Studies of the “Chain Reversal Regions” of the Avian Sarcoma/Leukosis Virus (ASLV) and Ebolavirus Fusion Proteins: Analogous Residues Are Important,and a His Residue Unique to EnvA Affects the pH Dependence of ASLV Entry

Most class I fusion proteins exist as trimers of dimers composed of a receptor binding and a fusion subunit. In their postfusion forms, the three fusion subunits form trimers of hairpins consisting of a central coiled coil (formed by the N-terminal helices), an intervening sequence, and a region containing the C helix (and flanking strands) that runs antiparallel to and packs in the grooves of the N-terminal coiled coil. For filoviruses and most retroviruses, the intervening sequence includes a “chain reversal region” consisting of a short stretch of hydrophobic residues, a Gly-Gly pair, a CX₆CC motif, and a bulky hydrophobic residue. Maerz and coworkers (A. L. Maerz, R. J. Center, B. E. Kemp, B. Kobe, and P. Poumbourios, J. Virol. 74:6614-6621, 2000) proposed a model for this region of human T-cell leukemia virus type 1 (HTLV-1) Env in which expulsion of the final bulky hydrophobic residue is important for early conformational changes and specific residues in the chain reversal region are important for forming the final, stable trimer of hairpins. Here, we used mutagenesis and pseudovirus entry assays to test this model for the avian retrovirus avian sarcoma/leukosis virus (ASLV) and the filovirus ebolavirus Zaire. Our results are generally consistent with the model proposed for HTLV-1 Env. In addition, we show with ASLV EnvA that the bulky hydrophobic residue following the CX₆CC motif is required for the step of prehairpin target membrane insertion, whereas other residues are required for the foldback step of fusion. We further found that a His residue that is unique to the chain reversal region of ASLV EnvA controls the pH at which ASLV entry occurs.Class I fusion proteins are trimeric glycoproteins that project from the viral membrane surface. Most harbor the host cell receptor binding and membrane fusion functions within distinct subunits (16, 41). Each class I fusion subunit contains a hydrophobic fusion peptide (or fusion loop), two heptad repeats separated by an intervening sequence, a membrane-spanning sequence, and a cytoplasmic tail. Class I fusion proteins are primed for fusion by one or more proteolytic events that, in most cases, separate the receptor binding and fusion subunits, leaving the fusion peptide/loop at or near the N terminus of the fusion subunit, and most sit on the viral membrane surface in a metastable state in which the receptor binding subunit “clamps” the fusion subunit (21, 42). Receptor binding, decreasing pH (during endocytosis), disulfide exchange, or a combination of these factors triggers release of this clamp (41). Once triggered, the fusion subunit undergoes conformational changes that first extend the fusion peptide/loop for interaction with the target membrane and then bend the fusion subunit roughly in half, forming a trimer of hairpins that brings the fusion peptides and the membrane-spanning domains, and hence the two membranes they are tethered to, together to create a fusion pore. For class I hairpins, the N-heptad repeats form a trimeric coiled coil, and C-terminal regions containing the helical C-heptad repeats pack (antiparallel) in the grooves of the N-terminal coiled coil. Additional zippering of the N- and C-terminal regions likely facilitates fusion pore formation (26).The intervening sequence between the N- and C-heptad repeats is of variable length. For the fusion proteins of coronaviruses and paramyxoviruses, it is quite long (∼150 to 250 residues) (1, 43), whereas for the influenza virus HA2 subunit, it contains only 7 residues. The conversion of these 7 HA2 residues from a helix to a loop during fusion activation reverses the direction of the C-terminal end of HA2 (1, 43). For the human T-cell leukemia virus type 1 (HTLV-1) Env glycoprotein, the intervening sequence is 30 residues long and is comprised of a “chain reversal region” at the apex of the bend and a strand that extends to the C helix (20). The chain reversal region contains a CX₆CC motif (20, 23) in which the first two Cys residues form an intramolecular disulfide bond, whereas the last Cys is bonded to the last Cys of a CXXC thiol-disulfide exchange motif in the receptor binding subunit. Reduction of the intersubunit disulfide bond by the free Cys of the CXXC motif is an integral part of the fusion-triggering process (22, 30). Many retroviral and all filoviral fusion subunits contain a CX₆CC motif (Fig. ​(Fig.1A)1A) (32). However, the filovirus glycoproteins (GPs) and alpharetrovirus Envs lack a thiol exchange motif, and at least for the avian sarcoma/leukosis virus subtype A (ASLV-A) Env (EnvA), the receptor binding and fusion subunits remain covalently attached throughout the fusion process (35).Open in a separate windowFIG. 1.Alignment of residues in the chain reversal regions of the ASLV-A, ZEBOV, HTLV-1, and MoMLV fusion subunits. The sequence of ASLV EnvA is available at http://home.ncifcrf.gov/hivdrp/RCAS/RCASBPa_provisional_sequence.txt. ZEBOV, accession number {"type":"entrez-nucleotide","attrs":{"text":"U31033","term_id":"1141778","term_text":"U31033"}}U31033; HTLV-1, accession number {"type":"entrez-protein","attrs":{"text":"NP_955621","term_id":"40796176","term_text":"NP_955621"}}NP_955621; MLV, accession number {"type":"entrez-protein","attrs":{"text":"NP_955589","term_id":"40796136","term_text":"NP_955589"}}NP_955589. The numbers of the first and last residues in the sequence are given. For ASLV EnvA, the numbering begins after signal peptide cleavage; for all others, the numbering is from the start Met. Residues were aligned using the Clustal W PBIL multiple-alignment algorithm; output symbol designations are as follows: *, identical; :, strongly similar; ., weakly similar. The last residue of the N helix is denoted by an inverted triangle above the sequences. The chain reversal region as defined by Maerz, et al. (23) is underlined. The two unique His residues in the ASLV chain reversal region are denoted by arrows. Backbone sequences are in green. The three Cys residues of the CX₆CC motif are colored black and highlighted in yellow. The Gly-Gly pair is highlighted in gray. Mutated residues are underlined. Residues for which infectivity compared to the wild type is decreased by 1 to 2 log units (this study) or cell-cell fusion is 20% of wild type (23) are colored orange; those for which infectivity is decreased by ≥2 log units (this study) or cell-cell fusion is less than 10% of wild type (23) are colored red. Corresponding untested residues in the other sequences are colored blue. Residues that were tested but were nearly wild type in infectivity are left green. The His residue that affects the pH of ASLV entry is denoted with a red asterisk. (B) Postfusion structures of CX₆CC-bearing fusion subunits. The cores of the postfusion structures of the ZEBOV GP (1EBO), HTLV-1 Env (1MG1), and MoMLV Env (1MOF) are presented, together with a model of the postfusion ASLV TM core obtained by threading the corresponding EnvA sequence onto the ZEBOV GP postfusion structure using SwissPDB Viewer. Residues are colored as in panel A, except that the Cys residues of the CX₆CC motif are colored yellow and the blue residues in panel A are now cyan. Residues with relevance to this study are shown as spheres. The first Gly of the Gly-Gly pair is denoted by a star. The structures were rendered in Pymol.Maerz and coworkers (23) assessed the role of the chain reversal region for HTLV-1 Env (which contains a thiol exchange motif in its receptor binding subunit) by analyzing the ability of mutant HTLV-1 Env proteins to induce cell-cell fusion. Based on their findings, they presented a model in which a hydrophobic residue immediately after the CX₆CC motif is extracted from a hydrophobic pocket during fusion triggering and then a Gly-Gly pair preceding the CX₆CC motif acts as an “activation hinge” redirecting the C-terminal portion of the molecule so that it runs antiparallel to the N helix. It was further postulated that a salt bridge (between a charged residue within the CX₆CC motif and an Arg residue near the C terminus of the N helix), as well as hydrophobic interactions among aromatic residues at the C terminus of the N helix, stabilizes the hairpin (see Fig. ​Fig.66 in reference 23).Open in a separate windowFIG. 6.Location of critical residues at the base of the postfusion structures of ZEBOV GP2 and HTLV-1 TM. (A) An enlarged view of the base of the ZEBOV GP postfusion trimer of hairpins. The three chains are colored green. Residues on chain 1 are colored as in Fig. ​Fig.11 and, where applicable, shown as spheres. (B) Enlarged and reoriented view of the neighborhood of R587 (box B in panel A) showing its interaction with the backbone carbonyl of the second Cys of the CX₆CC motif on its own chain and N586 of a neighboring chain. (C) Enlarged and reoriented view of the interchain hydrophobic pocket (box C in panel A) showing the interactions of F592 with L594, W597, and I603 of a neighboring chain. Note that R587 and F592 interact with different neighboring chains. (D) Interaction of the HTLV-1 TM L382, which aligns with ZEBOV GP2 F592 (Fig. ​(Fig.1A),1A), with F384 (which aligns with L592) on a neighboring chain. The figures were generated in Pymol using PDB 1EBO (A to C) and PDB 1MG1 (D).In this study, we asked what elements of the model proposed by Maerz and coworkers (23) extend to CX₆CC motif-bearing fusion proteins that do not contain a thiol exchange motif in their receptor binding subunits. We did this by analyzing analogous and additional mutations in the Zaire ebolavirus (ZEBOV) GP and in the ASLV EnvA. Each system provided an advantage. The structure of ZEBOV GP is known in both its native and postfusion states (21, 40), but its fusion trigger has not yet been elucidated. Conversely, no structures are available for ASLV EnvA, but its “2-step” fusion-triggering mechanism has been characterized in detail (2, 6, 25, 28, 36). Our findings are generally consistent with the proposed model (23) and reveal requirements for analogous and additional residues in the chain reversal regions of ASLV EnvA and ZEBOV GP. In particular, we show, for EnvA, that the bulky hydrophobic residue following the CX₆CC motif is required for receptor-triggered target membrane association of the prehairpin, whereas specific residues in the chain reversal region are critical for forming the hairpin. In addition we identify a His residue, unique to the chain reversal region of EnvA, as being a critical determinant of the low-pH dependence of ASLV entry.     

Stigma structure and variation in Bromeliaceae—Neglected taxonomic characters

Three distinct types of stigma architecture, designated as “simple-erect,” “conduplicate-spiral”, and “convolute-blade” were identified for Bromeliaceae. Structure of the conduplicate-spiral and convolute-blade stigma types is highly complex and previously unreported. Stigma morphology, largely neglected in Tillandsioideae until recently, promises to yield new characters valuable in interpreting systematic relationships in this subfamily. Data indicate that the putatively plesiomorphic state (simple-erect) is retained in some members of all three subfamilies. We found convolute-blade-type stigmas only in Tillandsioideae, though not in all members of the subfamily. Our sample indicates that Tillandsioideae stigmas are more like those of Pitcairnioideae than of Bromelioideae, which is consistent with Smith’s hypothesis of phylogenetic proximity of Tillandsioideae and Pitcairnioideae. Stigma architecture is proving to be a rich source of data for phylogenetic reconstruction and for classification at several infrafamilial levels.     

Dynamics of phytoplankton in the Great Barrier Reef Lagoon

The characteristics of the phytoplankton crop in the centralregion of the Great Barrier Reef were analyzed through two annualcycles together with basic oceanographic parameters. Chlorophylla standing crop and primary production were size fractionatedinto nanoplankton and microplankton components. Community compositionwas determined using the Utermöhl settling technique andcommunity diversities estimated by the Shannon-Weaver equation.The data and analysis are the most comprehensive in existencefor the region, and the first detailed study since the 1928–29Great Barrier Reef expedition. A marked seasonal cycle was identified,contrary to most assumptions, closely associated with precipitationpatterns and nutrients introduced by land drainage. The regionwould rank as mesotrophic with some eutrophic areas in the innerreaches of the Lagoon. Oscillatoria spp. accounted for a largefraction of the majority of phytoplankton maxima and were inverselyrelated to diatom crop densities even under conditions favoringdiatom growth. Microplankton crop species diversities usuallydecreased during extended Oscillatoria blooms. The reduced diversitypersisted after the bloom suggesting that Oscillatoria spp.were the source of extracellular metabolites and/or decompositionproducts adversely influencing diatom microplankton.     

Phytoplankton biomass and primary production in semi-enclosed reef lagoons of the central Great Barrier Reef,Australia

Phytoplankton biomass and primary production rates within semi-enclosed reef lagoons of the central Great Barrier Reef were compared with adjacent shelf waters. Chlorophyll concentrations and surface primary production rates were usually higher in lagoons although seasonal differences were only significant during the summer. Nitrate concentrations were higher in lagoons than in shelf waters year-round. Nano- (<20 m size fraction) or pico-phytoplankton (<2 m size fraction) dominated phytoplankton biomass and production within reef lagoons throughout the year. Net phytoplankton (>10–20 m size fraction), however, were relatively more important in both reef lagoons and open shelf waters during the summer. Biomass-specific production within lagoons (range 41–90 mg C mg chl^–1 day^–1) was high, regardless of season. Lagoonal phytoplankton production (range 0.2–1.6 g C m^–2 day^–1) was directly correlated with standing crop and inversely related to lagoon flushing rates. Phytoplankton blooms develop within GBR reef lagoons during intermittent calm periods when water residence times exceed phytoplankton generation times.     

Activation of morphogenesis and proliferation by low specificity chemical effectors

Activation of highly specific biochemical processes by simple chemical agents is demonstrated for morphogenesis (anlage and development of female gametophyte in cereal) and mitosis (in cell cultures and animal and plant tissues). The effects of these agents are tissue-specific. Structure--activity relationship is analyzed in this group of compounds. Thus, the phenomenon reveals the exact pathways of the influence of allelopathic and anthropogenic chemical agents on evolution of plant biocenoses.     

Gene regulatory divergence among species estimated by altered developmental patterns in interspecific hybrids

Disturbances in the schedules of gene expression in developing interspecific fish hybrids have been used to draw inferences about the extent of gene regulatory divergence between species and about the degree to which this gene regulatory divergence is correlated with structural gene divergence, as estimated by genetic distance. Sperm from each of 10 different species representing six genera within the family Centrarchidae was used to fertilize eggs of the Florida largemouth bass (Micropterus salmoides floridanus). The genetic distances (D; Nei 1978) between the parental species used to form the hybrids ranged from 0.133 to 0.974. The developmental success and temporal patterns of gene expression of each of the hybrids were compared with those of the Florida largemouth bass. As the genetic distance between the paternal species and the Florida largemouth bass increased, there was a general decline in developmental success in the hybrid embryos as demonstrated by the observed reductions in the percentage of hatching and by progressively earlier and more extensive morphological abnormalities. Concomitantly, progressively more marked alterations in developmental schedules of expression of 15 enzyme loci occurred in the hybrids as the genetic distance between parental species increased. However, observed deviations from this trend for a few species may represent an uncoupling of the rates and modes of evolution of structural genes from those for genes regulating developmental processes.