized with the smallpox vaccine make antibodies against B5 (5, 22, 62, 82). It is also known that animals receiving the smallpox vaccine generate antibodies against B5 (7, 20, 27, 70). Furthermore, previous neutralization assays have indicated that antibodies generated against B5 are primarily responsible for neutralization of VACV EV (5, 83). Recently Chen at al. generated chimpanzee-human fusion MAbs against B5 and showed that the MAbs can protect mice from lethal challenge with virulent VACV (17). We recently reported, in connection with a study using murine monoclonal antibodies, that neutralization of EV is highly complement dependent and the ability of anti-B5 MAbs to protect in vivo correlated with their ability to neutralize EV in a complement-dependent manner (7).The focus of the study described here was to elucidate the mechanisms of EV neutralization, focusing on the human antibody response to B5. Our overall goal is to understand underlying immunobiological and virological parameters that determine the emergence of protective antiviral immune responses in humans.     

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.     

Microbial Community Structure and Denitrification in a Wetland Mitigation Bank

Wetland mitigation is implemented to replace ecosystem functions provided by wetlands; however, restoration efforts frequently fail to establish equivalent levels of ecosystem services. Delivery of microbially mediated ecosystem functions, such as denitrification, is influenced by both the structure and activity of the microbial community. The objective of this study was to compare the relationship between soil and vegetation factors and microbial community structure and function in restored and reference wetlands within a mitigation bank. Microbial community composition was assessed using terminal restriction fragment length polymorphism targeting the 16S rRNA gene (total bacteria) and the nosZ gene (denitrifiers). Comparisons of microbial function were based on potential denitrification rates. Bacterial community structures differed significantly between restored and reference wetlands; denitrifier community assemblages were similar among reference sites but highly variable among restored sites throughout the mitigation bank. Potential denitrification was highest in the reference wetland sites. These data demonstrate that wetland restoration efforts in this mitigation bank have not successfully restored denitrification and that differences in potential denitrification rates may be due to distinct microbial assemblages observed in restored and reference (natural) wetlands. Further, we have identified gradients in soil moisture and soil fertility that were associated with differences in microbial community structure. Microbial function was influenced by bacterial community composition and soil fertility. Identifying soil factors that are primary ecological drivers of soil bacterial communities, especially denitrifying populations, can potentially aid the development of predictive models for restoration of biogeochemical transformations and enhance the success of wetland restoration efforts.Wetlands provide more ecosystem services (e.g., flood control, water purification, nutrient cycling, and habitat for wildlife) per hectare than any other ecosystem (16). Riparian wetlands, in particular, are sites of intense biogeochemical activity and play an important role in improving water quality, recycling nutrients, and detoxifying chemicals (41). Changing patterns of land use over the last century have resulted in the loss of over half of the wetlands in the contiguous United States (17) and about 60% of wetlands in the Midwestern United States (82). The loss of ecosystem services through conversion of wetlands to alternative (primarily agricultural) land uses exacerbates nutrient pollution and eutrophication of downstream ecosystems (57). Declines in wetland acreage have continued despite a federal policy goal of no-net-loss of wetland acreage and function adopted in 1990 (7, 55). Wetland mitigation projects provide compensation for impacted wetlands and aim to replace the critical functions provided by wetlands. Despite decades of wetland mitigation, however, restoration efforts frequently fail to reestablish desired levels of ecosystem services. Restoration outcomes remain uncertain, and more information is necessary in order to improve monitoring and assessment of wetland development (13, 18, 50, 80).One approach to wetland compensation is through mitigation banks. These sites are areas that are restored, established, enhanced, or preserved for replacement of wetlands that will be affected by future land use change. Mitigation banks are considered “third-party” compensatory mitigation, where the permittee (e.g., developer planning to destroy a wetland) is responsible for purchasing wetland credits in acreage, but the wetland bank is established and managed by another party (24). Wetland mitigation banks have unique characteristics that distinguish them from smaller individual restoration projects (7, 69, 81). Due to their size, wetland mitigation banks are especially heterogeneous and may have a great deal of within-site variability in hydrology and nutrient status, making it challenging to implement a single restoration design. Thus, wetland mitigation banks require intense management and monitoring for improved success (7, 69, 81).Restoration efforts such as mitigation banks aim to replace chemical, physical, and biological ecosystem functions of wetlands that have been lost through anthropogenic disturbance (24). Monitoring of wetland mitigation sites has largely focused on measures of macro-scale community structure (e.g., vegetation surveys) (52) along with measures of hydrology and soil type (24). Measurement of vegetation is a common proxy for wetland performance but does not provide an accurate assessment of wetland function (6, 52). Quantitative assessment is achievable, however, for ecosystem services such as water quality improvement through nitrate removal, where well-characterized microbial mechanisms underlie denitrification processes.The link between microbial community structure and function in a restoration context is a topic of current interest (33). Relating microbial community composition and dynamics to chemical, physical, and biological variables can help to reveal important ecological drivers of microbial communities and their activities (26, 35, 42). Conserved bacterial functional genes related to specific biogeochemical transformations allow evaluation of the community structure of microbial populations directly involved in these processes (49, 60, 63, 77, 79). Assessing the diversity of microorganisms that are specifically involved in denitrification is possible through amplification of the nosZ gene, which encodes the catalytic subunit of nitrous oxide reductase, the enzyme responsible for the final step of denitrification (60, 63, 66). Phylogenetically diverse microorganisms can carry out denitrification though the majority of previously described denitrifiers belong to subphyla within the Proteobacteria (53, 56, 60, 61). Denitrification is a facultative process that occurs only under anaerobic conditions (53, 75). Complete denitrification to N₂ is more prevalent in anaerobic, saturated wetland ecosystems (14, 76), and incomplete denitrification to N₂O is the less desirable, more common endpoint of denitrification under more aerobic, drier conditions (14, 62). While the environmental factors (e.g., oxygen, carbon, nitrate, and pH) that influence bulk denitrification rates have been well characterized (31, 72), the influence of these factors on the composition of denitrifier communities, particularly in a restoration context, is unclear. Understanding the relationship between the microbial populations responsible for nitrogen transformations and easily measured environmental parameters (e.g., soil chemical and physical measures) could lead to assessment metrics that are linked directly to ecosystem functions such as denitrification and bridge the current gap in functional assessment methods (36, 60, 70).The objectives of this study were (i) to compare the microbial and plant community composition in restored wetlands to the composition in adjacent reference floodplain forest wetlands; (ii) to assess the relationship between microbial community composition (based on terminal restriction fragment length polymorphism [T-RFLP]) and potential denitrification activity throughout the mitigation bank; and (iii) to examine soil factors correlated with microbial community composition using both phylogenetic and functional gene markers. As soil environmental conditions affect microbial community structure and activity, we expected that sites where wetland hydrology and soil chemistry have been successfully restored would harbor microbial assemblages that are similar in composition and denitrification function to those observed in reference wetlands within this mitigation bank.     

Divergent Bro1 Domains Share the Capacity To Bind Human Immunodeficiency Virus Type 1 Nucleocapsid and To Enhance Virus-Like Particle Production

To promote the release of infectious virions, human immunodeficiency virus type 1 (HIV-1) exploits the endosomal sorting complex required for transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in p6 Gag. An LYPx_nL motif in p6 serves as docking site for the central V domain of ALIX and is required for its ability to stimulate HIV-1 budding. Additionally, the nucleocapsid (NC) domain of Gag binds to the N-terminal Bro1 domain of ALIX, which connects ALIX to the membrane-deforming ESCRT-III complex via its CHMP4 subunits. Since the isolated Bro1 domain of ALIX is sufficient to markedly stimulate virus-like particle (VLP) production in a minimal Gag rescue assay, we examined whether the Bro1 domains of other human proteins possess a similar activity. We now show that the Bro1 domain-only protein Brox and the isolated Bro1 domains of HD-PTP and rhophilin all bind to HIV-1 NC. Furthermore, all shared the capacity to stimulate VLP production by a minimal HIV-1 Gag molecule, and Brox in particular was as potent as the Bro1 domain of ALIX in this assay. Unexpectedly, Brox retained significant activity even if its CHMP4 binding site was disrupted. Thus, the ability to assist in VLP production may be an intrinsic property of the boomerang-shaped Bro1 domain.Retroviruses engage an endosomal budding machinery via so-called late assembly (L) domains in Gag to promote virus budding at the plasma membrane (4, 17, 33). In the case of human immunodeficiency virus type 1 (HIV-1), the C-terminal p6 domain of Gag harbors a conserved P(T/S)AP motif, which binds to the host protein Tsg101 and functions as the primary L domain (18, 29, 44). Additionally, HIV-1 p6 contains an auxiliary L domain of the LYPx_nL type, which serves as a docking site for ALIX (28, 41, 45). Tsg101 and ALIX are both components of a protein network that is required for the biogenesis of multivesicular bodies (MVB) (22, 38). These compartments are formed through the budding of vesicles from the limiting membrane of endosomes into their lumen, a process that is topologically equivalent to virus budding at the plasma membrane. Recently, it emerged that the protein network essential for MVB formation also functions in cytokinesis, which requires a membrane fission event of similar topology (7, 32).Most of the components of the protein network that mediates these events are subunits of heteromeric endosomal sorting complexes required for transport (ESCRT) (3, 22, 38). For instance, Tsg101 is a subunit of the heterotetrameric ESCRT-I complex (22, 38). ESCRT-I and the downstream ESCRT-II are stable complexes, whereas ESCRT-III assembles only upon membrane binding (38). ESCRT-III is formed by the structurally related human CHMP proteins, which exist in an autoinhibited monomeric conformation in the cytosol (40, 46). A conformational change from a closed to an open conformation is thus likely required for the activation of CHMP proteins and the assembly of ESCRT-III. Interestingly, the uncontrolled activation of CHMP proteins through the removal of autoinhibitory C-terminal sequences results in the potent inhibition of HIV-1 budding, indicating a central role for ESCRT-III in retroviral release (46).ALIX consists of a boomerang-shaped N-terminal Bro1 domain, a central ligand binding domain that is shaped like a V, and a C-terminal proline-rich region (16). While ALIX is essential for equine anemia virus budding, its role in HIV-1 budding is less critical than that of Tsg101 (8, 16, 28, 41). However, ALIX can clearly support efficient HIV-1 budding, because its overexpression potently rescues the release defect of Tsg101 binding site mutants (16, 43). This effect of ALIX depends on the interaction between its central V domain and the LYPx_nL motif in HIV-1 p6 (16, 43), confirming that this motif constitutes a functional L domain.The Bro1 domain of ALIX interacts tightly with ESCRT-III subunit CHMP4B and less avidly with CHMP4A and CHMP4C (25, 28, 41, 45). The ability of ALIX to rescue HIV-1 L domain mutants depends on the interaction between its Bro1 domain and CHMP4, indicating that CHMP4 is of particular importance in viral budding (16, 43). Interestingly, human CHMP4A assembles into membrane-attached filaments if overexpressed in mammalian cells, and these filaments can be induced to form circular arrays that drive the formation of buds and tubules with the same topology as that of a retroviral bud (21). Also, the single yeast ortholog of the mammalian CHMP4 proteins forms homo-oligomeric filaments on endosomes that appear to drive MVB sorting and biogenesis (42).By binding to membranes with its convex surface, the Bro1 domain of ALIX could also contribute directly to the generation of negative curvature required for budding away from the cytosol. In support of this notion, we recently observed that the isolated Bro1 domain of ALIX can potently enhance the formation of virus-like particles (VLP) by a minimal HIV-1 Gag construct that retains the primary L domain but lacks certain assembly domains and thus is presumably defective in its ability to deform membranes (37). We also observed that the Bro1 domain of ALIX physically interacts with the nucleocapsid (NC) region of HIV-1 Gag and that mutations in NC that interfere with the interaction induce a phenotype that resembles that of L domain mutants (37).Despite limited sequence homology between human ALIX and a yeast counterpart, the structures of their Bro1 domains are largely superimposable (16, 26), suggesting that all Bro1 domains have a shape that would be compatible with a membrane-deforming function. We therefore asked whether the ability to stimulate VLP production is unique to the Bro1 domain of ALIX or a property of Bro1 domains in general. We now show that widely divergent Bro1 domains share the ability to associate with HIV-1 Gag in an NC-dependent manner and to enhance VLP production by a minimal Gag molecule. In particular, a human Bro1 domain-only protein termed Brox (23) was as potent as the ALIX Bro1 domain in stimulating VLP production, and even forms of Brox that did not bind to CHMP4 retained significant activity. We thus propose that Bro1 domains are inherently capable of promoting budding events away from the cytosol.     

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.In animals, RNA interference (RNAi) was first described for Caenorhabditis elegans (27). The production or introduction of double-stranded RNA (dsRNA) in cells leads to the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of mRNAs. Central to RNAi is the production of 21- to 26-nucleotide small interfering RNAs (siRNAs) from dsRNA and the assembly of an RNA-induced silencing complex (RISC), followed by the degradation of the target mRNA (23, 84). RNAi is a known antiviral strategy of plants (3, 53) and insects (21, 39, 51). Study of Drosophila melanogaster in particular has given important insights into RNAi responses against pathogenic viruses and viral RNAi inhibitors (31, 54, 83, 86, 91). RNAi is well characterized for Drosophila, and orthologs of antiviral RNAi genes have been found in Aedes and Culex spp. (13, 63).Arboviruses, or arthropod-borne viruses, are RNA viruses mainly of the families Bunyaviridae, Flaviviridae, and Togaviridae. The genus Alphavirus within the family Togaviridae contains several mosquito-borne pathogens: arboviruses such as Chikungunya virus (16) and equine encephalitis viruses (88). Replication of the prototype Sindbis virus and Semliki Forest virus (SFV) is well understood (44, 71, 74, 79). Their genome consists of a positive-stranded RNA with a 5′ cap and a 3′ poly(A) tail. The 5′ two-thirds encodes the nonstructural polyprotein P1234, which is cleaved into four replicase proteins, nsP1 to nsP4 (47, 58, 60). The structural polyprotein is encoded in the 3′ one-third of the genome and cleaved into capsid and glycoproteins after translation from a subgenomic mRNA (79). Cytoplasmic replication complexes are associated with cellular membranes (71). Viruses mature by budding at the plasma membrane (35).In nature, arboviruses are spread by arthropod vectors (predominantly mosquitoes, ticks, flies, and midges) to vertebrate hosts (87). Little is known about how arthropod cells react to arbovirus infection. In mosquito cell cultures, an acute phase with efficient virus production is generally followed by the establishment of a persistent infection with low levels of virus production (9). This is fundamentally different from the cytolytic events following arbovirus interactions with mammalian cells and pathogenic insect viruses with insect cells. Alphaviruses encode host response antagonists for mammalian cells (2, 7, 34, 38).RNAi has been described for mosquitoes (56) and, when induced before infection, antagonizes arboviruses and their replicons (1, 4, 14, 15, 29, 30, 32, 42, 64, 65). RNAi is also functional in various mosquito cell lines (1, 8, 43, 49, 52). In the absence of RNAi, alphavirus and flavivirus replication and/or dissemination is enhanced in both mosquitoes and Drosophila (14, 17, 31, 45, 72). RNAi inhibitors weakly enhance SFV replicon replication in tick and mosquito cells (5, 33), posing the questions of how, when, and where RNAi interferes with alphavirus infection in mosquito cells.Here we use an A. albopictus-derived mosquito cell line to study RNAi responses to SFV. Using reporter-based assays, we demonstrate that SFV cannot avoid or efficiently inhibit the establishment of an RNAi response. We also demonstrate that the RNAi signal can spread between mosquito cells. SFV cannot inhibit cell-to-cell spread of the RNAi signal, and spread of the virus-induced RNAi signal (dsRNA/siRNA) can inhibit the replication of incoming SFV in neighboring cells. Furthermore, we show that SFV expression of a siRNA-binding protein increases levels of virus replication mainly by enhancing virus spread between cells rather than replication in initially infected cells. Taken together, these findings suggest a novel mechanism, cell-to-cell spread of antiviral dsRNA/siRNA, by which RNAi limits SFV dissemination in mosquito cells.