Amino acid substitutions at positions 242, 255 and 268 in rabies virus glycoprotein affect spread of viral infection

Rabies virus Nishigahara strain kills adult mice after intracerebral inoculation, whereas the derivative RC‐HL strain does not. It has previously been reported by us that the R(G 242/255/268) strain, in which amino acids at positions 242, 255 and 268 on the G protein have been replaced by those from the Nishigahara strain in the genetic background of the RC‐HL strain, kills adult mice. This indicates that these three amino acids of G protein are important for pathogenicity of the Nishigahara strain. In order to obtain insights into the mechanism by which these amino acids affect pathogenicity, in this study spread of viral infection and apoptosis‐inducing ability of the attenuated RC‐HL strain and the virulent R(G 242/255/268) strain were compared. RC‐HL infection spread less efficiently in the mouse brain than did R(G 242/255/268) infection. However, the apoptosis‐inducing abilities of both viruses were almost identical, as shown by both in vitro and in vivo experiments. It was demonstrated that cell‐to‐cell spread of RC‐HL strain was less efficient than that of R(G 242/255/268) strain in mouse neuroblastoma cells. These results indicate that the three amino acid substitutions affect efficiency of cell‐to‐cell spread but not apoptosis‐inducing ability, probably resulting in the distinct distributions of RC‐HL and R(G 242/255/268) strain‐infected cells in the mouse brain and, consequently, the different pathogenicities of these strains.     

Modeling a sialic acid binding pocket in the external loops of JC virus VP1

JC virus (JCV) is a common human polyomavirus that infects over 70% of the population worldwide. JCV has a restricted cell tropism that is caused partly by the initial interaction between the virus and sialic acid-containing host cell receptors. To identify the molecular interactions between the virus and its cellular receptor, we used a combined approach of site-directed mutagenesis and homology-based molecular modeling. A model of the major viral capsid protein VP1 based on sequence alignment with other closely related polyomaviruses allowed us to target specific amino acids in the extracellular loops of VP1 for mutagenesis. An analysis of the growth rates of 17 point mutants led to the identification of VP1 amino acids that are critical in virus-host cell receptor interactions. Molecular dynamics simulations were then used to build and confirm a model of the interaction between VP1 and the sialic acid component of the JCV receptor.     

Specificity and affinity of sialic acid binding by the rhesus rotavirus VP8* core

Nuclear magnetic resonance spectroscopy demonstrates that the rhesus rotavirus hemagglutinin specifically binds alpha-anomeric N-acetylneuraminic acid with a K(d) of 1.2 mM. The hemagglutinin requires no additional carbohydrate moieties for binding, does not distinguish 3' from 6' sialyllactose, and has approximately tenfold lower affinity for N-glycolylneuraminic than for N-acetylneuraminic acid. The broad specificity and low affinity of sialic acid binding by the rotavirus hemagglutinin are consistent with this interaction mediating initial cell attachment prior to the interactions that determine host range and cell type specificity.     

Amino acid sequence of sialic acid binding lectin from frog (Rana catesbeiana) eggs

The complete amino acid sequence of sialic acid binding lectin from frog (Rana catesbeiana) egg is presented. The 111-residue sequence was determined by the analysis of peptides generated by digestion of the S-carboxymethylated protein with Achromobacter protease I, chymotrypsin, or cyanogen bromide. The sequence is unique and not homologous to any known protein sequence. The protein may represent a new type of lectin.     

Persistent vesicular stomatitis virus infection mediates base substitutions in viral RNA termini.

We have sequenced (via a product RNA) the 3' RNA terminus of a defective interfering particle that was generated from the standard virus isolated from a culture of BHK-21 cells persistently infected with vesicular stomatitis virus for over 5 years. By hybridization and RNA sequencing, seven mutations were identified in the 46 nucleotides at the terminus of this defective-interfering-particle RNA. It is likely that these mutations are a reflection of altered protein-nucleic acid interactions that the virus has evolved to maintain its persistently infected carrier state in vitro.     

Direct correlation between sialic acid binding and infection of cells by two human polyomaviruses (JC virus and BK virus)

For the human polyomaviruses JC virus (JCV) and BK virus (BKV), the first step to a successful infection involves binding to sialic acid moieties located on the surfaces of host cells. By stripping and then reconstituting specific sialic acid linkages on host cells, we show that JCV uses both α(2,3)-linked and α(2,6)-linked sialic acids on N-linked glycoproteins to infect cells. For both JCV and BKV, the sialic acid linkages required for cell surface binding directly correlate with the linkages required for infection. In addition to sialic acid linkage data, these data suggest that the third sugar from the carbohydrate chain terminus is important for virus binding and infection.     

Amino acid substitutions that reduce the affinity of penicillin-binding protein 3 of Escherichia coli for cephalexin

The location of amino acid substitutions that allow an enzyme to discriminate between the binding of its normal substrate and a substrate analogue may be used to identify regions of the polypeptide that fold to form the substrate binding site. We have isolated a large number of cephalexin-resistant mutants of Escherichia coli in which the resistance is due to the production of altered forms of penicillin-binding protein 3 that have reduced affinity for the antibiotic. Using three mutagens, and a variety of selection procedures, we obtained only five classes of mutants which could be distinguished by their patterns of cross-resistance to other beta-lactam antibiotics. The three classes of mutants that showed the highest levels of resistance to cephalexin were cross-resistant to several other cephalosporins but not to penicillins or to the monobactam, aztreonam. The penicillin-binding protein 3 gene from 46 independent mutants was cloned and sequenced. Each member of the five classes of cephalexin-resistant mutants had the same amino acid substitution in penicillin-binding protein 3. The mutants that showed the highest levels of resistance to cephalexin had alterations of either Thr-308 to Pro, Val-344 to Gly, or Asn-361 to Ser. The Thr-308 to Pro substitution had occurred within the beta-lactam-binding site since the adjacent residue (Ser-307) has been shown to be acylated by benzylpenicillin. The Asn-361 to Ser change occurred in a region that showed substantial similarity to regions in both penicillin-binding protein 1A and 1B and may also define a residue that is located within the beta-lactam-binding site in the three-dimensional structure of the enzyme.     

Amino acid residues involved in substrate binding and catalysis in an insect digestive beta-glycosidase

A beta-glycosidase (M(r) 50000) from Spodoptera frugiperda larval midgut was purified, cloned and sequenced. It is active on aryl and alkyl beta-glucosides and cellodextrins that are all hydrolyzed at the same active site, as inferred from experiments of competition between substrates. Enzyme activity is dependent on two ionizable groups (pK(a1)=4.9 and pK(a2)=7.5). Effect of pH on carbodiimide inactivation indicates that the pK(a) 7.5 group is a carboxyl. k(cat) and K(m) values were obtained for different p-nitrophenyl beta-glycosides and K(i) values were determined for a range of alkyl beta-glucosides and cellodextrins, revealing that the aglycone site has three subsites. Binding data, sequence alignments and literature beta-glycosidase 3D data supported the following conclusions: (1) the groups involved in catalysis were E(187) (proton donor) and E(399) (nucleophile); (2) the glycone moiety is stabilized in the transition state by a hydrophobic region around the C-6 hydroxyl and by hydrogen bonds with the other equatorial hydroxyls; (3) the aglycone site is a cleft made up of hydrophobic amino acids with a polar amino acid only at its first (+1) subsite.     

Involvement of interaction between viral VP466 and host tropomyosin proteins in virus infection in shrimp

The accumulating evidence indicates that the viral structural proteins play critical roles in virus infection. However, the interaction between the viral structural protein and host cytoskeleton protein in virus infection remains to be addressed. In this study, the viral VP466 protein, one of the major structural proteins of shrimp white spot syndrome virus (WSSV), was characterized. The results showed that the suppression of VP466 gene expression led to the inhibition of WSSV infection in shrimp, indicating that the VP466 protein was required in virus invasion. It was found that the VP466 protein was interacted with the host cytoskeleton protein tropomyosin. As documented, the VP466–tropomyosin interaction facilitated the WSSV infection. Therefore our findings revealed a novel molecular mechanism in the virus invasion to its host, which would be helpful to better understand the molecular events in virus infection in invertebrate.     

Involvement of interaction between viral VP466 and host tropomyosin proteins in virus infection in shrimp

The C3 component of complement has different roles in kidney disease and its local production in donor kidney may affect allograft function and rejection after organ transplantation. A single base substitution in c3 gene (rs2230199), defines two common allelic variants with different mobility on gel electrophoresis: S (Slow) and F (Fast). In order to evaluate the effect of this polymorphism on acute renal allograft rejection, one hundred samples of donor and recipients were collected and genotyping was done by PCR-RFLP method. The allelic frequencies were: C3S=0.791, C3F=0.209. There was no significant association between recipient's genotype and acute rejection (p value<0.05). No correlation between donor genotype and acute rejection was also present. Patients were divided into four groups, according to the recipient and donor genotypes: SS recipients and FS or FF donor, SS recipient and donor, FF or FS recipient and SS donor and FS or FF recipient and donor. There was no significant difference in rate of acute rejection between groups. Although the results didn't find any association between C3 complement polymorphisms and acute allograft rejection, there was no acute rejection in FS or FF donors and SS recipient group.     

Amino acid residues constituting the agonist binding site of the human P2X3 receptor

Homomeric P2X3 receptors are present in sensory ganglia and participate in pain perception. Amino acid (AA) residues were replaced in the four supposed nucleotide binding segments (NBSs) of the human (h) P2X3 receptor by alanine, and these mutants were expressed in HEK293 cells and Xenopus laevis oocytes. Patch clamp and two-electrode voltage clamp measurements as well as the Ca(2+) imaging technique were used to compare the concentration-response curves of the selective P2X1,3 agonist α,β-methylene ATP obtained at the wild-type P2X3 receptor and its NBS mutants. Within these NBSs, certain Gly (Gly-66), Lys (Lys-63, Lys-176, Lys-284, Lys-299), Asn (Asn-177, Asn-279), Arg (Arg-281, Arg-295), and Thr (Thr-172) residues were of great importance for a full agonist response. However, the replacement of further AAs in the NBSs by Ala also appeared to modify the amplitude of the current and/or [Ca(2+)](i) responses, although sometimes to a minor degree. The agonist potency decrease was additive after the simultaneous replacement of two adjacent AAs by Ala (K65A/G66A, F171A/T172A, N279A/F280A, F280A/R281A) but was not altered after Ala substitution of two non-adjacent AAs within the same NBS (F171A/N177A). SDS-PAGE in the Cy5 cell surface-labeled form demonstrated that the mutants appeared at the cell surface in oocytes. Thus, groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the hP2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor.     

Effects of amino acid substitutions in the VP2 B-C loop on antigenicity and pathogenicity of serotype Asia1 foot-and-mouth disease virus

ABSTRACT: BACKGROUND: Foot-and-mouth disease virus (FMDV) exhibits a high degree of antigenic variability. Studies of the antigenic diversity and determination of amino acid changes involved in this diversity are important to the design of broadly protective new vaccines. Although extensive studies have been carried out to explore the molecular basis of the antigenic variation of serotype O and serotype A FMDV, there are few reports on Asia1 serotype FMDV. METHODS: Two serotype Asia1 viruses, Asia1/YS/CHA/05 and Asia1/1/YZ/CHA/06, which show differential reactivity to the neutralizing monoclonal antibody (nMAb) 1B4, were subjected to sequence comparison. Then a reverse genetics system was used to generate mutant versions of Asia1/YS/CHA/05 followed by comparative analysis of the antigenicity, growth property and pathogenicity in the suckling mice. RESULTS: Three amino acid differences were observed when the structural protein coding sequences of Asia1/1/YZ/CHA/06 were compared to that of Asia1/YS/CHA/05. Site-directed mutagenesis and Immunofluorescence analysis showed that the amino acid substitution in the B-C loop of the VP2 protein at position 72 is responsible for the antigenic difference between the two Asia1 FMDV strains. Furthermore, alignment of the amino acid sequences of VP2 proteins from serotype Asia1 FMDV strains deposited in GenBank revealed that most of the serotype Asia1 FMDV strains contain an Asn residue at position 72 of VP2. Therefore, we constructed a mutant virus carrying an Asp-to-Asn substitution at position 72 and named it rD72N. Our analysis shows that the Asp-to-Asn substitution inhibited the ability of the rD72N virus to react with the MAb 1B4 in immunofluorescence and neutralization assays. In addition, this substitution decreased the growth rate of the virus in BHK-21 cells and decreased the virulence of the virus in suckling mice compared with the Asia1/YS/CHA/05 parental strain. CONCLUSIONS: These results suggest that variations in domains other than the hyper variable VP1 G-H loop (amino acid 140 to 160) are relevant to the antigenic diversity of FMDV. In addition, amino acid substitutions in the VP2 influenced replicative ability and virulence of the virus. Thus, special consideration should be given to the VP2 protein in research on structure-function relationships and in the development of an FMDV vaccine.     

Second sialic acid binding site in Newcastle disease virus hemagglutinin-neuraminidase: implications for fusion

Paramyxoviruses are the leading cause of respiratory disease in children. Several paramyxoviruses possess a surface glycoprotein, the hemagglutinin-neuraminidase (HN), that is involved in attachment to sialic acid receptors, promotion of fusion, and removal of sialic acid from infected cells and progeny virions. Previously we showed that Newcastle disease virus (NDV) HN contained a pliable sialic acid recognition site that could take two states, a binding state and a catalytic state. Here we present evidence for a second sialic acid binding site at the dimer interface of HN and present a model for its involvement in cell fusion. Three different crystal forms of NDV HN now reveal identical tetrameric arrangements of HN monomers, perhaps indicative of the tetramer association found on the viral surface.     

Amino acid substitutions within the analogous nucleotide binding loop (P-loop) of aminoglycoside 3'-phosphotransferase-II

• 1.1. Oligonucleotide-directed mutagenesis of APH(3')-II was used to investigate the functions of key amino acids in the P-loop analogous motif of the enzyme.
• 2.2. The mutations of Gly205 → Glu, Gly210 → Ala and Arg211 → Pro considerably reduced the resistance of the resulting strains to KM and to related drugs, e.g. G418.
• 3.3. Similarly, enzyme activity in the crude extracts of these mutants was substantially reduced as well as the enzyme's affinity for Mg²⁺ ATP.
• 4.4. Alternatively substitutions at a highly conserved basic residue (Arg211 → Lys and Arg211 → His) were not sufficient for the enzyme to sustain the activity at a level comparable to that of the wildtype.
• 5.5. Moreover, an Arg211 → His mutation drastically reduced affinity of the enzyme for Mg²⁺ ATP.
• 6.6. This argues the importance of Arg211 residue in contributing to the formation of the P-loop structure in addition to its involvement in phosphoryl transfer reaction.
• 7.7. Computer analysis of the secondary structure predicted that the APH(3')-II loop connects a β -strand to an α-helix and that the above mutations caused varying degrees of structural distortions at the corresponding regions of the protein.

     

The histochemical demonstration of sialic acid residues in pancreatic islets

Summary Using a modified colloidal iron reaction in connection with neuraminidase extraction test 3 different sialic acid-containing components have been demonstrated in pancreatic islets comprising golgi region and glycocalyx layer of islet cells and intrainsular capillary walls. The colloidal iron positive cationophilia increased markedly after treatment with alkali; an effect which might be due to deesterification, thus exposing additional free carbonyl groups of sialic acid residues.     

Amino acid substitutions in the S2 subunit of mouse hepatitis virus variant V51 encode determinants of host range expansion

We previously described mouse hepatitis virus (MHV) variant V51 derived from a persistent infection of murine DBT cells with an expanded host range (R. S. Baric, E. Sullivan, L. Hensley, B. Yount, and W. Chen, J. Virol. 73:638-649, 1999). Sequencing of the V51 spike gene, the mediator of virus entry, revealed 13 amino acid substitutions relative to the originating MHV A59 strain. Seven substitutions were located in the amino-terminal S1 cleavage subunit, and six were located in the carboxy-terminal S2 cleavage subunit. Using targeted RNA recombination, we constructed a panel of recombinant viruses to map the mediators of host range to the six substitutions in S2, with a subgroup of four changes of particular interest. This subgroup maps to two previously identified domains within S2, a putative fusion peptide and a heptad repeat, both conserved features of class I fusion proteins. In addition to an altered host range, V51 displayed altered utilization of CEACAM1a, the high-affinity receptor for A59. Interestingly, a recombinant with S1 from A59 and S2 from V51 was severely debilitated in its ability to productively infect cells via CEACAM1a, while the inverse recombinant was not. This result suggests that the S2 substitutions exert powerful effects on the fusion trigger that normally passes from S1 to S2. These novel findings play against the existing data that suggest that MHV host range determinants are located in the S1 subunit, which harbors the receptor binding domain, or involve coordinating changes in both S1 and S2. Mounting evidence also suggests that the class I fusion mechanism may possess some innate plasticity that regulates viral host range.     

Amino acid substitutions in alphaA and alphaC of Cyt2Aa2 alter hemolytic activity and mosquito-larvicidal specificity

Cyt2Aa2 produced by Bacillus thuringiensis subsp. darmstadiensis exhibits in vitro cytolytic activity against broad range of cells but shows specific in vivo toxicity against larvae of Dipteran insects. To investigate the role of amino acids in alphaA and alphaC of this toxin, 3 single-point mutants (A61C, S108C and V109A) were generated. All 3 mutant proteins were highly produced as inclusion bodies that could be solubilized and activated by proteinase K similar to that of the wild type. Hemolytic activity of A61C and S108C mutants was significantly reduced whereas the V109A mutant showed comparable hemolytic activity to the wild type. Interestingly, the A61C mutant exhibited high larvicidal activity to both Aedes aegypti and Culex quinquefasciatus. S108C and V109A mutants showed low activity against C. quinquefasciatus but relatively high toxicity to A. aegypti. These results demonstrated for the first time that amino acids in alphaA and alphaC are involved in the selectivity of the Cyt toxin to the targeted organism.     

Identification of amino acid residues in BK virus VP1 that are critical for viability and growth

BK virus (BKV) is a ubiquitous pathogen that establishes a persistent infection in the urinary tract of 80% of the human population. Like other polyomaviruses, the major capsid protein of BKV, virion protein 1 (VP1), is critical for host cell receptor recognition and for proper virion assembly. BKV uses a carbohydrate complex containing alpha(2,3)-linked sialic acid attached to glycoprotein and glycolipid motifs as a cellular receptor. To determine the amino acids important for BKV binding to the sialic acid portion of the complex, we generated a series of 17 point mutations in VP1 and scored them for viral growth. The first set of mutants behaved identically to wild-type virus, suggesting that these amino acids were not critical for virus propagation. Another group of VP1 mutants rendered the virus nonviable. These mutations failed to protect viral DNA from DNase I digestion, indicating a role for these domains in capsid assembly and/or packaging of DNA. A third group of VP1 mutations packaged DNA similarly to the wild type but failed to propagate. The initial burst size of these mutations was similar to that of the wild type, indicating that there is no defect in the lytic release of the mutated virions. Binding experiments revealed that a subset of the BKV mutants were unable to attach to their host cells. These motifs are likely important for sialic acid recognition. We next mapped these mutations onto a model of BKV VP1 to provide atomic insight into the role of these sites in the binding of sialic acid to VP1.     

Role of sialic acid in bovine sperm-zona pellucida binding

Sperm binding activity has been detected in zona pellucida (ZP) glycoproteins and it is generally accepted that this activity resides in the carbohydrate moieties. In the present study we aim to identify some of the specific carbohydrate molecules involved in the bovine sperm-ZP interaction. We performed sperm binding competition assays, in vitro fecundation (IVF) in combination with different lectins, antibodies and neuraminidase digestion, and chemical and cytochemical analysis of the bovine ZP. Both MAA lectin recognising alpha-2,3-linked sialic acid and neuraminidase from Salmonella typhimurium with catalytic activity for alpha-2,3-linked sialic acid, demonstrated a high inhibitory effect on the sperm-ZP binding and oocyte penetration. These results suggest that bovine sperm-ZP binding is mediated by alpha-2,3-linked sialic acid. Experiments with trisaccharides (sialyllactose, 3'-sialyllactosamine and 6'-sialyllactosamine) and glycoproteins (fetuin and asialofetuin) corroborated this and suggest that at least the sequence Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc is involved in the sperm-ZP interaction. Moreover, these results indicate the presence of a sperm plasma membrane specific protein for the sialic acid. Chemical analysis revealed that bovine ZP glycoproteins contain mainly Neu5Ac (84.5%) and Neu5GC (15.5%). These two types of sialic acid residues are probably linked to Galbeta1,4GlcNAc and GalNAc by alpha-2,3- and alpha-2,6-linkages, respectively, as demonstrated by lectin cytochemical analysis. The use of a neuraminidase inhibitor resulted in an increased number of spermatozoa bound to the ZP and penetrating the oocyte. From this last result we hypothesize that a neuraminidase from cortical granules would probably participate in the block to polyspermy by removing sialic acid from the ZP.