Host-selected amino acid changes at the sialic acid binding pocket of the parvovirus capsid modulate cell binding affinity and determine virulence

The role of receptor recognition in the emergence of virulent viruses was investigated in the infection of severe combined immunodeficient (SCID) mice by the apathogenic prototype strain of the parvovirus minute virus of mice (MVMp). Genetic analysis of isolated MVMp viral clones (n = 48) emerging in mice, including lethal variants, showed only one of three single changes (V325M, I362S, or K368R) in the common sequence of the two capsid proteins. As was found for the parental isolates, the constructed recombinant viruses harboring the I362S or the K368R single substitutions in the capsid sequence, or mutations at both sites, showed a large-plaque phenotype and lower avidity than the wild type for cells in the cytotoxic interaction with two permissive fibroblast cell lines in vitro and caused a lethal disease in SCID mice when inoculated by the natural oronasal route. Significantly, the productive adsorption of MVMp variants carrying any of the three mutations selected through parallel evolution in mice showed higher sensitivity to the treatment of cells by neuraminidase than that of the wild type, indicating a lower affinity of the viral particle for the sialic acid component of the receptor. Consistent with this, the X-ray crystal structure of the MVMp capsids soaked with sialic acid (N-acetyl neuraminic acid) showed the sugar allocated in the depression at the twofold axis of symmetry (termed the dimple), immediately adjacent to residues I362 and K368, which are located on the wall of the dimple, and approximately 22 A away from V325 in a threefold-related monomer. This is the first reported crystal structure identifying an infectious receptor attachment site on a parvovirus capsid. We conclude that the affinity of the interactions of sialic-acid-containing receptors with residues at or surrounding the dimple can evolutionarily regulate parvovirus pathogenicity and adaptation to new hosts.     

Four amino acids in the alpha subunits determine the gamma-aminobutyric acid sensitivities of GABAA receptor subtypes

GABA(A) receptors, mediators of fast inhibitory neurotransmission, are heteropentameric assemblies from a large array of subunits. Differences in the sensitivity of receptor subtypes to endogenous GABA may permit subunit-dependent finely tuned responsiveness to the same GABAergic inputs. Using both radioligand binding and electrophysiology combined with mutagenesis, we identified a domain of four amino acids within the alpha subunits that mediates the distinct sensitivities to GABA allowing their selective switch between alphabeta3gamma2 combinations. Replacing this domain in alpha3 by the corresponding segments of alpha1-alpha5 resulted in mutant receptors displaying the GABA EC(50) values of the respective wild-type receptors. Vice versa, the alpha3 motif forced the low sensitivity to GABA of alpha3 upon alpha1beta3gamma2, alpha4beta3gamma2, and alpha5beta3gamma2. Binding of the GABA agonist [(3)H]muscimol was not affected by the exchange of the motif between alpha1 and alpha3 subunits. Thus, the equilibrium binding pocket is maintained upon replacement of the four amino acids. Taken together our data suggest that the identified motifs contribute to a structure involved in the transduction of the binding signal rather than to the binding itself.     

Multiple amino acids in the capsid structure of canine parvovirus coordinately determine the canine host range and specific antigenic and hemagglutination properties.

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are over 98% similar in DNA sequence but have specific host range, antigenic, and hemagglutination (HA) properties which were located within the capsid protein gene. In vitro mutagenesis and recombination were used to prepare 16 different recombinant genomic clones, and viruses derived from those clones were analyzed for their in vitro host range, antigenic, and HA properties. The region of CPV from 59 to 91 map units determined the ability to replicate in canine cells. A complex series of interactions was observed among the individual sequence differences between 59 and 73 map units. The canine host range required that VP2 amino acids (aa) 93 and 323 both be the CPV sequence, and those two CPV sequences introduced alone into FPV greatly increased viral replication in canine cells. Changing any one of aa 93, 103, or 323 of CPV to the FPV sequence either greatly decreased replication in canine cells or resulted in an inviable plasmid. The Asn-Lys difference of aa 93 alone was responsible for the CPV-specific epitope recognized by monoclonal antibodies. An FPV-specific epitope was affected by aa 323. Amino acids 323 and 375 together determined the pH dependence of HA. Amino acids involved in the various specific properties were all around the threefold spikes of the viral particle.     

Retroviral capsid determinants of Fv1 NB and NR tropism

The specificity determinants for susceptibility to resistance by the Fv1 n and b alleles map to amino acid 110 of the murine leukemia virus CA protein. To study the interaction between Fv1 and CA, we examined changes in CA resulting in the loss of susceptibility to Fv1 resistance in naturally occurring NB- and NR-tropic viruses. A variety of amino acid changes affecting Fv1 tropism were identified, at CA positions 82, 92 to 95, 105, 114, and 117, and they all were mapped to the apparent exterior of virion-associated CA. These amino acids may form a binding surface for Fv1.     

The molecular architecture of an insect midgut brush border cytoskeleton.

The cytoskeletal apparatus of the vertebrate intestinal brush border (BB) has served as a model system for the actin-based cytoskeleton of nonmuscle cells. In this study, we examine the structural organization and molecular architecture of the BB cytoskeleton expressed in the midgut of lepidopteran larvae, Manduca sexta. Electron microscopy of the midgut of the 5th instar larvae revealed enterocytes with an apical BB surface comparable to that in the vertebrate intestine, with both microvillar (MV) and terminal web (TW) domains, the latter defined by a zone of organelle exclusion directly beneath the MV. As reported previously for the larval dragon fly, the MV contain a bundle of actin filaments, as determined by staining with rhodamine phalloidin (Kukulies, J., et al., Protoplasma 121, 157-162 (1984)) and heavy meromyosin decoration (Komnick, H., J. Kukulies, Zoomorphology 107, 241-253 (1987)). Two-dimensional gel analysis revealed the presence of multiple isoelectric variants of actin with the major isoform corresponding to the non-muscle actin isoform II, expressed in Drosophila. Like the vertebrate BB, the Manduca BB can be isolated intact from enterocytes by mechanical shear. Immunochemical analysis of isolated BB fractions or whole homogenates of midgut revealed proteins of appropriate molecular weight immunoreactive with antibodies to the MV core proteins: BB myosin I, villin and fimbrin, and the TW components: spectrin, myosin II and tropomyosin. Immunocytochemical localization of a subset of these proteins at the light microscopic (spectrin) and electron microscopic (actin, villin, spectrin, myosin II, and tropomyosin) level reveals that the molecular architecture of the Manduca BB cytoskeleton is homologous to that found in vertebrates.     

Primary culture of insect midgut cells

This protocol describes the preparation of primary cell cultures from Lepidopteran midgut. These cultures have been used to identify factors that control midgut growth and differentiation, cell responses to these factors, effects of toxins on midgut growth, and the regulation of cell physiology. The protocol is divided into (1) procedures for cell collection, (2) composition of the culture, and (3) assay methods used for cell health, proliferation, and differentiation. Collection and setup require 4–6 h. Once established, a culture can survive several months at 25°C, be kept a year or longer at 4°C, or be frozen for indefinite storage.     

Brush-border formation in the midgut of an insect,Calliphora erythrocephala meigen

Summary The embryonic development of the brush-border of anterior midgut cells of Calliphora was studied by electron microscopy. Dense surface-forming vesicles, as described by Bonneville (1970), are found prior to microvillus formation. These dense vesicles provide membranous and coating material for the moulding of the microvilli. The number of dense vesicles increases rapidly to a maximum just before brush-border formation, after which it decreases very rapidly, accompanied by an increase in the number of microvilli. Formation of microvilli proceeds in essentially the same way as in Xenopus. First, some of the vesicles fuse with the apical cell membrane, resulting in an increase of the cell surface, part of which is coated with filamentous material deriving from the dense vesicles. This in turn leads to bulging, and short irregular microvilli appear. These are erected and elongated.Prefabricated tubular elements are believed to play a part in this erection and elongation, probably due to the unwinding of spirally coiled strands.Microvillus formation proper lasts 2 to 3 hours in Calliphora. Almost the entire amount of membranous and coating material is prefabricated prior to the formation of microvilli.     

Subsite substrate specificity of midgut insect chymotrypsins

Insect chymotrypsins are distinctively sensitive to plant protein inhibitors, suggesting that they differ in subsite architecture and hence in substrate specificities. Purified digestive chymotrypsins from insects of three different orders were assayed with internally quenched fluorescent oligopeptides with three different amino acids at P1 (Tyr, Phe, and Leu) and 13 amino acid replacements in positions P1', P2, and P3. The binding energy (DeltaG(s), calculated from K(m) values) and the activation energy (DeltaG(T)++, determined from k(cat)/K(m) values) were calculated. The hydrophobicities of each subsite were calculated from the efficiency of hydrolysis of the different amino acid replacements at that subsite. The results showed that except for S1, the other subsites (S2, S3, and S1') vary among chymotrypsins. This result contrasts with insect trypsin data that revealed a trend along evolution, putatively associated with resistance to plant inhibitors. In spite of those differences, the data suggested that in lepidopteran chymotrypsins S2 and S1' bind the substrate ground state, whereas only S1' binds the transition state, supporting aspects of the present accepted mechanism of catalysis.     

Active transport of potassium by insect midgut

Midguts isolated from fifth-instar larvae of the insert Hyalophora cecropia actively transport potassium in the hemolymph to lumen direction. No specific co- or counter-ion is required and other alkali ions are actively transported in the same direction as potassium. No specific inhibitor of K+ active transport has been found although most metabolic inhibitors reduce the net K+ flux, potential difference, and short-circuit current to zero. The site of the epithelial active transport of potassium has been identified by microelectrode measurements of intracellular resistance as the goblet cell, one of the two major cell types in the single-layered midgut. Under certain external conditions, the neighboring columnar cells are added to the goblet cell transport route through intercellular electrical coupling that occurs after application of external depolarizing current. Tracer influx kinetics were used to establish that the fraction of exchangeable K involved in the transport route under open-circuit conditions is small, corresponding to a goblet cell pathway. Under depolarizing current conditions, virtually all of the exchangeable midgut K is involved in the transport route, corresponding to a goblet and columnar cell pathway. These results and others are used to construct a model for rheogenic active transport of potassium in insect midgut.     

Absorption pathways of amino acids in the midgut of Manduca sexta larvae

The unidirectional transport of leucine and aspartic acid by midgut epithelium cells of the tobacco ornworm, Manduca sexta, was investigated using brush border membrane vesicles. Both K⁺-stimulated and K⁺-insensitive transport pathways for these two amino acids were identified. Kinetic analyses and profiles of cis-inhibition by other amino acids demonstrated that leucine and aspartic acid are transported by separate pathways and exhibit different sensitivity to potassium ion.     

The intracellular pathway and kinetics of digestive enzyme secretion in an insect midgut cell

The opaque zone cells of the midgut of the stablefly, Stomoxys calcitrans display a cyclical series of ultrastructural events in response to feeding, which it has been suggested are related to the synthesis and secretion of digestive enzymes. These cells have been studied in vivo using a combination of biochemical, morphometric and electron microscopical autoradiographic techniques. The cyclical nature, timing and relationship of the ultrastructural events to enzyme secretion has been confirmed. The autoradiographic data presented is in good agreement with the classical synthetic pathway for exported proteins. The kinetics of the cellular process have been described: transfer of newly synthesized product from the rough endoplasmic reticulum to Golgi apparatus begins ca. 13-14 min after labelling of the fly and from the Golgi apparatus to secretory granules after ca. 24-26 min. Secretion of this newly synthesized material begins before 60 min and possibly as early as 30 min after labelling of the fly. The data are discussed in relation to the comparable studies in other tissues.     

Timing of transmission and the evolution of virulence of an insect virus

We used the nuclear polyhedrosis virus of the gypsy moth, Lymantria dispar, to investigate whether the timing of transmission influences the evolution of virulence. In theory, early transmission should favour rapid replication and increase virulence, while late transmission should favour slower replication and reduce virulence. We tested this prediction by subjecting one set of 10 virus lineages to early transmission (Early viruses) and another set to late transmission (Late viruses). Each lineage of virus underwent nine cycles of transmission. Virulence assays on these lineages indicated that viruses transmitted early were significantly more lethal than those transmitted late. Increased exploitation of the host appears to come at a cost, however. While Early viruses initially produced more progeny, Late viruses were ultimately more productive over the entire duration of the infection. These results illustrate fitness trade-offs associated with the evolution of virulence and indicate that milder viruses can obtain a numerical advantage when mild and harmful strains tend to infect separate hosts.     

Three-dimensional structure determination of capsid of Aedes albopicus C6/36 cell densovirus

The three-dimensional structure of capsid of Aedes albopictus C6/36 densovirus was determined to 14-(A) resolution by electron cryomicroscopy and computer reconstruction. The triangulation number of the capsid is 1. There are 12 holes in each triangular face and a spike on each 5-fold vertex. The validity of the capsid and nucleic acid densities in the reconstructions was discussed.     

The amino acid/K symporters for neutral amino acids along the midgut of lepidopteran larvae: Functional differentiations

The functional properties of the K⁺-dependent symporter for neutral amino acids have been investigated in brush border membrane vesicles prepared from the anterior, middle and posterior portions corresponding to the three morphologically distinguishable regions of the midgut of Bombyx mori larvae. An intravesicular accumulation of leucine was driven by a K⁺-gradient in the three preparations, but vesicles from the posterior tract displayed much higher uptake and accumulation values. Kinetic analysis of leucine uptake, performed in experimental conditions which mimic as closely as feasible experimentally those occurring in vivo (Δψ = −90 mV, pH_in7.2/pH_out8.7, [K⁺]_out100 mM), evidenced that the affinity for the amino acid was similar along the midgut (150 μM), but V_max in the posterior region was more than 11-fold higher than that of the anterior-middle tract (11.3 ± 0.7 and 0.98 ± 0.07 nmol/7s/mg protein, respectively). Leucine uptake was remarkably influenced by extravesicular pH and by Δψ only in vesicles from the posterior midgut: a lowering of pH to 7.2 caused a sevenfold increase of K_m, whereas in the absence of Δψ, V_max decreased threefold. The selectivity sequence for the alkali cations was somewhat different in the two midgut regions, but K⁺ remained the most effective. In the posterior midgut, the selectivity for K⁺ was greatly enhanced when a transmembrane electrical potential was present. Leucine kinetics as a function of external potassium concentration was hyperbolic in the posterior and sigmoidal in the anterior-middle part. Inhibition of leucine uptake induced by a 20-fold excess of different amino acids suggested the presence in both midgut tracts of a broad specificity system for neutral amino acids, with many-but not all-features in common with the B^o system of mammal intestinal and renal epithelial brush borders. However, there are differences between the two midgut regions as regard to the ability of the symporters to recognize the different amino acids, which concern the side chain and the presence of the aromatic ring. Altogether these data suggest that two kinds of symporters for neutral amino acids, with different functional properties, are expressed in the anterior-middle and posterior regions of the lepidopteran midgut.     

Use of sequence-specific tri-block copolymers to determine the helix-forming tendencies of amino acids

In order to examine the helix-coil transition of water-insoluble polypeptides, without requiring interspersion of charged or polar residues within the sequence, a tri-block copolymer strategy has been developed to determine the helix propensity of amino acids in short (15-residue) peptides. The method is also well suited to evaluate specific interactions that contribute to helix formation. In this approach, measurement is made of the helix content of the central block of tri-block copolymers of the type Lys₁₅-X-Lys₁₅, where X is the 15-residue peptide whose helix content is being investigated. The suitability of tri-block copolymers of this type has been verified experimentally by using the water-soluble peptide (Ala₂GlnAla₂)₃ as the central block. The CD spectrum of the central block in the tri-block copolymer and of Ac-(Ala₂GlnAla₂)₃-NH₂ are indistinguishable within experimental error. © 1996 John Wiley & Sons, Inc.     

The hemagglutinin-neuraminidase protein of Newcastle disease virus determines tropism and virulence

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a crucial role in the process of infection. However, the exact contribution of the HN gene to NDV pathogenesis is not known. In this study, the role of the HN gene in NDV virulence was examined. By use of reverse genetics procedures, the HN genes of a virulent recombinant NDV strain, rBeaudette C (rBC), and an avirulent recombinant NDV strain, rLaSota, were exchanged. The hemadsorption and neuraminidase activities of the chimeric viruses showed significant differences from those of their parental strains, but heterotypic F and HN pairs were equally effective in fusion promotion. The tissue tropism of the viruses was shown to be dependent on the origin of the HN protein. The chimeric virus with the HN protein derived from the virulent virus exhibited a tissue predilection similar to that of the virulent virus, and vice versa. The chimeric viruses with reciprocal HN proteins either gained or lost virulence, as determined by a standard intracerebral pathogenicity index test of chickens and by the mean death time in chicken embryos (a measure devised to classify these viruses), indicating that virulence is a function of the amino acid differences in the HN protein. These results are consistent with the hypothesis that the virulence of NDV is multigenic and that the cleavability of F protein alone does not determine the virulence of a strain.     

Increased insect virulence in Beauveria bassiana strains overexpressing an engineered chitinase

Entomopathogenic fungi are currently being used for the control of several insect pests as alternatives or supplements to chemical insecticides. Improvements in virulence and speed of kill can be achieved by understanding the mechanisms of fungal pathogenesis and genetically modifying targeted genes, thus improving the commercial efficacy of these biocontrol agents. Entomopathogenic fungi, such as Beauveria bassiana, penetrate the insect cuticle utilizing a plethora of hydrolytic enzymes, including chitinases, which are important virulence factors. Two chitinases (Bbchit1 and Bbchit2) have previously been characterized in B. bassiana, neither of which possesses chitin-binding domains. Here we report the construction and characterization of several B. bassiana hybrid chitinases where the chitinase Bbchit1 was fused to chitin-binding domains derived from plant, bacterial, or insect sources. A hybrid chitinase containing the chitin-binding domain (BmChBD) from the silkworm Bombyx mori chitinase fused to Bbchit1 showed the greatest ability to bind to chitin compared to other hybrid chitinases. This hybrid chitinase gene (Bbchit1-BmChBD) was then placed under the control of a fungal constitutive promoter (gpd-Bbchit1-BmChBD) and transformed into B. bassiana. Insect bioassays showed a 23% reduction in time to death in the transformant compared to the wild-type fungus. This transformant also showed greater virulence than another construct (gpd-Bbchit1) with the same constitutive promoter but lacking the chitin-binding domain. We utilized a strategy where genetic components of the host insect can be incorporated into the fungal pathogen in order to increase host cuticle penetration ability.